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Universidade Federal do Rio Grande do Sul
Biologia de células-tronco mesenquimais pós-natais
Lindolfo da Silva Meirelles
Tese submetida ao Programa de Pós-
Graduação em Genética e Biologia
Molecular da UFRGS como requisito
parcial para a obtenção do grau de
Doutor em Ciências
Orientadora: Profa. Dra. Nance Beyer Nardi
Orientador no exterior: Prof. Dr. Arnold I. Caplan
Porto Alegre
Abril de 2007
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Este trabalho foi desenvolvido no Laboratório de Imunogenética do Departamento de
Genética, Instituto de Biociências da Universidade Federal do Rio Grande do Sul, e no
Skeletal Research Center da Case Western Reserve University (Cleveland, OH, EUA) com
o auxílio financeiro do CNPq.
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Dedicada a minha esposa, Adriana, e a meus pais, Sônia e Telmo.
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Agradecimentos
Nance, obrigado por me abrir as portas de um laboratório onde pude exercer minhas
atividades, e também por permitir que eu pudesse colocar em prática minha criatividade e
meu entusiasmo no intuito de fazer Ciência. Obrigado também por permitir e incentivar
minha busca por independência científica.
Manifesto aqui minha gratidão ao Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) por ter-me concedido uma bolsa de doutorado. Devo também ao
CNPq a chance de permanecer por seis meses no exterior, adquirindo conhecimentos em
um laboratório de excelência em minha área de pesquisa.
My thanks are also due to The Company of Biologists that, by granting me a Travelling
Fellowship through Journal of Cell Science, helped me visit Dr. Arnold I. Caplan’s
laboratory in the US.
Arnold, thanks for accepting me as a visiting student, for the help with my installation and
for helping Adriana find a place where she could do some shadowing while we were there.
Most importantly, thanks for sharing your scientific experience with me.
Thanks also to everyone in the Caplan lab during my stay there, namely Andreas, Dave,
Debbie, Don, Jean, Kitsie, Lisa, Margie, Marilyn, Michael and Paul, for helping me out
with everything.
Andrés, Elmo, Luisa, Melissa, Pedro, Renata e Zéca: obrigado por todas as vezes em que
me ajudaram, em todos os sentidos.
And last, but not least, I didn’t forget you, Pam. Thanks for your help with everything not
only for the period of our stay there in the US, but also during all this time.
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Sumário
Lista de abreviaturas.................................................................................................. 6
Resumo....................................................................................................................... 7
Abstract...................................................................................................................... 8
Capítulo 1 – Introdução.............................................................................................. 9
Células-tronco...................................................................................................... 10
A medula óssea e o estroma medular................................................................... 10
A célula-tronco mesenquimal.............................................................................. 11
Obtenção e caracterização da MSC...................................................................... 12
Fontes adicionais de MSCs.................................................................................. 13
Aplicações da MSC.............................................................................................. 14
Capítulo 2 – Objetivos............................................................................................... 16
Capítulo 3 – Mesenchymal stem cells reside in virtually all post-natal organs and
tissues.................................................................................................... 18
Capítulo 4 – Manuscrito em preparação: Tetraploidy in long-term cultured murine
mesenchymal stem cells: a link between cultured mesenchymal stem
cells and the perivascular niche?........................................................... 29
Capítulo 5 – Manuscrito em preparação: Towards a unifying concept of the
identity and natural distribution of mesenchymal stem cells................ 43
Capítulo 6 – Manuscrito em preparação: Human mesenchymal stem cells take up
perivascular locations when implanted in ceramic cubes in vivo......... 78
Capítulo 7 – Discussão............................................................................................... 96
Capítulo 8 – Referências bibliográficas..................................................................... 102
Capítulo 9 – Anexos................................................................................................... 109
Mesenchymal stem cells: isolation, in vitro expansion and characterization...... 110
Functional characterization of cell hybrids generated by induced fusion of
primary porcine mesenchymal stem cells with an immortal murine cell line...... 145
Polyethylene glycol-mediated fusion between primary mouse mesenchymal
stem cells and mouse fibroblasts generates hybrid cells with increased
proliferation and altered differentiation............................................................... 161
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Lista de abreviaturas
CFU-F - colony forming unit-fibroblast, unidade formadora de colônia de fibroblastos
DNA - deoxyribonucleic acid, ácido desoxiribonucléico
FGF-2 - fibroblast growth factor 2, fator de crescimento de fibroblasto 2
G-CSF - granulocyte colony-stimulating factor, fator estimulatório de colônia de
granulócitos
GM-CSF – granulocyte macrophage colony-stimulating factor, fator estimulatório de
colônia de granulócitos macrófagos
HSC - hematopoietic stem cell, célula-tronco hematopoiética
LIF - leukemia inhibitory factor, fator inibitório de leucemia
M-CSF – monocyte colony-stimulating factor, fator estimulatório de colônia de monócitos
mMSC - murine mesenchymal stem cell, célula-tronco mesenquimal murina
MSC - mesenchymal stem cell, célula-tronco mesenquimal
OI - osteogenesis imperfecta
SCF - stem cell factor, fator de célula-tronco
VLA-1 - very late activation antigen 1, antígeno de ativação tardia 1 (integrina composta
pelas cadeias α1 e β1)
VLA-2 - very late activation antigen 1, antígeno de ativação tardia 2 (integrina composta
pelas cadeias α2 e β1)
VLA-3 - very late activation antigen 1, antígeno de ativação tardia 3 (integrina composta
pelas cadeias α3 e β1)
VLA-4 - very late activation antigen 1, antígeno de ativação tardia 4 (integrina composta
pelas cadeias α4 e β1)
VLA-5 - very late activation antigen 1, antígeno de ativação tardia 5 (integrina composta
pelas cadeias α5 e β1)
VLA-6 - very late activation antigen 1 antígeno de ativação tardia 6 (integrina composta
pelas cadeias α6 e β1)
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Resumo
Células-tronco mesenquimais (MSCs) são um tipo de célula-tronco pós-natal que se
mostram muito promissoras como ferramentas terapêuticas porque elas exibem grande
plasticidade, e podem ser isoladas e manipuladas de modo reprodutível e com poucos ou
nenhum problema ético. Elas foram inicialmente descritas há mais de 30 anos, sob a
designação de unidades formadoras de colônia de fibroblasto, e a maior parte do nosso
conhecimento sobre elas advém de estudos in vitro. Compreender o comportamento das
MSCs in vivo. é um fator chave para o desenvolvimento de terapias celulares eficientes e
para engenharia tecidual. Atualmente, as localização e função reais de MSCs in vivo ainda
são pouco compreendidas. Em uma tentativa de melhor compreender a biologia da MSC,
células apresentando características de tronco mesenquimal foram isoladas de vários
tecidos diferentes de camundongos adultos, e foram caracterizadas in vitro. Os resultados
obtidos, conjuntamente com dados da literatura, indicaram que as populações celulares
obtidas eram derivadas da vasculatura, mais especificamente da região perivascular.
Conseqüentemente, um modelo em que células perivascular ao longo dos vasos sangüíneos
constituem uma reserva de células tronco/progenitoras para os tecidos a que pertencem foi
concebido. Constatou-se que o conteúdo de DNA das células cultivadas era, em geral,
tetraplóide, e esse resultado foi tomado como mais uma evidência a favor da visão de
MSCs como células perivasculares, uma vez que tetraploidização em células
perivasculares in vivo foi relatada como sendo usual em roedores. Uma análise das
evidências indicando ligações entre MSCs e pericitos também foi realizada. Finalmente,
constatou-se que MSCs humanas inseridas em cubos de cerâmica e implantadas em
camundongos imunocomprometidos assumem uma localização perivascular, além de gerar
tecido ósseo, dando mais embasamento para a visão de que MSCs cultivadas in vitro
descendem de células perivasculares. Tomados em conjunto, as informações obtidas
indicam que o compartimento perivascular abriga células tronco/progenitoras ao longo de
toda sua extensão, e que MSCs isoladas classicamente da medula óssea são provavelmente
um subtipo de célula-tronco perivascular.
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Abstract
Mesenchymal stem cells (MSCs) are a type of post-natal stem cell that holds great promise
as therapeutic tools because they exhibit great plasticity, and can be isolated and
manipulated in a reproducible fashion with little or no ethical issues. They were initially
described more than 30 years ago, under the designation of colony-forming unit-
fibroblasts, and most of our current knowledge on them comes from in vitro studies.
Understanding the behavior of MSCs in vivo is a key factor for the development of
efficient cell-based therapies and for tissue engineering. To date, the actual location and
function of MSCs in vivo are still poorly understood. In an attempt to better understand
MSC biology, cells bearing mesenchymal stem characteristics were isolated from several
different tissues of adult mice and were characterized in vitro. The results obtained, along
with data from the literature, indicated the cell populations obtained were derived from the
vasculature, more specifically from the perivascular region. As a consequence, a
theoretical model in which perivascular cells along the blood vessels constitute a reservoir
of stem/progenitor cells for the tissues where they belong was drawn. The DNA content of
the cultured cells was found to be generally tetraploid, and this finding was taken as one
more evidence towards the view of MSCs as perivascular cells, since tetraploidization in
perivascular cells in vivo has been reported as usual in rodents. An analysis of the
evidences indicating links between MSCs and pericytes was also performed. Finally,
human MSCs loaded in ceramic cubes and implanted into immunocompromised mice were
found to take up perivascular locations in addition to generate osseous tissue, providing
further support for the view that in vitro cultured MSCs descend from perivascular cells.
Taken together, the informations obtained indicate that the perivascular compartment
harbors stem/progenitor cells throughout its extent, and that MSCs classically isolated from
bone marrow are probably one subtype of perivascular stem cell.
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Capítulo 1
Introdução
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Células-tronco
Células-tronco podem ser definidas como aquelas capazes de auto-renovação
ilimitada ou prolongada e que podem também dar origem a pelo menos um tipo celular em
estágio de diferenciação mais avançado (revisado por Morrison et al., 1997; Watt e Hogan,
2000). A célula-tronco embrionária é totipotente e dá origem a todas as células do
organismo (revisado por Odorico et al., 2001; van der Kooy e Weiss, 2000). Indivíduos
adultos também possuem células-tronco, mas estas não são totipotentes como a
embrionária. Em humanos adultos, a primeira célula-tronco relatada foi a hematopoiética
(hematopoietic stem cell, HSC) (revisado por Weissman, 2000). Esta célula localiza-se na
medula óssea, e já foi extensivamente caracterizada, demonstrando ser uma célula
multipotente que dá origem às diferentes células do sangue (revisado por Nardi e Alfonso,
1999).
Além da HSC, outras células-tronco pós-natais já foram descritas. As células-tronco
epitelial, neural e mesenquimal podem ser apontadas como exemplos. A primeira é
encontrada no intestino e na epiderme, e dá origem a células em camadas epiteliais
(revisado por Slack, 2000). A célula-tronco neural situa-se no cérebro, e origina neurônios,
astrócitos, oligodendrócitos e, surpreendentemente, células sangüíneas (McKay, 1997;
Bjornson et al., 1999; Gage, 2000). A célula-tronco mesenquimal (mesenchymal stem cell,
MSC), é encontrada na medula óssea e gera ossos, tendão, cartilagem, tecidos adiposo e
muscular, estroma medular e até mesmo células com características neurais (Caplan, 1991;
Prockop, 1997; Pittenger et al., 1999; Kopen et al., 1999).
A medula óssea e o estroma medular
O compartimento medular é muitas vezes descrito como composto basicamente por
três sistemas celulares: hematopoiético, endotelial e estromal. O termo estroma refere-se ao
conjunto composto pelo sistema celular estromal – que inclui fibroblastos, células
endoteliais, células reticulares, adipócitos e osteoblastos – conjuntamente com a matriz
extracelular a ele associada, bem como outros tipos celulares, tais como macrófagos
(revisado por Deans e Moseley, 2000). Estes, embora sejam de origem hematopoiética, são
considerados componentes estromais funcionais (Dexter, 1982). O referido sistema celular
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estromal foi proposto por Owen em 1985 e baseia-se em uma analogia com o sistema
hematopoiético. Neste modelo, células-tronco estromais residem na medula óssea, mantêm
um determinado grau de auto-renovação, e dão origem a células que podem diferenciar-se
em várias linhagens de tecido conjuntivo e em tecidos estromais. Além dos sistemas
celulares mencionados acima, é importante ressaltar que o compartimento medular abriga
também um sistema nervoso, que desempenha funções importantes na integração da
hematopoiese com o sistema nervoso central (Elenkov et al.,, 2000).
As células do estroma medular garantem suporte mecânico às células
tronco/precursoras hematopoiéticas, além de produzirem matriz extracelular e fatores
solúveis que regulam a hematopoiese (Dexter, 1982). Proteínas da matriz, tais como
fibronectina, colágeno, vitronectina e tenascina atuam em conjunto com fatores solúveis
como o fator de célula-tronco (stem cell factor, SCF), citocinas como o fator estimulatório
de colônia de granulócitos-macrófagos (granulocyte-macrophage colony-stimulating
factor, GM-CSF) e o fator estimulatório de colônia de granulócitos (granulocyte colony-
stimulating factor, G-CSF), e moléculas de adesão tais como as da superfamília das
integrinas (VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6) na constituição do nicho da
célula-tronco hematopoiética (revisado por Whetton e Grahan, 1999).
O contato físico direto entre as células do estroma também é relevante para a
regulação desse microambiente. Embora já tenha sido demonstrado que esse contato não é
fundamental para que a hematopoiese ocorra (Verfaillie, 1992), existem evidências de que
ele esteja relacionado com a qualidade das células hematopoiéticas produzidas (Breems et
al., 1998).
A célula-tronco mesenquimal
A primeira evidência direta de que a medula óssea contém células precursoras de
tecidos mesenquimais não hematopoiéticos advém do trabalho de Friedenstein e
colaboradores, iniciado em Moscou nos anos 60 - 70 (revisado por Phinney, 2002). Em
seus experimentos, Friedenstein dispensava amostras de medula óssea suspensas em meio
de cultura em frascos de cultura de tecidos, e a fração aderente era cultivada. Por volta do
terceiro ao quinto dia, focos discretos de dois a quatro fibroblastos surgiam nas culturas,
entre histiócitos e células mononucleares (Friedenstein et al., 1976). Em uma revisão sobre
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o assunto, Prockop (1997) acrescenta que a característica mais marcante dessas células era
sua capacidade de se diferenciarem em colônias que lembravam pequenos depósitos de
osso ou cartilagem. Nos anos 80, diversos estudos estabeleceram que células isoladas pelo
método de Friedenstein eram multipotentes e capazes de se diferenciarem em osteoblastos,
condroblastos, adipócitos, e até mioblastos (revisado por Prockop, 1997). Em 1991,
Caplan, tomando como base modelo do sistema estromal proposto por Owen em 1985,
criou o termo célula-tronco mesenquimal para designar a célula que dá origem aos
diferentes tipos de tecidos mesenquimais da medula óssea.
Obtenção e caracterização da MSC
MSCs são encontradas na medula óssea de indivíduos adultos, onde estão imersas
no estroma (Pittenger et al., 1999). Sua freqüência neste tecido é baixa. Wexler et al.
(2003) estimaram que há 1 MSC em cada 34.000 células nucleadas da medula óssea
humana por meio de ensaios de unidade formadora de colônia de fibroblastos (CFU-F). Em
um estudo preliminar, a freqüencia dessas células na medula óssea murina foi estimada ser
1 em uma faixa de 11.300 – 27.000 células nucleadas (Meirelles e Nardi, 2003).
Os protocolos utilizados para a obtenção da MSC envolvem basicamente o cultivo
das células aderentes da medula óssea em placas plásticas (Haynesworth et al., 1992;
MacKay et al., 1998; Pittenger et al., 1999, Muraglia et al., 2000; Makino et al., 1999;
Wakitani et al., 1995), embora protocolos envolvendo imunodepleção de contaminantes
hematopoiéticos antes do cultivo primário já tenham sido utilizados (Kopen et al., 1999;
Badoo et al., 2003).
A expansão in vitro prolongada das células humanas obtidas pelas técnicas
convencionais tem sido difícil de ser obtida (Sekiya et al., 2002), e por isso ainda existem
poucos dados abrangentes retratando o quão capazes de proliferação e diferenciação
hMSCs são. Há, no entanto, trabalhos que demonstram que a proliferação de MSCs
humanas in vitro pode ser aumentada por transdução retroviral de construções contendo o
cDNA codificante da subunidade catalítica da telomerase humana (Okamoto et al., 2002;
Mihara et al., 2003), ou por adição de fator de crescimento de fibroblasto 2 (fibroblast
growth factor 2, FGF-2) (Bianchi et al., 2003). MSCs de camundongo, por sua vez, podem
ser expandidas in vitro por período de tempo ainda indeterminado, sem manipulação
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genética ou adição de fatores de crescimento além daqueles presentes no soro fetal bovino
(Meirelles e Nardi, 2003).
Vários marcadores moleculares já foram descritos para progenitores mesenquimais
comprometidos e para os estágios fenotípicos finais de células originadas da MSC (Seshi et
al., 2000; Long, 2001; Bruder et al., 1994). Marcadores moleculares candidatos para a
definição de MSCs humanas estão incluídos numa longa lista publicada em uma revisão
feita por Deans e Moseley (2000). Dentre estes, podem-se destacar as moléculas de
superfície CD44, CD29, CD90, e os fatores secretados LIF (leukemia inhibitory factor,
fator inibitório de leucemia), M-CSF (monocyte-colony-stimulating factor, fator
estimulador de colônia de monócitos), e SCF. Até pouco tempo, a verificação dos
marcadores específicos da MSC murina (mMSC) ainda não era possível devido à
dificuldade de obtenção de uma população pura através das técnicas convencionais
(Phinney et al., 1999). Trabalhos recentes têm colaborado para reverter esta situação
(Wieczorek et al., 2003; Badoo et al., 2003, Meirelles e Nardi, 2003).
A caracterização dos marcadores da MSC observados durante seu cultivo in vitro,
conforme descrito no parágrafo anterior, não teria validade se a população de células em
estudo não tivessem características funcionais de tronco e mesenquimal confirmadas, ou
seja: células em cultura podem ser operacionalmente consideradas MSCs se forem capazes
de auto-renovação e também de dar origem, em condições adequadas de cultivo, a
diferentes tipos celulares mesenquimais, tais como adipócitos, condrócitos e osteócitos.
Diferenciação neuronal (Woodbury et al., 2000; Zuk et al., 2002) e suporte à hematopoiese
(Majumdar et al., 1998) são características também atribuídas a MSCs, e também devem
ser levadas em consideração. Finalmente, para que o termo “operacional” possa ser
deixado de lado é necessária, ainda, a demonstração de que tais células contribuem para a
formação de tecidos mesenquimais quando infundidas in vivo (revisado por Verfaillie,
2002).
Fontes adicionais de MSCs
Além de estar presente na medula óssea de indivíduos adultos, a MSC também pode
ser encontrada em outros tecidos e fases do desenvolvimento (Tabela 1).
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Tabela 1. Ocorrência de células com características de tronco mesenquimal em diferentes
órgãos/tecidos, durante o período pós-embrionário.
Local Espécie Período de vida Referência (s)
Tecido adiposo
Homo sapiens
pós-natal
Zuk et al., 2001; Zuk et
al., 2002
Tecido adiposo
Mus musculus
pós-natal Safford et al., 2002
Pâncreas
Homo sapiens
fetal Hu et al.,2003
Medula óssea
Homo sapiens
fetal Campagnoli et al., 2001
Fígado
Homo sapiens
fetal Campagnoli et al., 2001
Sangue
Homo sapiens
fetal Campagnoli et al., 2001
Tendão
Mus musculus
pós-natal
Salingcarnboriboon et al.,
2003
Membrana sinovial
Mus musculus
pós-natal de Bari et al., 2003
Líquido amniótico
Homo sapiens
fetal in ‘t Anker et al., 2003
Sangue periférico
Homo sapiens
pós-natal
Zvaifler et al., 2000;
Kuwana et al., 2003
Sangue de cordão
umbilical
Homo sapiens
fetal/pós-natal Alfonso et al., 2000
A distribuição de células com características de MSC no organismo pós-natal foi
estudada em maior detalhe no primeiro artigo desta tese (Capítulo 3).
Aplicações da MSC
MSCs já vêm sendo exploradas há algum tempo com o propósito de se obter
regeneração esquelética (Bruder et al., 1994). Seu potencial de diferenciação em múltiplas
linhagens, sua sensibilidade elevada a moléculas sinalizadoras específicas e uma relativa
facilidade de manuseio in vitro (Caplan e Bruder, 2001; Beyer Nardi e da Silva Meirelles,
2006) as torna ferramentas importantes para a engenharia de tecidos, e para terapias
celulares.
O potencial de utilização de MSCs para engenharia tecidual é reforçado ainda por
dois fatores: a) elas podem diferenciar-se em tipos celulares não-mesenquimais in vitro
(neurônios – Woodbury et al., 2000) e in vivo (células neurais – Kopen et al., 1999;
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hepatócitos – Sato et al., 2005); e b) elas exibem a tendência de adquirir características
tecido-específicas quando co-cultivadas com tipos celulares especializados ou quando
expostas a extratos teciduais in vitro (células de câncer gástrico – Houghton et al., 2004;
células secretoras de insulina – Choi et al., 2005; hepatócitos – Lange et al., 2005).
A MSC também apresenta potencial para o tratamento de doenças caracterizadas
pela deficiência do produto de algum gene, de duas formas: a) utilizando-se a MSC per se,
quando a deficiência genética puder ser suprida por seu patrimônio genético, ou b)
modificando-se a MSC para que ela expresse altos níveis do produto gênico em questão,
quando a primeira estratégia não é eficaz. Insere-se no primeiro caso a tentativa bem
sucedida de correção da osteogenesis imperfecta (OI) – doença genética causada pela
deficiência da produção de colágeno tipo I - em crianças utilizando-se transplante de
medula óssea de doadores compatíveis (Horwitz et al., 1999; Horwitz et al., 2001). Esse
trabalho baseou-se no fato de que a medula óssea contém MSCs capazes de inserir-se em
vários pontos do organismo quando injetadas na circulação, de diferenciar-se em células
envolvidas na produção de ossos e cartilagem e músculo, e que podem auxiliar a
regeneração desses tecidos por suprirem a deficiência de colágeno tipo I nos indivíduos
receptores.
Outras características que fazem da MSC uma boa candidata a agente terapêutico
são seus efeitos imunomodulatórios, demonstrados quando usadas no tratamento de um
paciente com doença do enxerto contra o hospedeiro (Le Blanc et al, 2004), e também seus
efeitos tróficos (Caplan e Dennis, 2006), que podem ser úteis para o tratamento de
acidentes vasculares cerebrais.
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Capítulo 2
Objetivos
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Em vista das características da MSC aqui apresentadas, este trabalho teve os
seguintes objetivos:
Conhecer melhor a biologia da MSC, através da caracterização de MSCs obtidas de
órgãos/tecidos de camundongos adultos que não a medula óssea, e da comparação das
mesmas entre si, utilizando os critérios já estabelecidos para a mMSC derivada da medula
óssea (Meirelles e Nardi, 2003);
Checar mMSCs obtidas quanto a alterações genéticas grosseiras, tais como aneuploidias,
através da observação de seu conteúdo de DNA;
Compreender a relação da MSC com o nicho perivascular in vivo.
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Capítulo 3
Mesenchymal stem cells reside in virtually all post-natal
organs and tissues
Lindolfo da Silva Meirelles, Pedro Pedro Cesar Chagastelles & Nance Beyer Nardi
J Cell Sci. 2006 Jun 1;119(Pt 11):2204-13.
18
2204 Research Article
Introduction
Stem cells are defined as having the capacity for extensive self-
renewal and for originating at least one type of highly
differentiated descendant (Watt and Hogan, 2000). Post-natal
tissues have reservoirs of specific stem cells which contribute
to maintenance and regeneration. Examples include epithelial
stem cells in epidermis and intestinal crypts (Slack, 2000),
neural stem cells in the central nervous system (McKay, 1997)
and satellite cells in muscle (Charge and Rudnicki, 2004). The
adult bone marrow shelters different types of stem cells,
including hematopoietic (Weissman, 2000) and mesenchymal
(Prockop, 1997; Nardi and da Silva Meirelles, 2006) stem cells.
Experiments using bone marrow cells have raised the issue
of phenotypic plasticity (Herzog et al., 2003; Wagers and
Weissman, 2004), because they have shown the consequent
generation of specialized cells derived from bone marrow in
the central nervous system (Eglitis and Mezey, 1997; Mezey et
al., 2000), skeletal muscle (Ferrari et al., 1998), liver (Petersen
et al., 1999) and heart (Orlic et al., 2001). The specific cell
type(s) involved in these phenomena is not clear. However,
reports describing mesenchymal stem cell (MSC)
differentiation capabilities suggest that they may contribute to
the results observed: they can differentiate into specific cell
types in vitro and in vivo (Woodbury et al., 2000; Kopen et al.,
1999; Sato et al., 2005), and have a tendency to acquire tissue-
specific characteristics when co-cultured with specialized cell
types or exposed to tissue extracts in vitro (Houghton et al.,
2004; Choi et al., 2005; Lange et al., 2005). In addition, the
capacity to differentiate into mesodermal (Pittenger et al.,
1999), ectodermal (Kopen et al., 1999) and endodermal (Sato
et al., 2005) cell lineages characterizes MSCs as pluripotent
cells, suggesting that the term ‘mesenchymal’ stem cell might
be inappropriate to describe this particular stem cell. Since
different methodologies are used to cultivate and characterize
MSC-related cell types, there is still a lack of consensus on the
hierarchy intrinsic to the MSC compartment (Nardi and da
Silva Meirelles, 2006), reflected by the existence of similar
cells such as multipotent adult progenitor cells (MAPCs)
(Reyes et al., 2001), marrow-isolated adult multilineage
inducible (MIAMI) cells (D’Ippolito et al., 2004), and
recycling stem cells (RS-1, RS-2) (Colter et al., 2000).
The exact nature and localization of MSCs in vivo remain
poorly understood; growing evidence indicates a relationship
with pericytes (Doherty et al., 1998; Farrington-Rock et al.,
2004). Approaches so far include the use of selected markers
known to be expressed by MSCs in vitro to seek positive cells
in vivo (Bianco et al., 2001; Shi and Gronthos, 2003) and the
infusion of marked cultured cells in vivo to analyze their tissue
distribution (Anjos-Afonso et al., 2004). The first type of
approach, though sensitive, may be nonspecific as the majority
of cell markers are specific only in a given context. The second
strategy may be less accurate to study MSC natural distribution
in vivo because the cells might engraft nonspecifically in
different locations. The systematic isolation of MSCs from
different organs and tissues and the evaluation of their
characteristics could represent an alternative approach. Studies
describing the isolation of post-natal mesenchymal stem cells
from different sources – e.g. adipose tissue (Zuk et al., 2001),
Mesenchymal stem cells (MSCs) are multipotent cells
which can give rise to mesenchymal and non-mesenchymal
tissues in vitro and in vivo. Whereas in vitro properties
such as (trans)differentiation capabilities are well known,
there is little information regarding natural distribution
and biology in the living organism. To investigate the
subject further, we generated long-term cultures of cells
with mesenchymal stem cell characteristics from different
organs and tissues from adult mice. These populations have
morphology, immunophenotype and growth properties
similar to bone marrow-derived MSCs. The differentiation
potential was related to the tissue of origin. The results
indicate that (1) cells with mesenchymal stem
characteristics can be derived and propagated in vitro from
different organs and tissues (brain, spleen, liver, kidney,
lung, bone marrow, muscle, thymus, pancreas); (2) MSC
long-term cultures can be generated from large blood
vessels such as the aorta artery and the vena cava, as well
as from small vessels such as those from kidney glomeruli;
(3) MSCs are not detected in peripheral blood. Taken
together, these results suggest that the distribution of MSCs
throughout the post-natal organism is related to their
existence in a perivascular niche. These findings have
implications for understanding MSC biology, and for
clinical and pharmacological purposes.
Key words: Mesenchymal stem cell, Pericyte, CFU-F, Mouse, In
vitro cultivation
Summary
Mesenchymal stem cells reside in virtually all
post-natal organs and tissues
Lindolfo da Silva Meirelles, Pedro Cesar Chagastelles and Nance Beyer Nardi*
Departamento de Genética, Universidade Federal do Rio Grande do Sul, Av Bento Gonçalves 9500, 91540-970 Porto Alegre, RS, Brazil
*Author for correspondence (e-mail: [email protected])
Accepted 13 February 2006
Journal of Cell Science 119, 2204-2213 Published by The Company of Biologists 2006
doi:10.1242/jcs.02932
Journal of Cell Science
19
2205
In vivo distribution of mesenchymal stem cells
tendon (Salingcarnboriboon et al., 2003), periodontal ligament
(Seo et al., 2004), synovial membrane (De Bari et al., 2001)
and lungs (Sabatini et al., 2005) – can be found in the literature;
however, these isolated studies do not allow the consistent
visualization of the distribution of MSCs in the post-natal
organism.
Here, we analyzed the in vivo distribution of murine post-
natal MSCs through the establishment, long-term culture and
functional characterization of MSC populations from different
tissues and organs. The possibility that MSC cultures were
partially or entirely derived from circulating blood was
excluded by perfusing the animals intravascularly before
organ collection. Moreover, no MSC long-term culture could
be established from blood when a controlled protocol to
minimize vessel rupture was used. Our data demonstrate that
the MSC compartment is more widely distributed than
previously thought and we present evidence that MSCs are
resident in vessel walls. Variations in immunophenotype and
osteogenic or adipogenic differentiation potential according to
the site of origin suggest that functional roles are at least
partially organ specific. These findings provide insight on the
biology of MSCs in vivo, and add new information to be
considered when developing clinical protocols involving the
MSC compartment.
Results
Establishment and morphological characterization of
long-term cultures
The establishment of long-term cultures was highly dependent
on the conditions used to set up the primary culture. For
instance, the use of DMEM with 10 mM HEPES instead of
common buffered saline to dissolve collagenase, and a
digestion time limited to up to 1 hour at 37°C proved to be
essential for reproducibility (not shown). Other important
factors were the elapsed time between euthanasia and tissue
processing – the shorter the better, particularly for bone
marrow and liver – and the temperature of the culture medium,
which should not be lower than room temperature. The amount
of medium used during the collagenase digestion step was also
important for the establishment of brain-derived cultures,
because this organ rapidly acidifies small volumes of medium.
Furthermore, when establishing primary cultures from
perfused animals, we observed that using DMEM containing
10 mM HEPES and HB-CMF-HBSS (1:1), rather than culture
medium or saline alone, seemed to improve the quality of the
MSC-like cells in primary culture.
The MSC long-term cultures generated during this study are
described in Table 1. Not all cultures, in particular the earlier
ones, were generated using the optimized conditions described
above. To minimize eventual differences due to different
starting conditions, the analyses were mainly focused on
population sets established under similar conditions, even
though gross differences, as judged by flow cytometric
analyses, morphological and functional characteristics, were
not observed among long-term cultures generated in any of the
conditions (not shown).
Using the optimized conditions mentioned above, cells that
morphologically resembled characterized MSCs could be seen
as early as 24 hours post-plating (Fig. 1A). In the case of
glomerular cell culture, adherent cell outgrowths could be
observed from the third or fourth day onwards (Fig. 1B).
Depending on the starting amount and on which tissue was
used to establish the cultures, confluence could be reached
within 5 days. Individual glomerular cultures did not reach
confluence even when cultured for over a month despite robust
initial cell growth (Fig. 1B,C), possibly because of the contact
inhibition among the cells in each colony after some cell
divisions; on the other hand, cultures containing 20 glomeruli
or more per well could become confluent within three weeks
or so, if the outgrowths were evenly distributed on the bottom
of the dish. During the initial culture period (passages 1 to 5)
there was some morphological heterogeneity in the adherent
fraction, particularly in bone marrow and spleen cultures,
possibly because of the presence of hematopoietic
contaminants (Fig. 1D). As the cultures were passaged,
morphological homogeneity was gradually achieved in that flat
cells bearing a large nucleoli-rich nucleus predominated; this
was independent of the origin of the culture (Fig. 1E,F).
Glomeruli-derived explants, on the other hand, showed this
morphology right from the start of culture (Fig. 1B). The
expression of surface markers was analyzed by flow cytometry
preferably at this time (see below).
During the establishment of primary cultures, special care
was taken to avoid contamination by adjacent tissues. Muscle
was thoroughly removed from femora and tibiae before bone
Table 1. MSC populations generated and donor animals
Age MSC populations
Animal Strain (weeks) Gender Perfused generated
001B BALB/c 14 Male No 001Bbm
002B BALB/c 15 Male No 002Bs
004B BALB/c 8 Female No 004Bbm, 004Bs,
004Bt, 004Bk
005B BALB/c 9 Male No 005Bs
006B BALB/c 11 Male No 006Bb
009B BALB/c 16 Female No 009Bl
010B BALB/c 16 Female No 010Bb
011B BALB/c 10 Male No 011Bt
012B BALB/c 37 Male No 012Ba
013B BALB/c 37 Male No 013Ba1, 013Ba2
014B BALB/c 30 Male No 014Ba1, 014Ba2
015B BALB/c 31 Male No 015Ba1, 015Ba2,
015Bs, 015Bl
016B BALB/c 30 Male Yes 016Ba1, 016Ba2,
016Bs, 016Bm,
016Bk
017B BALB/c 43 Male Yes 017Bk
018B BALB/c 18 Female Yes 018Ba1, 018Ba2,
018Bs, 018Bm,
018Bk, 018Bl
019B BALB/c 20 Female Yes 019Bs, 019Bbm,
019Blu, 019Bk
021B BALB/c 27 Female No 021Bkg
001C C57Bl/6 39 Female No 001Ck
002C C57Bl/6 9 Male No 002Cbm
008C C57Bl/6 35 Female No 008Clu, 008Cvc
009C C57Bl/6 39 Female Yes 009Ck
001R ROSA26 20 Male No 001Rlu
003R ROSA26 13 Male No 003Rbm, 003Rp
001G GFP 22 Male No 001Gvc
In the right column, lowercase letters indicate the tissue or organ from
which the culture was derived: a, aorta; b, brain; bm, bone marrow; k, kidney;
kg, kidney glomeruli; l, liver; lu, lungs; m, muscle; p, pancreas; s, spleen; t,
thymus; vc, vena cava. In the case of aorta-derived cultures, the numbers after
the ‘a’ indicate the first and second fraction-derived populations (see
Materials and Methods for details).
Journal of Cell Science
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2206
marrow extraction, to avoid contamination with muscle-
derived cells; adipose tissue was likewise separated from the
aorta through serial enzymatic dissociation. In this particular
case, the two fractions obtained could generate long-
term cultures exhibiting the same morphological,
immunophenotypic and kinetic characteristics (not shown),
indicating that the enzymatic fractionation procedure is not
necessary. On the other hand, contamination from surrounding
tissue proved to be a serious issue for blood. The
establishment of MSC-like long-term cultures from blood
collected through cardiac puncture, or from the thoracic
cavity, was possible but not easily reproducible (not shown).
However, attempts to establish MSC cultures from blood
collected from the portal vein were consistently unsuccessful.
We consider this a very important observation, because of two
facts: first, MSC long-term cultures can be generated from
only one cell (see the Cloning section below); second, it is
possible that a few MSCs detach from the walls of ruptured
vessels and are collected with the blood. In this case, the
results would suggest that blood can originate MSC long-term
cultures. The insertion of an intravenous catheter cranially into
the portal vein helps minimize this problem. As the catheter
is introduced contrary to the blood flow, the few cells that
eventually detach from the vessel wall during the needle
insertion are likely to be lost in the circulation before blood
collection starts.
Journal of Cell Science 119 (11)
Immunophenotyping
The analysis of surface markers indicated that the MSC
populations originating from multiple sources have a very
similar immunophenotype. Examples of surface molecule
profiles for selected markers are shown in Fig. 2. All the
populations studied expressed CD29 (integrin 
1
chain) and
CD44 (hyaluronan receptor). The expression of molecules such
as CD34 (hematopoietic progenitor marker), Sca-1 (stem cell
antigen-1) and CD49e (integrin ␣
5
chain) was variable among
the different populations, and could also show variation during
extended subculture (Fig. 3). CD90.2 expression, when
present, was high; however, the proportion of positive cells was
variable, ranging from ~30% to nearly 100%. High expression
of CD117, the stem cell factor receptor, was infrequent; some
populations however seemed to express it at very low levels,
and a small percentage of positive cells could sometimes be
detected. CD117 expression showed a tendency to decrease
during extended serial passage, reaching control levels (Fig. 3),
as reported previously for bone-marrow-derived murine MSCs
(da Silva Meirelles and Nardi, 2003). The granulocyte marker
Gr-1 was expressed in low levels by some populations (not
shown). The hematopoietic markers CD45 and CD11b were
not expressed by MSCs. They were only observed during
the initial passages in cultures derived from bone marrow
and spleen, presenting a small proportion of cells with
morphological characteristics of macrophages, indicating
hematopoietic contamination. MSC cell populations were also
negative for the monocyte marker CD13, the leukocyte markers
CD18 and CD19, the endothelial marker CD31 and surface Ig.
The expression of CD49d (integrin ␣
4
chain) remains to be
analyzed in more detail. When some populations were
collected using either trypsin-EDTA or EDTA alone, CD49d
was observed on cells collected with EDTA but not on those
treated with trypsin, indicating its sensitivity to the enzyme
(not shown).
The immunophenotyping of MSCs at early stages revealed
the heterogeneity within cell populations before an eventual
subpopulation selection owing to extensive cultivation. The
expression of ␣SMA, a vascular smooth muscle cell marker,
was analyzed in cultures that had been tested for their
differentiation potential. All the MSC populations examined
expressed ␣SMA as exemplified by representative results in
Fig. 4, suggesting their relationship to perivascular cells.
Growth kinetics
Growth curves describing culture kinetics were generated as
previously described (da Silva Meirelles and Nardi, 2003).
Representative examples of growth curves for MSC cultures
established from each of the organs or tissues are shown in Fig.
5. The culture kinetics varied depending on the origin of the
cells and the culture stage. In general, the growth rate was low
during the early passages, and increased with serial subculture
(and time) until a stable value was reached. This behavior was
previously reported for bone-marrow-derived murine MSCs
alone (da Silva Meirelles and Nardi, 2003). However,
differences in the kinetics of the cultures related to the tissue of
origin of the MSCs could be observed. The growth rate of brain-
derived MSC cultures, for instance, was slow for a longer period
when compared with the other cultures. In addition, the stable
growth ratios achieved by the different MSC populations,
reflected in the inclination of their growth curves, differed.
Fig. 1. Morphology of MSC cultures derived from different organs
and tissues. (A) Phase-contrast micrographs of MSC-like cells in
primary culture of aorta 24 hours after plating. Glomerulus
outgrowth on the fourth (B) and sixth (C) day post-plating.
(D) Heterogeneity among bone marrow-derived cells at passage 4,
with MSC-like cells (lower portion), spindle-shaped and round cells.
(E) Pancreas-derived MSCs at passage 30. (F) Vena-cava-derived
MSCs at passage 22. Magnifications, ϫ100 (B-F); ϫ200 (A).
Journal of Cell Science
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2207
In vivo distribution of mesenchymal stem cells
Fig. 2. Immunophenotypic profile of MSCs derived from different sources. Flow cytometry histograms show the expression (shaded) of
selected molecules (CD34, Sca-1, CD29, CD44, CD49e, CD90.2 and CD117) by different MSC populations compared with controls (unshaded
peaks). Kidney-derived MSCs and kidney glomerulus-derived MSCs share essentially the same surface profile.
Journal of Cell Science
22
2208 Journal of Cell Science 119 (11)
The growth curves shown in Fig. 5 are based on the area
occupied by the MSCs. To estimate the expansion in terms of
population doublings (PDs), a simple correlation can be made
in that 2
50
, which represents 50 PDs, corresponds to
~1.13ϫ10
15
times the initial population. On the same basis,
2
100
equals ~1.27ϫ10
30
times the initial population, and so on.
The MSC populations depicted in Fig. 5 underwent over 50
PDs, some of them reaching 100-200 PDs. We have not
observed replicative senescence in any murine MSC long-term
cultures. The cultures were generated at different time points
and some of them were cultured longer than others. Most of
them were cryopreserved by the end of this study.
Differentiation
Functional assays to confirm the MSC identity of the
populations studied were preferably performed later (see
below), to evaluate the effect of prolonged cultivation on the
capacity of the cells to differentiate. When subjected to
osteogenic or adipogenic differentiation conditions, the MSC
populations confirmed their mesenchymal stem characteristics
by depositing a calcium-rich mineralized matrix as evidenced
by Alizarin Red S staining, or by acquiring intracellular lipid
droplets, evidenced by Oil Red O staining (Fig. 6).
Differences in the frequency of differentiated cells, as well
as in the degree of differentiation, could be observed among
the cultures originating from different tissues. Vena-cava-
derived MSCs, for instance, were very efficient at depositing
mineralized matrix, whereas muscle-derived MSCs showed
little efficiency (Fig. 6C,G). On the other hand, muscle-derived
MSCs were easily induced to differentiate into mature
Fig. 3. Effect of long-term culture on the expression of CD117,
CD34, Sca-1 and CD49e. Thymus-derived MSCs were analyzed by
flow cytometry at passages 11 and 42. Whereas the level of
expression of Sca-1 was around 2 log values (versus 1 log control),
CD117 expression decreased to nearly control levels, CD34
expression was lost, and expression of CD49e increased 1 log value.
Fig. 4. ␣SMA expression by MSCs. The histograms represent levels
of expression of ␣SMA (shaded) in MSCs from vena cava, bone
marrow, muscle, pancreas and kidney glomeruli compared with
controls (unshaded).
Fig. 5. Comparative growth kinetics of cultures originated from
different organs and tissues. Growth curves of representative MSC
populations from each source (as presented in Table 1) are plotted.
The threshold for 50 population doublings (50 Pds) is shown as a
dashed line. Lowercase letters indicate the tissue or organ from
which the culture was derived: a, aorta; b, brain; bm, bone marrow;
k, kidney; l, liver; lu, lungs; m, muscle; p, pancreas; s, spleen; t,
thymus; vc, vena cava. Numbers and capital letters refer to the
animal used. Growth area is represented as a multiple of the area
occupied by a confluent primary culture, arbitrarily set to 1.
Journal of Cell Science
23
2209
In vivo distribution of mesenchymal stem cells
adipocytes whereas the vena-cava-derived cultures presented
small, poorly developed lipid vacuoles (Fig. 6H,D). Those
differences were maintained even when the cultures were
exposed to differentiation conditions for longer periods. The
differentiation properties of aorta- and bone-marrow-derived
MSCs were similar in terms of efficiency and quality (Fig.
6A,B,E,F). Spleen-, thymus-, lung- and kidney-derived MSCs
exhibited mineralized nodules when subjected to osteogenic
differentiation rather than mineralization of the whole
monolayer (Fig. 6K,M,O,P), in contrast to bone-marrow-
or aorta-derived MSCs (Fig. 6A,E). The adipogenic
differentiation observed in lung-, brain- and kidney-derived
MSCs seemed to be less efficient (Fig. 6L,N; brain-derived
MSC adipogenic differentiation not shown), even though the
degree of adipogenic differentiation presented by kidney- and
lung-derived MSCs was comparable to that of bone-marrow-
derived MSCs (Fig. 6B). Furthermore, these populations
required a longer induction period to differentiate into
adipocytes as compared with bone marrow-derived MSCs.
Glomeruli-derived MSCs, tested at the fifth passage, differed
from the whole-kidney-derived ones regarding osteogenesis:
they deposited a rich mineralized matrix that could be detected
by the first week of differentiation (Fig. 6Q), and were
thus equivalent to bone-marrow-derived MSCs regarding
their osteogenic potential. Glomeruli-derived MSCs could
differentiate into adipocytes (Fig. 6R), similarly to the whole-
kidney-derived cells. Pancreas-derived MSCs also exhibited
osteogenic (Fig. 6S) and adipogenic (Fig. 6T) potential.
The protocols used in this study are quite simple, and the
use of more complex differentiation strategies (e.g. special
substrates and growth factors) might increase the efficiency of
differentiation process, so that gross differences may disappear.
The results obtained with the assays used in the present study,
however, show that the propensity of MSC cultures to respond
to different stimuli varies according to their in vivo location.
In addition to differentiation induced as described above, in
Fig. 6. Differentiation of
MSCs derived from
different sources, as
presented in Table 1. MSCs
were cultured in osteogenic
or adipogenic medium for
up to 2 months. Calcium
deposited in the
extracellular matrix is
stained red by Alizarin Red
S (A,C,E,G,I,K,M,O-Q,S).
Lipid vacuoles are stained
orange with Oil Red O
(B,D,F,H,J,L,N,R,T).
Magnifications and passage
number (P) are indicated
below each image. Cell
lines are identified as
described in the legend to
Fig. 5.
Journal of Cell Science
24
2210
some cases spontaneous differentiation was seen in primary
cultures. In cultures derived from aorta and muscle, for
instance, myotube-like cells and adipocytes were often
observed (Fig. 7). This phenomenon could be caused by the
short-term exposure to amphotericin-B present in antibiotic-
antimycotic solution (Phinney et al., 1999), or by the presence
of myogenic- and adipogenic-committed progenitors in the
primary culture. Spontaneous differentiation was not observed
in primary cultures originating from the other organs and
tissues studied.
Cloning
Two cloning processes were performed, with long-term MSC
cultures. In each of them, three 96-well plates were seeded with
individual cells and analyzed 2 weeks later. The results were
very similar, as presented in Table 2, and showed that around
50% of the cells were able to originate clones, with different
potentials for expansion. The morphology of the clones was
the same observed for the MSC cultures. Eight of the clones
Journal of Cell Science 119 (11)
were selected for further expansion, and were able to establish
cultures with normal growth kinetics, maintained for around 4
months. Two of these cultures were subcloned, and one of the
subclones was submitted to a further subcloning process, with
results similar to those of the initial cloning (Table 2). One
clone from each cloning procedure was tested for the ability to
differentiate along osteogenic or adipogenic pathways and
exhibited differentiation capabilities similar to those of
parental cultures in terms of both efficiency and quality.
Discussion
Mesenchymal stem cells have been conventionally isolated
from bone marrow (Pittenger et al., 1999; Kopen et al., 1999)
and, more recently, from some other tissues (Zuk et al., 2001;
De Bari et al., 2001; Seo et al., 2004; Sabatini et al., 2005).
This study was originally designed to investigate whether,
using the same conditions established for the cultivation of
murine bone-marrow-derived MSCs (da Silva Meirelles and
Nardi, 2003), these cells could be found in other organs.
Surprisingly, the results showed that long-term MSC cultures
could be established from all the organs and tissues studied,
irrespective or their embryonic origin. The cell populations
thus obtained can be operationally defined as MSCs, because
they exhibit the capacity of prolonged self-renewal and
differentiate along mesenchymal cell lineages. The possibility
that the long-term cultures might represent early progenitors
exhibiting multipotent capabilities was tested with cloning
experiments. The results showed that a percentage of cells
plated at the single-cell level were able to regenerate long-term
cell cultures indicating that, at a given time point, a proportion
of the cells in each population is committed to self-renewal
whereas the remainder are not. When clones were cultured
under osteogenic or adipogenic conditions, they exhibited
differentiation characteristics similar to those of parental
cultures. This indicates that the long-term cultures represent
populations containing stem cells, and suggests that population
asymmetry is responsible for the maintenance of the stem cell
pool (Watt and Hogan, 2000).
The MSC cell populations originating from brain, spleen,
liver, kidney, kidney glomeruli, lung, bone marrow, muscle,
thymus and pancreas presented similar morphology and, to a
certain extent, surface marker profile. On the other hand, the
differentiation assays showed some variation among the
cultures in the frequency of cells which actually differentiated
Fig. 7. Non-induced MSC differentiation in primary culture. Aorta
primary cultures exhibit myogenic (A) and adipogenic (B)
differentiation. The same happens in muscle primary cultures (C and
D, respectively). Magnifications are indicated on each image.
Table 2. Results of cloning and subcloning of long-term MSC cultures
Culture
Cloning 1 Cloning 2 Subcloning 1 Subcloning 2 Sub-subcloning
– 112 (38.8) 144 (50.0) 61 (31.8) 42 (21.9) 25 (26.0)
+ 36 (12.5) 12 (4.2) 34 (17.7) 53 (27.6) 16 (16.7)
++ 75 (26.0) 84 (29.2) 35 (18.2) 21 (10.9) n.a.
+++ 49 (17.0) 39 (13.5) 51 (26.6) 73 (38.0) 55 (57.3)
Aborted 12 (4.2) 6 (2.1) 9 (4.7) 3 (1.6) –
Differentiated 4 (1.5) 3 (1.0) 2 (1.0) – –
Osteogenic differentiation + (Clone H4) + (Clone 3D10) n.a. n.a. n.a.
Adipogenic differentiation + (Clone H4) + (Clone 3D10) n.a. n.a. n.a.
MSC long-term cultures were cloned by micromanipulation. Individual cells were plated in 96-well plates (three plates for each cloning process) with MSC-
conditioned medium and maintained for 2 weeks. Two well-developed clones were subcloned (two plates for each process), and one of the resulting cultures was
further subcloned (one plate prepared). The plates were analyzed for the number (and percentage) of negative (–) and positive wells which were classified
according to the size of the colonies (+, ++, +++). Aborted clones were those in which the cells proliferated and then spontaneously died. Differentiated clones
developed mature morphologies, which were not further investigated. n.a., not applicable.
Journal of Cell Science
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2211
In vivo distribution of mesenchymal stem cells
in the osteogenic or adipogenic phenotype, as well as on the
degree of differentiation, related to their site of origin. This
might be due to the influence of the local environment from
which they originate, reflecting the importance of the niche in
establishing the phenotype of the stem cells it interacts with
(Fuchs et al., 2004).
Whereas most other studies have approached the question
of the natural distribution of MSCs in the organism by
infusing cultured cells into the animal models and analyzing
their multi-site engraftment, this is, to our knowledge, the first
one to apply the opposite approach. The simultaneous analysis
of different organs and tissues for their MSC contents can
provide more accurate information regarding their natural in
vivo distribution. The wide distribution of MSCs observed
raised the question of the relationship among these
populations. Three hypotheses were then considered: first,
MSCs are tissue-resident cells, and can be collected from
individual tissues or organs; second, MSCs are resident in
tissues, and circulate in blood; third, MSCs are derived from
the circulating blood.
To test these hypotheses, the possibility that the MSC long-
term cultures were derived from cells circulating in the blood
was first analyzed by perfusing the animals before collection
of the tissues or organs. Cultures could be normally established
under these conditions. Since no long-term culture could be
derived from circulating blood collected under controlled
conditions, MSCs seem to be absent from the circulation under
normal physiological conditions, in agreement with Wexler et
al. (Wexler et al., 2003). The possibility that they circulate
systemically or locally under other circumstances, e.g. during
tissue injury, is not however excluded and was not tested in the
present study. Endothelial progenitor cells, for instance, can be
found at higher frequency in the peripheral blood of patients
with acute myocardial infarction (Shintani et al., 2001).
The characteristics of MSC populations obtained from the
different organs were however very similar, suggesting a closer
relationship between them. Since literature reports have
suggested that MSCs derive from perivascular cells (Doherty
et al., 1998; Bianco et al., 2001; Shi and Gronthos, 2003;
Farrington-Rock et al., 2004), it is possible that MSCs are
actually derived from the vasculature. To test this hypothesis,
the aorta and the vena cava were investigated and long-term
MSC cultures could be established from both tissues. We next
analyzed these and the MSC populations obtained from other
sources and found them positive for the vascular smooth
muscle cell marker ␣SMA (Owens, 1995). To demonstrate that
perivascular cells at the capillary level have mesenchymal stem
cell properties, we isolated a structure comprising capillaries
only – the kidney glomerulus. Decapsulated glomeruli are
composed of endothelial cells, podocytes and mesangial cells
– which are considered specialized pericytes (Schlondorff,
1987). Based on the expression of ␣SMA, reported as a marker
for activated mesangial cells (Johnson et al., 1991), lack of
expression of CD31 by the resultant population, and
morphology, the cultures can be regarded as originating from
mesangial cells within the glomerulus. Their osteogenic and
adipogenic differentiation capabilities, along with their self-
renewal capacity, allow them to be operationally defined as
MSCs. Taking these results as a whole, we conclude that the
MSC compartment extends through the whole post-natal
organism as a result of its perivascular location.
Early in vivo experiments have suggested that pericytes may
act as a source of undifferentiated cells during adipose
(Richardson et al., 1982) and osseous (Diaz-Flores et al., 1992)
tissue repair. These data, along with reports describing the
MSC differentiation capabilities (Pittenger et al., 1999; Kopen
et al., 1999; Woodbury et al., 2000; Sato et al., 2005; Choi et
al., 2005; Lange et al., 2005), and the functional differences
between the populations studied here according to their origin,
led us to propose the model depicted in Fig. 8. In this model,
MSCs act as a reservoir of undifferentiated cells to supply the
cellular (and non-cellular) demands of the tissue they belong
to, acquiring local phenotypic characteristics. When necessary,
and after signs from the microenvironment, they give rise to
committed progenitors that gradually integrate into the tissue
(Fig. 8A). Tissue injury can activate alternative processes (Fig.
8B). The model does not exclude the possible existence of
other tissue-specific stem cells; however, it suggests that a
portion of the apparent post-natal stem cell diversity may be
attributed to local MSCs behaving as tissue-specific stem cells.
Once again the term ‘mesenchymal’ stem cell seems
inappropriate, and possibly the term ‘perivascular stem cell’
might best represent this particular cell type.
We believe that, in addition to providing insight into MSC
biology, our findings and hypotheses can be useful for
designing therapeutic strategies for a range of diseases.
Irradiation, or drugs able to transiently destabilize the vessel
wall integrity, might facilitate cell engraftment in cell or cell-
Fig. 8. A proposed model of MSC contribution to tissue
maintenance. In this schematic representation of the transverse
section of a simple vessel, MSCs lie in the basement membrane (red
line), opposed to endothelial cells. Cues provided by the tissue-
specific microenvironment coordinate a gradual transition
(represented by green color gradient) from undifferentiated cells to
progenitor and mature cell phenotypes. This process can occur
naturally as represented by the dotted arrow (A). In case of tissue
injury, undifferentiated MSCs can be mobilized directly into the
tissue without the progenitor transition as represented by the curved
arrow (B).
Journal of Cell Science
26
2212
mediated therapies; also, drugs and genetic therapy vectors
could be directed to the perivascular compartment to achieve
tissue-specific activity, as perivascular-derived cells gradually
assume a tissue-specific phenotype. These approaches validate
the circulatory system and more specifically its stem cell
compartment as a vehicle for reaching the whole organism.
Materials and Methods
Reagents, culture media and solutions
Complete culture medium (CCM) was composed of Dulbecco’s modified Eagle’s
medium (DMEM) with HEPES (free acid, 2.5–3.7 g/l) and 10% fetal bovine serum
(Cultilab, Sao Paulo, Brazil). Ca
2+
- and Mg
2+
-free Hank’s balanced salt solution
containing 10 mM sodium HEPES (HB-CMF-HBSS) combined with DMEM (1:1)
was used as perfusion medium. All reagents used in this study were from Sigma
Chemical Co. (St Louis, MO), unless otherwise stated. Plasticware was from TPP
(Trasadingen, Switzerland).
Animals
Adult mice (8-43 weeks old) from the C57Bl/6 and BALB/c strains were used in
this study. ROSA26 (The Jackson Laboratory, Bar Harbor, ME) and eGFP mice
(green mouse FM131, kindly provided by M. Okabe, Osaka University, Japan),
derived from the C57Bl/6 strain, were also used. The animals were kept under
standard conditions (12 hours light/12 hours dark, water and food ad libitum) in our
animal house. All the experimental procedures were performed according to
institutional guidelines.
Perfusion
The animals were anesthetized with a combination of ketamine and xilazine (1.16
g and 2.3 g per kg body weight, respectively) delivered intraperitoneally. The
abdominal cavity was opened, the diaphragm was ruptured, and 100 units of
heparin in 200 l HB-CMF-HBSS were injected into the beating heart. The
ascending aorta was catheterized with a 27G intravenous catheter inserted through
the left ventricle. The caudal vena cava was cut, and around 50 ml of perfusion
medium were pumped in. For lung perfusion, the pulmonary artery was
catheterized instead.
MSC isolation and long-term culture
MSCs from bone marrow were isolated and cultured as previously described (da
Silva Meirelles and Nardi, 2003). MSCs from liver, spleen, pancreas, lung, kidney,
aorta, vena cava, brain and muscle were obtained as follows. Organs and tissues
were collected from perfused or non-perfused animals, rinsed in HB-CMF-HBSS,
transferred to a Petri dish and cut into small pieces. When dissecting organs, care
was taken to discard the portions containing visible vessels (e.g. the portal vein
and the vena cava in the liver). The dissected pieces (around 0.2-0.8 cm
3
) were
washed with HB-CMF-HBSS, cut into smaller fragments, and subsequently
digested with collagenase type I (0.5 mg/ml in DMEM/10 mM HEPES) for 30
minutes to 3 hours at 37°C. To separate the adipose layer surrounding the aorta,
the vessel was digested for around 30 minutes and subjected to vigorous agitation,
yielding a first cell fraction. The remnant of the vessel was then washed in 20 ml
HB-CMF-HBSS, and transferred to a new tube where digestion proceeded yielding
a second cell fraction. Both cell fractions were used to establish separate primary
cultures.
Whenever gross remnants persisted after collagenase digestion, they were
allowed to settle for 1 to 3 minutes, and the supernatant was transferred to a new
tube which was then completed with CCM. In some experiments, the cells were
further cleared from debris by centrifugation on Ficoll-Hypaque (Amersham
Pharmacia, Piscataway, NJ), followed by an additional washing step. After
centrifugation at 400 g for 10 minutes at room temperature (RT), the pellets were
resuspended in 3.5 ml CCM containing 1% antibiotic-antimycotic solution (GIBCO
BRL, Gaithersburg, MD), seeded in six-well dishes (3.5 ml/well) and incubated at
37°C in a humidified atmosphere containing 5% CO
2
. Three days later, if the
cultures were not confluent, the whole volume of CCM (with no antibiotics or
antimycotics) was replaced, and the adherent layer was refed every 3 or 4 days.
For subculture, the adherent layer was washed once and incubated with 0.25%
trypsin and 0.01% EDTA in HB-CMF-HBSS. The cultures were split whenever they
reached confluence, at ratios empirically determined for two subcultures a week at
most. Initial split ratios were 1:2 or 1:3, and as the culture kinetics accelerated the
ratios were set to values ranging from 1:6 to 1:25, until they stabilized at different
ratios as described below.
To evaluate the presence of MSCs in blood, animals were anesthetized, the
abdominal cavity was opened, and 100 units of heparin in 200 l HB-CMF-HBSS
were injected into the beating heart. Either a 27G intravenous catheter was
introduced cranially into the portal vein and 500-750 l blood were collected, or
the vessels arising from the heart were cut and 500-750 l blood were collected
from the thoracic cavity. Blood was also collected directly from the exposed heart
in some cases. The collected blood was either added to a 25 cm
2
flask containing
Journal of Cell Science 119 (11)
7 ml CCM incubated at 37°C, or fractionated on Ficoll-Hypaque. In this case,
mononuclear cells were collected, washed once in complete medium, resuspended
in 3.5 ml fresh complete medium, transferred to a well of a six-well dish and
incubated at 37°C. In either case, after 3 days, non-adherent cells were removed
along with the culture medium and fresh complete medium was added. The adherent
cells were then re-fed every 3 or 4 days.
To establish glomeruli-derived MSC cultures, kidneys were placed into a 15 ml
centrifuge tube containing 5 ml CCM, and mechanically disrupted by several rounds
of aspiration/expulsion using a 10 ml pipette. Single glomeruli devoid of the
Bowman’s capsule were isolated from the cell suspension by micromanipulation,
and transferred either individually or collectively to 12-well dishes containing CCM
and 1% antibiotic-antimycotic solution. Subsequent passages were performed as
described above.
Cell cloning
To clone MSCs to the single-cell level, cultures were trypsinized, resuspended in
MSC-conditioned medium which had been previously filtered through a 0.22 m
membrane, and individually transferred to 96-well dishes using a micromanipulator.
The number, morphology and kinetics of resulting clones were analyzed, and some
of them were selected for subcloning and differentiation assays.
Morphological analysis and photographs
MSC cultures were routinely observed with an inverted phase-contrast microscope
(Axiovert 25; Zeiss, Hallbergmoos, Germany). For detailed observation, cells were
rinsed with phosphate-buffered saline (PBS), fixed with ethanol for 5 minutes at RT
in some cases, and stained for 2.5 minutes with Giemsa. Photomicrographs were
taken with a digital camera (AxioCam MRc, Zeiss), using AxioVision 3.1 software
(Zeiss).
Flow cytometry
For detection of surface antigens the cells were trypsinized, centrifuged, and
incubated for 30 minutes at 4°C with phycoerythrin (PE)- or fluorescein
isothiocyanate (FITC)-conjugated antibodies against murine Sca-1, Gr-1, CD11b,
CD13, CD18, CD19, CD29, CD31, CD44, CD45, CD49d, CD49e, CD90.2, CD117
and IgG (Pharmingen BD, San Diego, CA). Excess antibody was removed by
washing.
For the detection of ␣-smooth muscle actin (␣SMA), the cells were collected,
washed once in HB-CMF-HBSS and fixed with 4% paraformaldehyde in PBS for
1 hour at RT. After centrifugation, the cells were kept for 15 minutes at RT in 5 ml
PBS containing 0.2% Triton X-100. Cells were collected, washed once in PBS, and
incubated with or without primary antibody against ␣SMA (Chemicon, Temecula,
CA) overnight at 4°C. The cells were then incubated with FITC-conjugated anti-
mouse IgG secondary antibody for 1 hour at 4°C.
The cells were analyzed using a FACScalibur cytometer equipped with 488 nm
argon laser (Becton Dickinson, San Diego, CA) with the CellQuest software. At
least 10,000 events were collected. The WinMDI 2.8 software was used for building
histograms.
MSC differentiation
Osteogenic differentiation was induced by culturing MSCs for up to 8 weeks in
CCM supplemented with 10
–8
M dexamethasone, 5 g/ml ascorbic acid 2-
phosphate and 10 mM -glycerophosphate (Phinney et al., 1999). To observe
calcium deposition, cultures were washed once with PBS, fixed with 4%
paraformaldehyde in PBS for 15-30 minutes at RT, and stained for 5 minutes at RT
with Alizarin Red S stain at pH 4.2. Excess stain was removed by several washes
with distilled water.
To induce adipogenic differentiation, MSCs were cultured for up to 8 weeks in
CCM supplemented with 10
–8
M dexamethasone, 2.5 g/ml insulin, 100 M
indomethacin and, in some experiments, 3.5 M rosiglitazone or 5 M 15-deoxy-
D
12,14
-prostaglandin J
2
. Later in this study, DMEM with 10 mM HEPES, heparin
and 20% platelet-free human plasma (Krawisz and Scott, 1982) was used to induce
kidney glomerulus-derived MSC adipogenic differentiation. Adipocytes were easily
discerned from the undifferentiated cells by phase-contrast microscopy. To further
confirm their identity, cells were fixed with 4% paraformaldehyde in PBS for 1 hour
at RT, and stained with either Oil Red O solution (three volumes of 3.75% Oil Red
O in isopropanol plus two volumes of distilled water) or Sudan Black B solution
(three volumes of 2% Sudan Black B in isopropanol plus two volumes of distilled
water) for 5 minutes at RT. When stained with Oil Red O, the cultures were
counterstained with Harry’s hematoxylin (1 minute at RT).
We thank Ricardo Ribeiro dos Santos (FIOCRUZ, Salvador, Brazil)
and Antonio Carlos Campos de Carvalho (Universidade Federal do
Rio de Janeiro, Brazil) for their assistance with the establishment of
the colonies of transgenic mice. This work was supported by Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and
Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul
(FAPERGS).
Journal of Cell Science
27
2213
In vivo distribution of mesenchymal stem cells
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Journal of Cell Science
28
Capítulo 4
Tetraploidy in long-term cultured murine mesenchymal stem
cells: a link between cultured mesenchymal stem cells and
the perivascular niche?
Lindolfo da Silva Meirelles & Nance Beyer Nardi
Manuscrito em preparação a ser submetido para Cell and Tissue Research
29
Tetraploidy in long-term cultured murine mesenchymal stem cells: a link between
cultured mesenchymal stem cells and the perivascular niche?
Lindolfo da Silva Meirelles & Nance Beyer Nardi
Departamento de Genética, Universidade Federal do Rio Grande do Sul
Av. Bento Gonçalves 9500
Porto Alegre, RS, Brazil
91501-970
Telephone: +55 51 33086737
Abstract
Mesenchymal stem cells are adult multipotent cells that are able to differentiate
along mesodermal and non-mesodermal pathways. They are distributed throughout the
organism, most probably due to their association to blood vessels. Furthermore, they have
been recently proposed to be progenitors for the mature cells of the tissues they are in.
Here, we analyzed by flow cytometry the DNA content of long-term cultured murine
mesenchymal stem cells derived from different anatomical locations. The results suggest
that long-term cultured murine MSCs progress to a tetraploid, or nearly tetraploid, state
upon extended cultivation in vitro. These results provide further evidence of the identity of
MSCs as perivascular cells, for which tetraploidization has been described.
Introduction
Nearly 35 years ago, A. Friedenstein and colleagues described fibroblastic
precursors present in post-natal bone marrow that could be detected using an in vitro
colony-forming assay (Friedenstein et al., 1974). The cells that originated such fibroblastic
colonies were thus called fibroblast colony-forming units (CFU-Fs). In the subsequent
years, it was found that the progeny of CFU-F was able to form bone, cartilage, and
adipose tissue cells in vitro and in vivo (reviewed by Prockop, 1997), and hence the term
mesenchymal stem cell (MSC) emerged (Caplan, 1991). Scientific evidence has
30
accumulated that indicate that the particular cell type currently referred to as MSC can
differentiate not only into mesenchymal cell types but also into non-mesenchymal cells
such as astrocytes (Kopen et al., 1999) and hepatocytes (Sato et al., 2005). Post-natal
MSCs, or MSC-like, cells have also been detected in other organs but the bone marrow (da
Silva Meirelles et al, 2006). The anatomical location of MSCs has also been a subject of
interest, and some studies have pointed to the perivascular space as their niche (Bianco et
al., 2001; Shi and Gronthos, 2003). Consequently, there is a possibility that cells known as
pericytes, mural cells, or Rouget cells, represent the in vivo counterparts of MSCs. We
have recently found evidence indicating that perivascular cells bear MSC properties and,
consequently, MSCs exist all over the post-natal organism due to their association with the
vasculature (da Silva Meirelles et al., 2006). Furthermore, a hypothesis has been drawn in
which MSCs are, in fact, perivascular cells that give rise to the mature cells of the tissues
they are embedded in (da Silva Meirelles et al, 2006).
So far, evidence exists indicating that the long-term cultivation of embryonic stem
cells (Maitra et al., 2005), and of human MSCs (Rubio et al., 2005), brings about
chromosomal alterations that might lead to problems if they were applied in vivo. On the
other hand, evidence also exists demonstrating that chromosomal anomalies such as
aneuploidies do occur naturally in vivo, suggesting that gain or loss of chromosomes may
have an effect on the differentiation of the resulting cells. To examine the chromosomal
integrity of cultured MSCs derived from different organs of mice, we have analyzed their
DNA content by flow cytometry. We have found that the cell lines studied were in general
tetraploid, or nearly tetraploid, and that tetraploidy came along with prolonged cell
cultivation. Furthermore, the MSC populations retained adipogenic differentiation
capabilities in spite of their ploidy. Since tetraploidization of rodent perivascular cells has
been reported, these observations suggest a link between cultured MSCs to the perivascular
niche, and provide further evidence favoring the perivascular stem cell hypothesis.
Material and methods
Mice
31
Mice used in this study were from C57Bl/6 or eGFP strains. eGFP mice (green
mouse FM131), derived from the C57Bl/6 strain, were gently provided by Dr M. Okabe,
Osaka University, Japan. The animals were kept under standard conditions (12 hours
light/12 hours dark, water and food ad libitum) in our animal house. All the experimental
procedures were performed according to institutional guidelines.
Cell culture
The cell populations used in this study are described in Table 1. 001Gvc, 014Ba1,
018Bm and 021Bkg were previously characterized (da Silva Meirelles and Nardi, 2006),
and were retrieved from frozen stocks. 004Gbm and 013Cbm were derived as previously
described (Meirelles and Nardi, 2003). The cells were cultured in low glucose Dulbecco’s
Modified Eagle’s Medium (Sigma Chemical Co., St Louis, MO) containing 10% fetal
bovine serum (Cultilab, Campinas, SP, Brazil) and 10 mM HEPES (free acid; Sigma).
Trypsin-EDTA (0.25% and 0.01%, respectively) was used to harvest the cells prior to each
passage, after rinsing with hepes-buffered Hank’s balanced salt solution.
Table 1 – Description of the MSC cell populations used.
Cell line Mouse strain Gender Age Anatomical origin
001Gvc eGFP male 22 weeks vena cava
004Gbm eGFP female 57 weeks bone marrow
013Cbm C57Bl/6 female 26 weeks bone marrow
014Ba1 BALB/c male 30 weeks aorta artery
018Bm BALB/c female 18 weeks skeletal muscle
021Bkg BALB/c female 27 weeks kidney glomeruli
Adipogenic differentiation
To induce adipogenic differentiation, MSCs were cultured in Iscove’s Modified
Dulbecco’s Medium added of 20% human platelet-free plasma, supplemented with 10
-7
M
dexamethasone, 2.5 μg/ml insulin, 50 μM indomethacin (all from Sigma), 5 μM
rosiglitazone (Avandia; GlaxoSmithKline, Middlesex, United Kingdom) dissolved in
32
DMSO (Sigma), and 10 units/ml sodium heparin. This protocol was adapted from Krawisz
and Scott (1982) and da Silva Meirelles et al. (2006). At the end of the differentiation
experiments, cells were fixed with 4% paraformaldehyde in phosphate buffered saline
(PBS) for 1 hour at room temperature (RT), stained with Oil Red O solution for 5 minutes
at RT, and counterstained with Harry’s hematoxylin for 1 minute at RT.
DNA content analysis
Cells were harvested with trypsin-EDTA, washed twice with PBS and resuspended
into 0.5 ml PBS in a 15-ml centrifuge tube. They were chilled in ice, vortexed gently, and
two ml of ice-cold methanol were added dropwise. The cells were stored at 4
o
C overnight
or for a maximum of 7 days, when they were used for the experiments. Adipocytes
differentiated from MSCs were harvested using collagenase type I (0.5 mg/ml in serum-
free medium). The whole contents were centrifuged at 400 g for 10 min, and the buoyant
fraction was collected using a micropipette. The volume was measured, and ice-cold
ethanol was added to it to a proportion of around 80%. The cells were kept at 4
o
C until
used. The fixation using methanol was modified from Rousselle et al. (1998). Fresh mouse
splenocytes were processed in the same ways as the cultured cells to provide a diploid
control for subsequent analyses.
The fixed cells were centrifuged at 400 g for 10 minutes, and washed twice with
PBS. They were resuspended into 0.9 ml PBS, and 0.1 ml of 1 mg/ml RNAse A was
added. After incubation at 37°C for 25 minutes, 30 μl propidium iodide solution (5 mg/ml
in ddH
2
O) were added to the cell suspensions, and they were incubated for 30 min at RT.
This protocol was modified from Juan and Darzynkiewicz (1998). The cells were washed
twice with PBS, and finally resuspended into 0.5 ml PBS.
DNA content was measured using a FACScalibur flow cytometer (Becton
Dickinson, San Jose, CA), equipped with a 488 nm laser beam. At least 10,000 events were
acquired. Data were collected using CellQuest software (Becton Dickinson), and were
analyzed using WinMDI 2.8 (freely available at http://facs.scripps.edu/software.html).
Graphics plotting the parameters FL-2 width versus FL-2 area were built. Quadrants were
used to mark G
0
/G
1
and G
2
regions of the diploid control (intersections), and to compare
these with those of the other samples. Images containing both quadrant intersections were
33
built by overlay using the GNU Image Manipulation Program (freely available at
http://www.gimp.org/).
Results
Long-term cultured mMSCs display a tetraploid or nearly tetraploid DNA content
Even though flow cytometry is a good method to analyze DNA content of different
types of cells, the application of this methodology on murine MSCs was difficult because
these cells are very large (around 20 μm diameter in suspension), and they tend to clog the
flow cytometer pipeline when fixed, particularly in the presence of propidium iodide, and
when a large number of events is acquired. As a consequence, sometimes the dots in the
graphics were dislocated during acquisition. To avoid this problem, the samples were read
at intervals, pausing acquisition at times and then allowing large amounts of PBS to flow
through the pipeline before continuing.
As shown in Figure 1, MSC populations derived from different organs of adult
mice generally display a nearly tetraploid DNA content. The MSC population 021Bkg
represented an exception to this general rule, as their DNA content was compatible with
that of a diploid cell line. The results indicating a tetraploid, or nearly tetraploid, DNA
content are validated by cytogenetic analysis of one bone marrow cell line generated in a
previous study (Islam et al., 2006).
The relative number of tetraploid cells increases along passages
We also observed that the proportion of diploid cells in relation to the remainder
decreases along the passages, as shown in Figure 2. Since murine bone marrow MSCs may
be considered free from hematopoietic contaminants by passage 8 using our standard
conditions (Meirelles and Nardi, 2003), the cells had their DNA content first assessed at
passage 9. At this point, the proportion of diploid cells in G
0
/G
1
plus S phases of cell cycle
corresponded to around 37% of the total, whereas this proportion decreased to around 27%
at passage 11, and to around 2% and less then 2% at passages 18 and 20, respectively.
34
Long-term cultured mMSCs retain their adipogenic differentiation characteristics
Since most of the cell populations used had been previously characterized,
adipogenic differentiation was performed for two reasons: first, to check if the cell
populations retained their capability of differentiation toward at least one mesenchymal
lineage; and second, because we wanted to check the ploidy status before and after
differentiation, and adipocytes are easier to dissociate than cells subjected to osteogenic
differentiation, where a mineralized extracellular matrix forms. As shown in Figure 3, the
MSCs retrieved from frozen stocks retained their adipogenic differentiation characteristics
previously described (da Silva Meirelles et al., 2006).
Adipogenic differentiation does not affect ploidy status
To check if adipogenic differentiation could somehow interfere with the ploidy
status, one MSC population that was known to have adequate adipogenic differentiation
capability (004Gbm, as judged from the adipogenic differentiation assay) was set up in two
parallel cultures. When these were nearly confluent, one of them had its DNA content
analyzed, whereas the other was subjected to adipogenic differentiation. When mature
adipocytes were visible (Figure 3E), the cells were harvested and their DNA content
compared with that of their undifferentiated counterparts. As seen in Figure 4, no
differences in DNA ploidy could be observed between undifferentiated and differentiated
cells. Differentiated cells displayed, however, a group of cells with an apparent DNA
content below the G
0
/G
1
limit, indicating that those were actually apoptotic cells (Figure
4B).
Discussion
In this work, we demonstrated that long-term cultured murine MSCs generally have
a tetraploid, or nearly tetraploid, DNA content. The proportion of diploid cells decreased
along serial passaging in one of the populations studied, and adipogenic differentiation did
not modify the ploidy status when one of the populations used was analyzed.
35
Even though it is difficult to relate data obtained from experiments in vitro with
other data from experiments in vivo, our results may be interpreted as an indication that
cultured murine MSCs undergo, in vitro, an increase in their ploidy that is comparable to
the tetraploidization that has been described as accompanying the hypertrophy of rodent
smooth muscle cells in vitro in response to transforming growth factor beta (Owens et al.,
1988) or angiotensin II (Geisterfer et al, 1988) and, more importantly, in vivo, as a
consequence of hypertension (Chobanian et al., 1987) or aging (Jones and Ravid, 2004). In
fact, the identity of cultured murine MSCs has been proposed to overlap with that of
perivascular cells based in part on the expression of alpha smooth muscle actin, a smooth
muscle cell marker (Owens, 1995). We interpret the results presented herein a further
evidence indicating the perivascular origin of the cultured cell populations operationally
defined as MSCs.
Aknowledgements
The authors are indebted to to Conselho Nacional de Desenvolvimento Cientifico e
Tecnologico (CNPq) for financial support. Thanks are due to Dr. Andrés Delgado Cañedo
for help with flow cytometry methods, and discussions.
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Chakravarti A (2005) Genomic alterations in cultured human embryonic stem cells. Nat
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Meirelles Lda S, Nardi NB (2003) Murine marrow-derived mesenchymal stem cell:
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38
splenocytes 001Gvc P30
014Ba1 P12
018Bm P63 021 Bkg P28
0013Cbm P20
A B
C D
E F
Figure 1 - DNA content of MSCs from different organs. Murine splenocytes (A)
were used to draw horizontal lines corresponding to cells in phases G
0
/G
1
or G
2
of
the cell cycle. A third horizontal line was placed twice the distance between those
two lines above the G
2
line, as a marker for 4n cells in the G2 phase. The dots to
the right of the line drawn in angle, represent cell clusters. B, vena cava MSCs at
passage 30 present a DNA content compatible with the presence of tetraploid and,
possibly, cells with even higher DNA content. C and D depict the DNA content of
bone marrow and aorta MSCs, respectively, at passages 20 and 12, and indicates
that both populations comprise a massive majority of tetraploid cells, and a minority
of diploid, or near diploid, cells. E, muscle MSCs at passage 63, displaying a DNA
content compatible with a near tetraploid state. F, kidney glomerulus MSCs at
passage 28, with a DNA content that indicates that the majority of the cells are
diploid, or nearly diploid, while a smaller portion of the population is tetraploid.
2n, G
2
2n, G
0
/G
1
4n, G
0
/G
1
4n, G
2
39
P9 P11
P18 P20
Figure 2 - Reduction of the proportion of diploid cells along passages. The bone
marrow MSC population 013Cbm had its DNA content analysed at passages (P) 9, 11,
18 and 20. As an estimate of the proportion of diploid cells, a gate (R2) was drawn that
comprehended diploid cells in G
0
/G
1
and S phases of the cell cycle, and it was com-
pared with a larger gate (R1) comprehending all non-clustered cells. The proportions
are expressed as a percentage of the total number of non-clustered cells.
40
021Bkg P28 200X021Bkg P28 200X
004Gbm P75 100X004Gbm P75 100X
001Gvc P30 400X001Gvc P30 400X
018Bm P63 100X 018Bm P63 100X
014Ba1 P12 100X014Ba1 P12 100X
Figure 3 - Adipogenic differentiation of the cell populations used. Adipocytic cells
differentiated from MSC populations 018Bm at passage 63 (A), 021Bkg at passage 28
(B), 001Gvc at passage 30 (C), and 014Ba1 at passage 12 (D), as evidenced by stain
-
ing with Oil Red O. E, phase contrast picture of live adipocytes differentiated from
004Gbm cells at passage 75.
41
004Gbm P75
004Gbm P75 A
A B
Figure 4 - DNA content in an MSC population subjected to adipogenic differentia-
tion. A, DNA content of the bone marrow MSC cell line 004Gbm at passage 75. B, DNA
content of 004Gbm cells subjected to adipogenic differentiation at passage 75 (same as
Figure 3E).
42
Capítulo 5
Towards a unifying concept of the identity and natural
distribution of mesenchymal stem cells.
Lindolfo da Silva Meirelles, Arnold I. Caplan & Nance Beyer Nardi
Manuscrito em preparação a ser submetido como artigo de revisão para Stem Cells and
Development
43
Manuscript to be submitted as a review to Stem Cells and Development
Towards a unifying concept of the identity and natural distribution of mesenchymal stem
cells
Lindolfo da Silva Meirelles
1
, Arnold I. Caplan
2
, and Nance Beyer Nardi
1
1
Departamento de Genetica, Universidade Federal do Rio Grande do Sul, Av Bento
Goncalves 9500, 91501-970, Porto Alegre RS, Brazil.
2
Department of Biology, Skeletal Research Center, Case Western Reserve University,
2080 Adelbert Road, Cleveland, OH 44106-7080, USA.
Address reprint requests to:
Departamento de Genetica, Universidade Federal do Rio Grande do Sul
Av Bento Goncalves 9500
91501-970, Porto Alegre RS, Brazil.
E-mail: [email protected]
Running title: Natural distribution of mesenchymal stem cells.
44
Lindolfo da Silva Meirelles
Departamento de Genetica, Universidade Federal do Rio Grande do Sul
Av Bento Goncalves 9500
91501-970, Porto Alegre RS, Brazil
Telephone: +55 51 33086737
E-mail: lindolfo_m[email protected]
Arnold I. Caplan
Department of Biology, Skeletal Research Center
Case Western Reserve University
2080 Adelbert Road, Cleveland, OH 44106-7080, USA
E-mail: [email protected]
Nance Beyer Nardi
Departamento de Genetica, Universidade Federal do Rio Grande do Sul
Av Bento Goncalves 9500
91501-970, Porto Alegre RS, Brazil
Telephone: +55 51 33086740
E-mail: [email protected]
45
ABSTRACT
In spite of the extraordinary advances on the investigation of adult stem cells seen
during the past few years, we are still far from understanding their true nature. Most
studies are conducted with cells isolated from their natural environment and subject to
artificial conditions during in vitro culture. This is more apparent when specific types of
adult SCs, such as the mesenchymal stem cells (MSCs), are considered. MSCs present a
high degree of plasticity, and have shown promising results in pre-clinical and clinical
studies for a number of diseases. These characteristics make them promising tools for cell
and cell-based therapies. MSCs, however, are only known in vitro, and the definition of
their niche in the organism would be of great importance in the rational design of
therapeutic approaches. The main cell types they originate – osteoblasts, chondrocytes, and
adipocytes – are present through the entire organism, so that their location should allow for
their progeny to be distributed to most or all tissues and organs. Multiple evidences now
suggest a perivascular location for MSCs. In this review, we focus on evidences showing
that MSCs share characteristics with pericytes, and discuss a model in which mesenchymal
stem cells have a perivascular niche and lie in the basement, opposed to endothelial cells.
According to this model, cues provided by the niche coordinate a gradual transition to
progenitor and mature cell phenotypes. This concept has implications for the design of new
therapeutic approaches for a range of diseases, targeting the perivascular compartment
through cell-mediated therapies.
46
INTRODUCTION
The past few years have witnessed an outpouring of new studies about the biology of
stem cells (SC) and their therapeutic potential. Although it is less than ten years since
James Thomson and coworkers derived the first human embryonic stem cell line (1), a
huge amount of new information has come to light. Embryo stem cells are collected and
cultured with well established techniques; their molecular characterization is well
advanced, and research is mainly focused on new methods to differentiate them into
specialized cells types. As for adult stem cells, many different types have been identified,
and research efforts concentrate on improving methods for their isolation, expansion and
characterization, and on investigating their potential for therapeutic uses. Much has already
been learned about their basic biology; nevertheless, we are not much closer to
understanding their true nature (2).
The main problem lies in the basic approach we use for studying the adult stem cell,
which is very similar to the approach used to investigate embryo stem cells – collect,
expand and characterize. Actually, however, besides sharing part of their names, there is
little in common between these two types of stem cells. Although recent reports show that
embryonic stem cells are not “like peas in a pod”, since even at the four-cell stage cells
from mouse embryos are different from one another (3), it is easy to know which cells to
collect from the blastocyst to establish a stem cell culture. The biology of the cells is
modified by in vitro culture, but the key property of embryo stem cells – pluripotency – is
maintained, as shown by their ability to form teratomas when injected into
immunodeficient mice (4). As for adult stem cells, one of the key issues is precisely which
cells (or cell fraction) to isolate. Since there are no definite markers for adult stem cells, the
47
cells discarded in the “negative fraction” resulting from isolation processes may contain
stem cells not identified by the current methods.
Embryo and adult stem cells are different in two other main points. First, embryo SCs
are meant to proliferate, at high rates. It does not go too much against their nature to force
them to proliferate in vitro. As for the differentiation process, they are also meant to
differentiate – more than we want to –, so the problem is to stop them from differentiating
too much, and direct the process into the lines we are more interested in (5). One of the key
properties of adult SC, on the other hand, is quiescence (6). This is of fundamental
importance for species of long life cycle such as ours, to avoid genetic damage to a cell
line that is meant to survive for many decades without reposition from external sources.
Working with adult SCs in vitro, however, means having to force them to proliferate, while
keeping their stemness in adequate levels. This difficulty is more apparent for some adult
SC types than for others. There is, for instance, a great interest in expanding hematopoietic
stem cells (HSCs) in vitro to increase their numbers in situation such as the use of cord
blood for transplantation (7). Expanding HSCs, while still keeping their undifferentiated
state, has proven a difficult goal to achieve, and a great number of studies have suggested
different methodologies (reviewed in ref. 8). The same situation applies to other adult stem
cells (9).
Second, the inner mass of blastocysts consists of a group of cells sharing the
characteristic of being stem cells, and the question of defining the appropriate culture
condition for maintaining their proliferation as embryo SCs has been more easily solved
(5). Adequate niches for the multiple types of adult stem cells, on the other hand, are
proving extremely hard to define, so that added to the difficulty of isolating tissue-specific
stem cells we have currently no clearly defined way of maintaining them in vitro.
48
The question then is: how to maintain and expand ex vivo cells that fundamentally
depend on their niche (that we can, possibly, replace with the adequate signals) and are
meant to be quiescent? By forcing them to divide, it is inevitable that their biology will be
strongly altered. Although many studies are dedicated to answering this question, what we
know about adult stem cells which have been manipulated ex vivo is probably very
different from their behaviour in vivo. This is still more apparent when specific types of
adult SCs, such as the mesenchymal stem cells (MSCs), are considered.
MESENCHYMAL STEM CELLS
Mesenchymal stem cells may be defined as cells capable of giving rise to a number
of unique, differentiated mesenchymal cell types (10,11). First described as fibroblast
precursors from bone marrow by Friedenstein et al. in 1970 (12), MSCs may be also
referred to as fibroblast colony-forming units (CFU-Fs) or marrow stromal cells (13).
There is evidence that MSCs exist not only in the bone marrow, but in virtually every
location of the body (14-16). Although no definitive markers are known for MSCs, their
cell-surface antigen profile is well explored (reviewed in ref. 17).
The literature presents evidence that cultured MSCs also exhibit a degree of
plasticity that goes beyond the mesenchymal limit, when maintained in vitro or implanted
in vivo (18-22). They have shown promising results in pre-clinical and clinical studies for a
number of diseases (reviewed in refs. 23-25), involving very different tissues, such bone or
cartilage defects, cardiac disorders, central nervous system or spinal cord injury, and lung
diseases. They may also hasten hematopoietic recovery after bone marrow transplantation,
and their unique immunologic properties might facilitate engraftment of transplanted
49
organs and reduce graft-vs-host disease. MSCs have also been proposed to exert paracrine
trophic effects through the secretion of bioactive molecules (26). These characteristics
make them promising tools for cell and cell-based therapies.
Protocols for the isolation of MSCs involve the selection of plastic-adherent cells (27-
29). Our ignorance on the basic biology of MSCs is reflected in the different types of
cultures derived from attempts of different groups to isolate them. These cell populations
have been named mesenchymal stem cells, marrow stromal cells, marrow-isolated adult
multilineage inducible (MIAMI) cells, recycling stem cells (RS-1, RS-2), or multipotent
adult progenitor cells (MAPCs) (reviewed in ref. 30). Gregory et al. in 2005 (31) analyzed
the multiple factors involved in MSC differentiation in vitro, suggesting that it is regulated
by a two-stage mechanism: preconditioning by factors in the culture microenvironment,
and response to soluble differentiating factors. An attempt to clarify the nomenclature for
MSCs has been recently put forward by the International Society for Cellular Therapy (32).
The fact is that MSCs are only known in vitro, and our inability to prospectively
identify them in their natural location in the organism results in that “MSC biology, at the
present time, remains biology out of context” (33). Several papers have described cell
populations derived from bone marrow or different locations, from human and other
species, that are able to differentiate into mesenchymal cell phenotypes in vitro (14,28,34).
On the other hand, there is evidence that even cells that have a mature phenotype in vivo
may be able to dedifferentiate to a more primitive phenotype when cultured (35) and
differentiate into other cell types in vitro (36,37). As with most other adult stem cells,
MSCs are operationally defined, after in vitro expansion, by their ability to self-renew and
to differentiate – in this case, in mesodermal (28), ectodermal (18) and endodermal (21)
cell lineages. How much does the in vitro expansion of plastic-adherent, self-renewing
50
cells modify the biology of what a “mesenchymal stem cell” is in vivo? Or: are the cultured
cells that display capacity to differentiate into mesenchymal cell types indeed MSCs?
WHERE ARE THE MESENCHYMAL STEM CELLS?
Adult, tissue-specific stem cells are found in specialized niches in the correspondent
tissue (38). Hematopoietic stem cells can be found in the bone marrow (reviewed in ref.
39), epidermal SCs in mammalian hair follicles (40), neural SCs in the subventricular zone
(41), and so on. Where are the mesenchymal stem cells to be found?
Very little is known about the ontogeny and developmental origin of the MSC
(reviewed in ref. 33). The main cell types they originate – osteoblasts, chondrocytes, and
adipocytes – are present through the entire organism. There are furthermore, as mentioned
above, signs of great plasticity of these cells. Their location in the organism must allow for
their progeny to be distributed to most or all tissues and organs. Three main situations
could be considered. In the first one, MSCs are located in one specific tissue or organ,
from where they circulate to other sites of the organism to replenish cell populations
entering apoptosis through physiological processes, or necrosis in case of lesions.
Although not formally denied, the great difficulty in establishing conventional MSC
cultures from peripheral blood (14) goes against this possibility. Furthermore, post-natal
mesenchymal stem cells have been isolated from different sources, besides the bone
marrow – e.g. adipose tissue (42), tendon (43), periodontal ligament (22), synovial
membrane (44) and lungs (45). We recently showed that MSC cultures with very similar
morphologic, immunophenotypic and functional properties can be established from the
51
brain, spleen, liver, kidney, lung, bone marrow, muscle, thymus, and pancreas of mice
(14).
These findings raise a second possibility, in which MSCs are embedded in many
different tissues and organs, as resident stem cell populations. The experimental approach
to the true location of MSCs within all these tissues is difficult, and it has not been
formally tested. However, the absolute dependence of adult stem cells on their niche makes
it hard to envisage a situation in which the appropriate microenvironment for MSCs would
be similarly available in the core of different types of tissues.
The third possibility, which constitutes the main theme of this work, has been put
forward by some authors, and suggests a relationship between MSCs and perivascular cells
(46-49). This situation would adequately (a) make MSCs available to all tissues, in their
role as a source of new cells for physiological turnover or for the repair of lesions, and (b)
explain the establishment of MSC cultures from virtually all tissues examined thus far (14).
THE PERICYTE
Pericytes – also referred to as periendothelial cells, Rouget cells, Ito/stellate cells (in the
liver) – are mural cells that lie on the abluminal side of blood vessels, immediately
opposed to endothelial cells (50-52). Pericytes are defined morphologically based on their
location in relation to the endothelial cells, especially in microvessels (arterioles,
capillaries and venules). There is evidence, however, that pericytes are also present in large
vessels. By staining tissue sections with the 3G5 antibody, a pericyte marker, Andreeva et
al., 1998 (53) could identify pericytes lying adjacent to endothelial cells not only in small,
52
but also in large vessels. They concluded that pericytes form a continuous subendothelial
network which spans the whole vasculature.
Pericyte markers
Whenever considering using marker molecules to detect pericytes, one should be aware
of the expression of the same molecules by other cell types, especially cells that are in
close proximity such as endothelial cells. Since there is evidence supporting the view that
MSCs and pericytes are in fact the same in the bone marrow, knowing what molecules are
expressed by MSCs is also highly recommended. A list of markers known to be expressed
or not by pericytes, ECs and MSCs is shown in Table 1. Markers ascribed to other cell
types, such as smooth muscle cells and hematopoietic cells, are included for comparison
whenever possible.
As stated by Armulik et al., 2005 (54), “the heterogeneous morphology and marker
expression make unambiguous identification of pericytes a challenge”. The NG2
proteoglycan is one of the molecules used to identify pericytes in vivo in rodents (55). In
rats, the NG2 proteoglycan has been shown not being consistently expressed by all
pericytes under normal circumstances, its expression being preferentially on the venular
(56). The human homologue of NG2, termed high molecular weight melanoma associated
antigen (HMW-MAA) and also known as human melanoma proteoglycan (57), is a marker
for activated (proliferative) human pericytes (58).
Alpha smooth muscle actin (αSMA) may also be used as a pericyte marker in spite of
being expressed by smooth muscle cells (59), and displaying expression differences
between species (60).
53
Angiopoietin-1 has also been reported to be expressed by human pericytes in vitro and
in vivo (61). Since this is a secreted factor, using it as a maker for the detection of pericytes
in vivo by immunohistochemistry might be difficult because it may be also found in the
basement membrane, and bound to its receptor on the surface of endothelial cells.
The monoclonal antibody SH-2, which has been raised against human MSCs (27) and
later found to recognize CD105 (62), can recognize endothelial and perivascular cells in
embryonic and young post-natal dermis. Its expression seems to be developmentally
controlled, and is possibly associated with active angiogenesis or neoangiogenesis (63).
The antigen defined by the STRO-1 antibody has been shown to react with stromal and
erythroid cells (64), and perivascular and endothelial cells (47) in human bone marrow.
The same antigen is expressed by perivascular and endothelial cells in human dental pulp
(48). Of interest, CFU-Fs have been observed only in the STRO-1+ positive fraction of
human bone marrow (64). In rats, the STRO-1+, adherent fraction of bone barrow can
differentiate into chondrocytes, osteoblasts and adipocytes (65).
Using a mouse developmental model, Brachvogel et al., 2005 (66) demonstrated that
annexin A5 is present in angioblasts during vasculogenesis, and its expression becomes
restricted to perivascular cells from embryonic day 10.5 on. The annexin A5+ cells sorted
from adult brain meninges expressed Sca-1, CD34 and CD117, similarly to brain-derived
murine MSCs (14). Furthermore, these sorted cells could be induced to differentiate along
osteogenic, chondrogenic and adipogenic pathways. Experiments aiming to test if annexin
A5 is expressed by human perivascular cells in a similar way it is expressed in mice might
be important to define a novel, and perhaps unique, marker for human pericytes.
To date, the ganglioside defined by the antibody 3G5 seems to be the best marker for
pericytes. Even though some specialized cell types are also recognized by this antibody, it
54
is highly specific for pericytes within the microvasculature (67). This same antibody has
been used to indicate that equivalent forms of microvascular pericytes are present in the
interface between the intima and the media layers of larger vessels, forming a continuous
pericytic network throughout the vasculature (53). In the same work, pericytes in the vasa
vasora were also found to be 3G5
+
. Human cells isolated from skin using the 3G5 antibody
express other pericyte-related markers such as HMW-MAA, desmin, α-SMA and
angiopoietin-1 when cultured shortly in vitro (61). Cell types such as fibroblasts and
smooth muscle cells do not express it (68). The 3G5 antibody has been used to identify
microvascular human dermal pericytes in skin biopsies (69). This antibody reacts with
human, bovine, rabbit and porcine antigens (70), but it does not react with murine cells.
Such a characteristic would be of interest for the detection of human pericytes transplanted
into mouse hosts.
Considering the information available on pericyte molecules, it seems that a good
combination of markers to detect human pericytes would be the 3G5 antibody-defined
ganglioside, and HMW-MAA. Endothelial cells can be distinguished from pericytes based
on their expression of CD31 (71).
Pericyte – endothelial cell interactions
Pericytes and endothelial cells exhibit an interdependent relationship, wherein soluble
factors and physical interactions synergistically contribute for blood vessel structure
maintenance (54). The secretion of angiopoietin-1 by pericytes followed by its uptake by
endothelial cells via the Tie-2 receptor is one example of soluble factor mediated
interaction (72). Endothelial cells secrete PDGF-BB, which becomes associated with
55
heparan sulfate proteoglycans in the basement membrane they produce. Upon binding to
its receptor on the cell surface of pericytes, this interaction provides them proliferation and
migration cues (54). Several other cell types express PDGF-BB (73), but in this particular
case, endothelial cells provide pericytes this growth factor associated with positional
information, similarly to lights in runways during the night. Furthermore, mice lacking
basement membrane-retaining motif for PDGF display defective investment of pericytes in
the microvasculature (74).
Physical contact also plays an important role in pericyte-endothelial cell
communication. The establishment of connexin 43-containing gap junctions between
murine mesenchymal precursors and bovine aortic endothelial cells has been shown to be
necessary for the production of the active form of TGF-β by the former. As a consequence,
the mesenchymal precursors used started expressing the pericyte/vascular smooth muscle-
related proteins aSMA, SM22a, and SM-myosin heavy chain (75). Another important
physical contact between pericytes and endothelial cells is that mediated by N-cadherin,
which provides vessel structure stabilization (54,76,77).
The intimate relationship between pericytes and endothelial cells shown above is
something that is often not considered when defining a pericyte. It is possible that what
ultimately tells pericytes from other cell types is the nature of their interaction with
endothelial cells. To be considered a pericyte, a given cell must a) establish physical
contact with endothelial cells by means of gap junctions; b) express at least one marker
attributed to pericytes; and c) not express pan-endothelial cell markers.
56
THE PERICYTE AS A LOCAL PROGENITOR FOR CHONDROCYTES AND
OSTEOCYTES IN BONE REPAIR: A POSSIBLE LINK BETWEEN THE IN
VITRO AND THE IN VIVO DIMENSIONS?
Endochondral bone formation has been studied during development, fracture repair,
and ectopic bone formation. In every case (see below), vascularization plays a key role in
bone formation.
During the formation of long bones, primitive cartilage becomes vascularized before
ossification. After birth, vasculature actively invades the growth plate at the level of the
hypertrophic chondrocytes, which express VEGF and undergo apoptosis. VEGF recruits
blood vessels to the site formerly occupied by the hypertrophic chondrocytes, and osseous
tissue formation takes place (78).
Aiming to establish an experimental model for the study of bone formation, early
experiments using demineralized bone matrices implanted subcutaneously in rats have
determined a sequence of stages that occur during ectopic bone formation. Such a sequence
comprises encystment by host’s cells, cartilage formation, cartilage hypertrophy and first
bone formation, vascular invasion, and finally massive bone formation (79). The
chronological histological characteristics, as described by Reddi and Anderson, 1976 (80),
are summarized in Table 2. According to this sequence of events, vascularization of the
demineralized bone matrices occurs after the appearance of hypertrophic chondrocytes,
and precedes bone formation. Hence, this model is compatible with the events described
above for post-natal bone formation in the growth plate.
Osseous tissue formation during fracture repair of long bones differs from the
processes described above in that there is no pre-existing cartilage. After fracture, and if it
57
is mechanically unstable, a blood clot forms, followed by an inflammatory response and
angiogenic activity. Fibro-cartilaginous tissue forms in the internal callus, while direct
osseous neoformation occurs in the external callus. The internal callus eventually becomes
mineralized and is replaced by osseous tissue. Finally, a remodelling process takes place,
replacing the callus with secondary lamellar bone. Notably, the hematoma that forms
initially has a strong angiogenic effect, and its removal attenuates fracture healing (81).
The role of pericytes as progenitors for cartilage and bone has been previously
suggested. Diaz-Flores et al., 1991, 1992 (82,83) tracked the fate of vascular and
perivascular cells labelled with Monastral Blue A during periosteal bone healing, and in
grafted perichondrium. As a result, mature osteocytes and chondrocytes displaying
cytoplasmic inclusions of the dye could be observed by means of light and electron
microscopy. The authors concluded that pericytes can give rise to osteocytes and
chondrocytes in vivo.
Brighton and Hunt, 1997 (84) studied the early the behaviour of periosteal vascular and
perivascular cells in response to bone fracture by means of light and electron microscopy.
They found that 24 hours after the fracture, both endothelial cells and pericytes were
hypertrophied and, at 48 hours, putative pericytes had divided and formed layers of stacked
cells. Five days after the fracture, chondroblasts that retained remnants of the basal lamina
could be observed in close proximity to the hypertrophied pericytes. At six days post-
fracture, hypertrophic chondrocytes, some of them showing signs of degeneration, could be
observed in the proximal zone of the inner layer of the periosteal callus. At this time,
woven bone was being replaced by lamellar bone in the distal zone of the inner layer. On
the seventh day, woven bone had been almost completely replaced by lamellar bone in the
distal zone of the inner layer of the callus, while endochondral bone formation was
58
occurring in the proximal zone. These results indicate that pericytes give rise to
chondrocytes, and that osteocytes are observed after that.
A PERIVASCULAR NICHE FOR MESENCHYMAL STEM CELLS
The components of the microenvironment with which stem cells interact are under
intense scrutiny, and much has been found about cells, extracellular matrix components,
and soluble factors that develop a two-way interaction with different types of adult stem
cells (6). As for mesenchymal stem cells as we know them (in vitro), very little is needed
for their maintenance in a state that allows them to be referred to as stem cells – with the
capacity of self-renewal and differentiation. All they need is culture medium, such as
Dulbecco’s modified Eagle’s medium supplemented with fetal calf serum, and a plastic
surface to attach to (85). Very little is known about their niche in the organism, yet to
comply with the current belief that links stemness to an adequate niche, they must have
their own location and microenvironment.
Multiple evidences now suggest a perivascular location for MSCs. Conventional MSC
cultures may be established from artery or vein walls (14,86,87). Cells with surface profile
similar to MSCs (positive for Stro-1 and CD146) were observed lining blood vessels in the
bone marrow and dental pulp (48). A functional relationship between MSCs and mural
cells has also become apparent. Pericytes are able to differentiate into different
mesenchymal cell types in vitro (46,49). Studies in vivo have suggested that pericytes can
give rise to adipocytes (66,88), chondrocytes (66,82,84), and osteoblasts (83,84). Vascular
smooth muscle cells have also been shown to differentiate into other mesenchymal
lineages such as osteoblasts, chondrocytes and adipocytes (reviewed in ref. 89).
59
Pericytes could thus be the in vivo representatives of in vitro MSCs. On the other hand,
MSCs could be a precursor for pericytes and vascular smooth muscle cells, or even to other
types of vascular cells. Recent evidences, for instance, show that MAPCs differentiate into
arterial and venous endothelial cells, with involvement of sonic hedgehog (Shh) and its
receptors, as well as Notch 1 and 3 receptors and some of their ligands in the process (90).
Although not completely identified with MSCs, MAPCs share a number of characteristics
with them, and further investigation should determine whether more conventional MSCs
show this differentiation potential.
Further information on the cellular and soluble components of the microenvironment
supporting MSCs in vivo are slowly gathering, as the studies progress (reviewed in ref. 17).
Based on these evidences as a whole, we suggested a model in which mesenchymal stem
cells have a perivascular niche and lie in the basement, opposed to endothelial cells (14).
According to this model, cues provided by the niche, composed by other cells, extracellular
matrix and signaling molecules including autocrine, paracrine, and endocrine factors (17),
coordinate a gradual transition to progenitor and mature cell phenotypes.
CONCLUSION
It is not yet possible to define if MSCs are identical to pericytes or are more primitive
cells which originate pericytes and vascular smooth muscle cells. Although still lacking
formal proof, however, evidences are advanced enough to give consistency to the concept
of a perivascular niche for the adult stem cell we call “mesenchymal stem cell”. The
elements of the niche include endothelial cells, soluble factors such as angiopoietin-1 and
PDGF-BB, and extracellular matrix components not well known as yet. As known for the
60
interaction of other types of adult stem cells and their niches, close contact with the basal
membrane keeps the cells undifferentiated. When detached from their niche, due to signs
received from the microenvironment and/or to internal programming, MSCs differentiate
into committed progenitors that gradually integrate into the tissue. According to this model
(14), a portion of the apparent post-natal stem cell diversity may be attributed to local
MSCs behaving as tissue-specific stem cells.
This concept makes the term “mesenchymal” stem cell seem inappropriate, and
possibly the term “perivascular stem cell” might best represent this cell type. The name is
however too well established to be changed, and may thus be maintained even if the
concept of the nature of the cell is modified. Finally, the concept has implications for the
design of new therapeutic approaches for a range of diseases, targeting the perivascular
compartment through cell-mediated therapies.
61
ACKNOWLEDGEMENTS
Thanks are due to Conselho Nacional de Desenvolvimento Cientifico e Tecnologico
(CNPq) for financial support, and to Dr. J. Michael Sorrell for discussions.
62
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73
Table 1. Markers expressed by pericytes and other cell types.
Molecule CD Pericytes SMCS MSCs ECs HCs Reference
3G5 antibody-defined
ganglioside
+
-
67,70
angiopoietin-1
+
-
61
angiopoietin-2
-
+
61
annexin A5
+
-
65
calponin
+
desmin
+ +
EGF receptor
+
+
nestin
+
+
NG-2 proteoglycan
+
55,56
PAL-E
+
PDGF-R
+
+
55
Sca-1 (mouse only)
+
+ + +
SM22-α
+
SM-myosin
+ + + + +
stem cell factor
+
STRO-1
+
+ +
Tie-1
-
+
61
Tie-2
-
+
61
VE-cadherin
+
VEGFR1
-; +
+
61
vWF
- - - + -
74
α-SMA
+ +
9
+
10
+
11a
-
11b
-
+
aminopeptidase N 13
+
+ + +
14
-
15
-
18
-
25
-
29
+
PECAM 31
- - - + -
34
-
+
85
36
+ +
44
+
45
- - -
+
49a
+
49b
+
49c
+
49d
-
49e
+
50
-
51
+
ICAM-1 54
+
58
+
75
61
+
62E
- +
62L
+
+
62P
-
71
+
73
+
Thy-1 90
+
+
+
102
+
TGFβRIII 105
+ +
106
+
117
-
+
85
119
+
120a
+
120b
+
121
+
123
+
124
+
126
+
127
+
prominin 133
+
PROW
PDGFRa 140a
+
+
PDGFRb 140b
+
+
55
CD, Cluster of Differentiation number; ECs, endothelial cells; HCs, hematopoietic cells; MSCs,
mesenchymal stem cells; PROW - Protein Reviews On The Web. http://mpr.nci.nih.gov/prow;
SMCs, smooth muscle cells. Empty cells indicate that marker expression is not known.
76
Table 2. Histological observations at different time points during the course of bone
formation in demineralized bone matrices.
Day Histological characteristics
1 Polymorphonuclear leukocytes +++
3
Polymorphonuclear leukocytes +
Fibroblasts ++++
5
Fibroblasts +++
Chondroblasts +
7 Chondrocytes +++
9
Hypertrophy and calcification of
chondrocytes
Capillary ingrowth
10
Chondrolysis
Osteoblasts +++
11 Bone +++
14
Bone +++
Early hemocytoblasts
18
Bone ++++
Bone marrow ++
21
Bone ++++
Bone marrow ++++
77
Capítulo 6
Cultured human mesenchymal stem cells take up
perivascular locations when implanted in ceramic cubes in
vivo.
Lindolfo da Silva Meirelles1, Donald P. Lennon, J. Michael Sorrell, Marilyn Baber, Nance
Beyer Nardi & Arnold I. Caplan.
Manuscrito em preparação a ser submetido para Cell and Tissue Research
78
Cultured human mesenchymal stem cells take up perivascular locations when
implanted in ceramic cubes in vivo.
Lindolfo da Silva Meirelles
1
*, Donald P. Lennon
2
, J. Michael Sorrell
2
, Marilyn
Baber
2
, Nance Beyer Nardi
1
and Arnold I. Caplan
2
.
1 – Departamento de Genética, Universidade Federal do Rio Grande do Sul, Brazil
Av Bento Goncalves 9500
Porto Alegre, RS, Brazil
91501-970
Telephone: +55 51 33086737
2 – Department of Biology, Skeletal Research Center
Case Western Reserve University
2080 Adelbert Road, Cleveland, OH, USA
44106-7080
* Bolsista do CNPq - Brasil
Abstract
Mesenchymal stem cells (MSCs) are defined as able to differentiate into mature
mesenchymal cells in vitro and in vivo. While much of the knowledge on MSC biology
derives from in vitro approaches, understanding their behavior in vivo is still a matter for
study. Experiments using injury models indicate that perivascular cells behave as local
progenitors during wound healing and, in addition, results from in vitro experiments
indicate that the cultured cells defined as MSCs derive from cells located in a perivascular
niche in vivo. We hypothesized that, if that is true, short-term cultured human MSCs would
take up a perivascular location when loaded into ceramic cubes and implanted
subcutaneously into immunocompromised mice. Using this approach, MSCs could be
detected around blood vessels from periods of time between 15 to 6 weeks, some of them
79
expressing the pericyte marker 3G5. These results strengthen the hypothesis that cultured
MSCs are perivascular cells in vivo.
Introduction
Mesenchymal stem cells (MSCs) may be defined as cells capable of giving rise to a
number of unique, differentiated mesenchymal cell types (Caplan, 1991; Prockop, 1997).
First described as fibroblast precursors from bone marrow by Friedenstein et al. (1970),
MSCs may be also referred to as fibroblast colony-forming units (CFU-Fs) or marrow
stromal cells (Phinney, 2002). To date, there is evidence that suggests MSCs may exist not
only in bone marrow, but in virtually every location of the body (da Silva Meirelles et al.,
2006; Prunet-Marcassus et al., 2006; Krampera et al., 2006). MSCs from human bone
marrow can be isolated and cultivated using standard methods (Haynesworth et al., 1992;
Pittenger et al., 1999; Lennon and Caplan, 2006). A variety of molecules is known to be
expressed by cultured MSCs (Beyer Nardi and da Silva Meirelles, 2006). The literature
presents evidence that cultured MSCs also exhibit a high degree of plasticity in vitro, and
when implanted in vivo, such plasticity going beyond the mesenchymal limit (Kopen et al.,
1999; Woodbury et al., 2000; Lange et al., 2005; Sato et al., 2005; Seo et al. 2005). MSCs
have also been proposed to exert paracrine trophic effects through the secretion of
bioactive molecules (Caplan and Dennis, 2006). These characteristics make them
promising tools for cell and cell-based therapies.
Although a growing body of information regarding the characteristics of cultured
MSCs has accumulated over the years, their exact identity in vivo is still a matter for study.
A careful examination of the literature indicates an identity overlap between MSCs and
pericytes because a) evidence indicates that pericytes can give rise to differentiated cells in
mesenchymal tissues in vivo (Richardson et al. 1982; Diaz-Flores et al., 1991; Diaz-Flores
et al., 1992; Brighton and Hunt, 1997); b) cultured pericytes can differentiate into cells of
mesenchymal lineages in vitro and when implanted in vivo (Doherty et al., 1998;
Farrington-Rock et al., 2004); c) MSCs have been traced to the perivascular space in
studies using defined MSC markers (Fleming et al., 1998; Bianco et al., 2001; Shi and
Gronthos, 2003; Brachvogel et al., 2005); and d) cells bearing MSC characteristics can be
80
isolated from virtually all tissues, as explained by a model that conceives that those are
perivascular cells in vivo (da Silva Meirelles et al., 2006).
Taking the above into account, we hypothesized that, if cultured human MSCs are
in fact perivascular cells in vivo, they would take up a perivascular location when loaded
into ceramic cubes and implanted subcutaneously into immunocompromised mice. To test
that hypothesis, the investment of ceramic cubes loaded with MSCs at early time points
was examined, and the implanted human cells could be observed throughout the pores of
the cubes, many of them in perivascular locations. Cultured human MSCs were found to
express the pericyte markers 3G5 and HMW-MAA in vitro, and 3G5-positive MSCs could
be detected in ceramic cubes implanted in vivo. Human brain vascular pericytes (HBVPs),
used as a positive control for immunofluorescence, were also found to express the human
MSC markers CD13, CD44 and CD105. These results are consistent with the hypothesis
that cultured MSCs are perivascular cells in vivo, and provide additional evidence of
identity overlap between MSCs and pericytes.
Materials and methods
Cell culture.
Bone marrow aspirates were obtained from healthy volunteers after informed
consent. MSCs were isolated and cultured as described elsewhere (Lennon and Caplan,
2006). Briefly, bone marrow was mixed with one volume of low glucose Dulbecco’s
Modified Eagle’s Medium (Sigma Chemical, Saint Louis, MO) added of 10% fetal bovine
serum (Gibco/Invitrogen, Carlsbad, CA) from selected lots. After centrifugation on a
Percoll gradient, the fraction above the density cut was cultured in the same medium
composition. For passaging, cells were rinsed with Tyrode’s Balanced Salt Solution
(TBSS, Sigma), and incubated with 0.25% trypsin-EDTA (Gibco). Cells from first or
second passages were used for the experiments. HBVPs were acquired from ScienCell
(San Diego, CA), and were cultured according to the supplier instructions.
Ceramic cube implantation
81
Implantation of MSC-loaded ceramic cubes into syngeneic or immunocompromised
host animals as an in vivo assay for the osteochondrogenic potential of these cells has been
described previously (Dennis et al., 1992, Dennis and Caplan, 1993). Briefly, blocks of
porous ceramic (mean pore size of 200 mm), consisting of 60% tricalcium phosphate and
40% hydroxyapatite, were cut into cubes measuring 3 mm per side. The ceramic cubes
were washed with water to remove ceramic dust, dried under a heating lamp, and sterilized
in an autoclave. To improve cell attachment, ceramic cubes were combined with a 100
mg/ml solution of human fibronectin (Collaborative Biomedical, Bedford,MA)in Tyrode's
salt solution in a 12x75-mm sterile, capped polystyrene tube (Becton Dickinson, Franklin
Lakes, NJ). A 20-gauge needle attached to a 30-ml syringe was inserted through the cap of
the tube, and the syringe plunger was retracted to evacuate air from the tube, thus
generating a partial vacuum and permitting the fibronectin solution to enter the pores of the
cubes. Cubes remained in the fibronectin solution for 2 h at room temperature (RT) and
were then air dried overnight in a laminar flow hood.
First or second-passage MSCs were tripsinized and resuspended at a concentration
of 5x10
6
cells/ml in serum-free medium, and transferred to a 12x75-mm sterile tube.
Fibronectin-coated ceramic cubes were introduced in the tube, and air was withdrawn with
a 20-ml syringe as for fibronectin coating.
Cell-loaded cubes were incubated at 37°C for 2h and then implanted
subcutaneously into pockets created by blunt dissection on the dorsal surface of SCID mice
(Charles River Laboratories, Wilmington, MA) anesthetized with a mixture of Ketamine
and Xylazine (100 and 10 mg/kg body weight, respectively). The incision on the skin was
closed with a steel wound clip. At defined times post-implant, the animals were euthanized
by anesthetic overdose, and the cubes were harvested. This protocol was approved by the
Institutional Animal Care and Use Committee.
Ceramic cube processing
After removal, the ceramic cubes were fixed with 10% neutral buffered formalin
overnight. The cubes were washed once in TBSS, and decalcified in a solution containing
tris (0.1M) and 10% (w/v) EDTA disodium salt, pH 7.0, for 2 weeks at 4°C, with 3
solution changes per week. This method was chosen because it has been shown to preserve
82
tissue antigens well as compared with others (Jonsson et al., 1986). For some experiments,
cubes were fixed in JB fixative (Beckstead, 1994), and then decalcified using the same
solution described above. Some cubes were included in paraffin. 5 μm sections were
prepared, and stained with Mallory-Heidenhain. For immunostaining, cubes were
embedded in optimal cutting temperature compound (OCT) and snap-frozen in liquid
nitrogen. 7 μm cryosections were prepared and transferred to gelatin-coated slides.
Antibodies and immunostaining
Commercially available primary antibodies used were anti-HMW-MAA (AbD
Serotec, Raleigh, NC), diluted 1:100, and anti-human CD31 (Chemicon, Temecula, CA),
also diluted 1:100. Antibodies against CD13 and CD44, and the antibodies SH-2
(Haynesworth et al., 1992b) and 6E2 were present in the supernatant of their respective
hybridomas, developed in this laboratory. The supernatants were used with no further
dilution for immunostaining. The SH-2 antibody has been shown to recognize CD105
(Barry et al., 1999). The antibody 6E2 recognizes an antigen present on the surface of most
human cells, and does not cross-react with murine cells. The 3G5 hybridoma was
purchased from American Type Culture Collection (Manassas, VA), and its supernatant
was used with no further dilution. A fluorescein isothiocyanate (FITC)-conjugated goat
anti-mouse Ig (SouthernBiotech, Birmingham, AL) was used as secondary antibody.
For immunostaining, cryosections were rinsed in TBSS at room temperature to
remove traces of OCT, washed once with phosphate-buffered saline (PBS) containing
0.1% bovine serum albumin (BSA), and incubated with the primary antibodies for one
hour at RT. The slides were then washed with PBS-BSA, and incubated with the secondary
antibody for one hour at RT. Some slides were incubated with the secondary antibody only
to provide a negative control. For immunocytochemistry, cells were plated on 35-mm
culture dishes or chamber slides at leas 24 hour before the experiment, and processed in the
same way as described for cryosections.
The samples were observed using either an upright microscope (Leica) or an
inverted microscope (Olympus), both equipped with filter sets for the detection of FITC.
Images were acquired using digital cameras attached to the microscopes, using software
83
provided by the respective manufacturers. Images were sharpened using Adobe Photoshop
CS2 software.
Results
Blood vessels invade MSC-loaded ceramic cubes
One of the first steps to examine if MSCs take up perivascular locations was to
observe vascular invasion in the MSC-loaded cubes. As shown in Figure 1A, cubes were
already invaded by the host’s vasculature at the first time point studied (8 days post-
transplant). Empty cubes, however, had undergone nearly no vascular invasion (Figure
1B). Many vessels could be observed near the osteoblastic layer that forms in the walls of
the pores at all time points analyzed (Figure 1). A noteworthy feature regarding the spatial
relation of blood vessels to the bone layer that forms inside the cube pores is that the
vasculature was never found inside the osseous tissue.
MSCs and HBVPs share surface markers
Since the molecules 3G5 and HMW-MAA are expressed in resting (Helmbold et al,
2004) and proliferative (Rajkumar et al., 1999) pericytes, respectively, in vivo, we
hypothesized that, if cultured MSCs are actually pericytes, only a small fraction of them
would express 3G5 after one passage, while a higher proportion would express HMW-
MAA. HBVPs were used as a positive control for the immunostaining. HBVPs had their
expression of some MSC markers checked to evaluate if they share some identity with
MSCs.
Virtually all cultured MSCs and HBVPs expressed HMW-MAA in a uniform
fashion (Figure 2A and B). Both populations presented subpopulations of 3G5-positive
cells (Figure 2C and D). 3G5-positive cells were also present as subpopulations among the
cells of the first colonies in primary culture (Figure 2E). All HBVPs expressed SH-2
(Figure 2F), CD13 (Figure 2G) and CD44 (Figure 2H).
Effect of fixation on the epitope defined by the 3G5 antibody
84
Locating 3G5-positive cells in the MSC-loaded cubes was of interest because the
3G5 antibody does not recognize murine antigens (Stramer et al. 2004), meaning that 3G5-
positive cells found would be human in origin. Furthermore, 3G5 expression in vivo was
expected only in pericytes (Andreeva, et al., 1998; Helmbold et al., 2004), meaning that
3G5-positive cells would represent human MSCs that became pericytes in vivo.
As a first step to check if 3G5-positive cell could be observed in cube cryosections,
the effect of formalin – the fixative routinely used – on the 3G5 antigen was assessed by
immunofluorescence on cultured HBVPs. As shown in Figure 3A and B, fixation with
formalin abolished the reactivity of the 3G5 antibody with its epitope. As an alternative, JB
fixative was tested with the same purpose, and was found to be effective (Figure 3C and
D). Ethanol was also tested not only because it might work as a fixative, but also because
embedding JB-fixed cubes in paraffin could be of interest. Ethanol also abolished 3G5
reactivity (Figure 3E).
The majority of cells inside the MSC-loaded cubes are human in origin
To observe what cells inside the ceramic cube were derived from the MSCs
implanted, cryosections were immunostained with the 6E2 antibody. As seen in Figure 4A
and B, most cells inside the ceramic cubes were human in origin. Immunolocalization of
3G5-positive cells was attempted in cryosections from cubes fixed with formalin and
subjected decalcification using Tris-EDTA because there was a possibility that this process
could work as an antigen retrieval protocol, since Tris-EDTA may be used as an antigen
retrieval method under other conditions (Torlakovic et al. 2005). Figure 4C shows a 3G5-
positive cell in a perivascular location.
MSCs take up perivascular locations
Even though the immunostainings performed using the 3G5 antibody allowed for
the visualization of some 3G5-positive cells, the signal was usually weak and sometimes
telling whether or not it was positive or artifactual was difficult. To seek for 3G5 positive
cells, cubes were fixed in JB fixative instead of formalin, and cryosectioned. To observe
85
human cells inside the cubes, cryosections were stained with the 6E2 antibody. In those
preparations, erythrocytes displaying some background fluorescence helped to locate the
blood vessels. Cells displaying a fluorescence level clearly higher than control sections or
erythrocytes could be identified around many blood vessels (Figure 5A). Cells expressing
human CD31 could not be located inside the cubes (Figure 5B). Immunoreactivity of the
anti-CD31 antibody used was checked on live and JB-fixed cultured human umbilical vein
endothelial cells (not shown). Finally, 3G5-expressing cells could also be located inside
JB-fixed ceramic cubes, but their frequency seemed to be far lower than the frequency of
human cells around blood vessels (not shown).
Discussion
This study differs from other where MSCs shown to engraft across the blood
vessels (Schmidt et al., 2006) in that here, MSCs were present in the target site prior to
vascularization and tissue formation, leading to a process that involves the interaction of
the pre-existing MSCs with the ceramic environment, and with the yet-to-come host
vasculature. In this regard, MSCs displayed the property of attracting the host vasculature,
as evidenced by the poor investment of empty ceramic cubes by blood vessels (Figure 1B).
The majority of the cells inside the pores of the cubes were found to be derived
from the MSCs at 15 days post-implant (Figure 4A), indicating that the osseous tissue
formed inside the cube was formed by a portion of the MSCs, and not by host’s cells taken
there along with, or by the blood vessels. Human cells could be detected also 6 weeks post-
implant, and in this case perivascular human cells were commonly observed (Figure 5A).
3G5 positive cells could be observed in the cubes 15 days (Figure 4C) and 6 weeks (Figure
5D-F) post-implant, but their frequency was very low as compared with the amount of
human cells present. Even though this may indicate that the frequency of MSCs that in fact
became pericytes was low, the possibility that this reflects the low frequency of 3G5-
positive cells in the short-term MSC cultures cannot be discarded. Since no CD31-positive
human cells could be found inside the cubes, the MSCs found around the blood vessels
may be considered pericytes on the basis of their location, independently of their
expression of 3G5.
86
The high frequency of MSC-derived pericytes may be considered as evidence that
the MSCs that were not in contact with the osteoinductive ceramic surface have a natural
tendency to remain as perivascular cells, which is consistent with the hypothesis that
cultured MSCs descend from pericytes (da Silva Meirelles et al., 2006). In addition to that,
the fact that both cultured MSCs and pericytes share markers associated with pericytes and
MSCs, respectively (Figure 2), reinforce this view.
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Figure 1 - Aspect of blood vessels inside ceramic cubes at different time
points. A, section from a cube harvested 8 days post-implant. Lines indicate the
areas formerly occupied by ceramic, and define the limits of the contents of a pore
(200X). B, section from an empty cube harvested 15 days post-implant. Note the
lack of vasculature and tissue in the center (50X). C, same as A, shown in a higher
magnification, evidencing an area where the blood vessels are in close proximity
with the osteoblastic layer on the pore wall (arrow) (400X). D and E, cubes
harvested 15 days post-implant. F, cube harvested 7 weeks post-implant. The
arrow on the osseous layer points to a transitional area between the perivascular
zone of a vessel and the osseous tissue (200X).
91
HBVPs
HMW-MAA
hMSCs
HMW-MAA
HBVPs
SH-2
HBVPs
CD13
MSC primary colony
3G5
hMSCs
3G5
HBVPs
3G5
200X 200X
100X
100x
100X 200X
100X 100X
HBVPs
CD44
A B
C D
E
F
G H
Figure 2 - Pericyte markers expression on MSCs, and MSC markers expression on
HBVPs. A and B, cultured MSCs and HBVPs uniformly express the activated pericyte
marker, HMW-MAA. C and D, fractions of cultured MSCs and HBVPs express the pericyte
marker 3G5. E, 3G5-positive cells are also present on the first MSC colonies in primary
culture. Round cells (one pointed by an arrow) represent false positives as compared with
the immunostaining control slide (not shown). F, G and H, cultured HBVPs express the
human MSC markers SH-2 (F), CD13 (G), and CD44 (H).
92
100X 100X
100X
unfixed formalin
unfixed
JB
ethanol
A B
C D
E
Figure 3 - Effect of different fixatives on the epitope defined by the 3G5 antibody. A
and B, pictures of a first experiment in which unfixed (A) and formalin-fixed (B) HBVPs
were immunostained with the 3G5 antibody. C, E and F, pictures from a second experi
-
ment in which unfixed (C), JB-fixed (E) and ethanol-fixed (F) HBVPs were immunostained
with the same antibody.
93
200X
200X
400X400X
A B
C D
6E2
3G5
Figure 4 - Immunohistochemistry on formalin-fixed cubes decalcified using tris-EDTA.
A, detail of one of the pores of a cube processed 15 days post-implant, after immunostaining
using the 6E2 antibody. B, negative control for A. C, immunolocalization of a 3G5-positive cell
(arrow) adjacent to a blood vessel, in a section of a cube harvested 3 weeks post-implant. D,
negative control for C.
94
A
B
C
D
E
F
6E2 3G5
3G5
3G5
CD31
100X 400X
400X 400X
400X 400X
control
Figure 5 - Immonohistochemistry on JB-fixed cube cryosections. A, immunostaining using
the 6E2 antibody. Background fluorescence from erythrocytes make blood vessels distinguish
-
able, and human cells can be seen around them (arrows). B, cryosection stained with anti-human
CD31 antibody. C, negative control. D - F, pictures of the same region on consecutive sections
subjected to immunostaining with the 3G5 antibody. Positive cells can be observed near the
osteoblastic layer on the pore wall (arrows).
95
Capítulo 7
Discussão
96
A MSC, por suas características de obtenção reprodutível por métodos
padronizados e alta plasticidade in vitro, vem atraindo a atenção de pesquisadores que
visam a sua utilização como agente terapêutico para o tratamento de várias doenças, ou
como componente de materiais biológicos que possam repor tecidos perdidos em
acidentes. É relevante, portanto, que se estudem as características de sua biologia, a fim de
que se obtenha maior sucesso nessas aplicações.
O primeiro artigo apresentado nesta tese (Capítulo 3) demonstra que células que
apresentam características de tronco mesenquimal in vitro podem ser obtidas de diferentes
órgãos e tecidos de camundongo. Nele, descartou-se a hipótese de que tais células eram
originárias do sangue circulante, e reforçou-se a hipótese de que as mesmas são
provenientes dos vasos sangüíneos, mais especificamente da região perivascular. Com base
também em outros dados da literatura, postulou-se um modelo no qual células
perivasculares seriam as responsáveis pela reposição de células dos órgãos em que estão
localizadas, podendo assumir um papel mais ativo no caso de lesão tecidual.
O segundo artigo (Capítulo 4) abordou o ponto específico do conteúdo de DNA das
mMSCs cultivadas in vitro, indicando que este encontra-se duplicado nas mesmas. Isso foi
considerado mais uma evidência na direção da origem perivascular das mesmas, posto que
a duplicação do conteúdo de DNA é uma característica de células perivasculares in vivo. A
condição tetraplóide de mMSCs cultivadas in vitro também levou a uma colaboração com
grupo estrangeiro, a fim de estudar-se a influência da fusão de tipos celulares maduros com
MSCs in vitro, uma vez que uma das hipóteses que poderiam explicar o surgimento de
células tetraplóides multipotentes seria a fusão, em cultura, de células-tronco com células
proliferativas indiferenciadas. (Anexos 2 e 3).
As conclusões do primeiro artigo, e também do segundo artigo, levaram a uma
reflexão a respeito de qual poderia ser o papel das células perivasculares in vivo, além de
sua função estrutural conhecida, uma vez que a abordagem utilizada no primeiro artigo não
permite uma conclusão definitiva acerca do comportamento de células perivasculares como
células-tronco in vivo. O terceiro artigo desta tese (Capítulo 5) sintetiza esta reflexão,
ressaltando aspectos de MSCs e de pericitos que apontam para uma sobreposição de
identidade entre ambos. O Capítulo 5 pode ser considerado, também, uma conseqüência
direta do primeiro trabalho resultante desta tese, apresentado como Anexo 1.
97
Já o quarto artigo (Capítulo 6) demonstra que MSCs cultivadas podem assumir uma
localização perivascular quando implantadas in vivo, utilizando-se um modelo
experimental de osteogênese. Essa característica foi interpretada como mais uma evidência
indicando a relação próxima entre MSCs definidas como tal in vitro e o espaço
perivascular.
Tomados em conjunto, os artigos apresentados indicam que o nicho perivascular é,
certamente, um objeto de estudo muito interessante para a obtenção de conhecimento
básico sobre a MSC. Além disso, o panorama que se vislumbra quando se pensa na região
perivascular como um nicho de células-tronco indica que não são MSCs que se encontram
distribuídas por todo o organismo pós-natal, mas sim um conjunto heterogêneo de células-
tronco associadas ao nicho perivascular e que, sob certas condições in vitro, podem
assumir fenótipos compatíveis com aqueles atribuídos à progênie da MSC. Uma evidência
que aponta nesta direção é o fato de que mMSCs derivadas de diferentes tecidos
apresentam propriedades de diferenciação distintas (Capítulo 3). O conceito de
heterogeneidade na tronquicidade das células perivasculares ao longo da vasculatura é
também coerente face à heterogeneidade no compartimento endotelial (Garlanda e Dejana,
1997).
As semelhanças entre MSCs obtidas de diferentes órgãos (Capítulo 3), por sua vez,
poderiam ser explicadas por suas origens embrionárias a partir de células derivadas da
região dorsal da aorta no período embrionário (dia 9,5 em camundongos) chamadas
mesoangioblastos (Minasi et al., 2002). Mesoangioblastos foram assim denominados por
darem origem a tecidos mesodérmicos não-hematopoiéticos, em contraste com
hemangioblastos, que são células derivadas da região ventral da aorta e que dão origem a
células hematopoiéticas e endoteliais; ambos seriam descendentes de um angioblasto
primitivo (Cossu e Bianco, 2003). Isso indica que células perivasculares, e por extensão
MSCs, têm muito em comum com células endoteliais, embora sejam tipos celulares
distintos. E, de fato, uma relação íntima entre células perivasculares e endoteliais,
remanescente de interações entre ambas durante o desenvolvimento embrionário, persiste
durante a vida adulta (Armulik et al., 2005).
O modelo proposto em que células perivasculares atuam como células-tronco nos
diferentes tecidos parece, em um primeiro momento, contrário a outro modelo que propõe
que células circulantes atuam como progenitoras para diferentes tecidos (Ratajczak et al.,
98
2004), exibido na Figura 1. A este modelo, somam-se evidências de que células circulantes
possuem potencial de diferenciação semelhante ao da MSC in vitro (Kuznetsov et al.,
2001; Kuwana et al., 2003) e também de que células da linhagem dos macrófagos
enxertam-se em áreas de angiogênese ativa (Rajantie et al., 2004).
Figura 1 – Modelo de células tronco/progenitoras circulantes. Células
originárias da medula óssea encontram-se na circulação, a partir da qual podem ser
mobilizadas para diferentes tecidos através de gradientes quimiotáticos, em determinadas
situações como, por exemplo, lesão tecidual. Retirado de Ratajczak et al. (2004).
Entretanto, dados de um estudo recente (Kajikawa et al., 2007) indicam que ambos
modelos relatados no parágrafo anterior não são mutuamente excludentes, mas sim
complementares. Naquele estudo, os autores utilizaram dois modelos para verificar a
contribuição de células circulantes e de células locais para o processo de reparo de lesão no
tendão patelar. Em um dos modelos (rato com células circulantes expressando GFP),
99
células circulantes foram encontradas no local da lesão 24 horas após a cirurgia, e a
quantidade de células derivadas da circulação reduziu-se para níveis muito baixos após 7
dias. No outro modelo (enxerto de tendão patelar de rato GFP em rato não-transgênico),
verificou-se que células locais tornaram-se proliferativas 3 dias após a lesão no tendão, e
que o número destas aumentou grandemente após 7 dias, mantendo sua proliferação. Os
autores concluíram que o reparo da lesão no tendão patelar ocorre em duas fases:
primeiramente, células circulantes migram para o local da lesão; em um segundo momento,
células locais passam a proliferar, e o número de células originárias da circulação é
reduzido a níveis muito baixos.
No primeiro artigo desta tese (Capítulo 3), utilizou-se o cultivo de glomérulos
renais de camundongo para a obtenção de células com características de MSC. Conclui-se
que as culturas derivadas dos glomérulos eram originárias das células mesangiais, que são
consideradas um tipo especializado de pericito (Schlondorff, 1987). Uma característica
marcante do estabelecimento de culturas a partir dos glomérulos isolados é o fato de que as
células aderentes derivadas dos glomérulos nunca foram observadas antes de 3 dias a partir
do início do cultivo. Acrescenta-se a isto uma proliferação impressionante a partir de
então. Em verdade, a transferência de glomérulos do organismo para condições artificiais
in vitro poderia ser considerada uma indução de lesão tecidual no glomérulo, mas sem a
presença de células circulantes.
A obtenção de uma resposta proliferativa de células glomerulares in vitro sem a
presença de células circulantes não descarta a hipótese de que as células circulantes
desempenham um papel importante neste processo in vivo. No entanto, essa informação,
em conjunto com os dados que apontam para as células mesangiais como as que originam
as culturas de células com características de MSC derivadas dos glomérulos, indicam que
células perivasculares podem estar envolvidas no processo de reparo tecidual bifásico
descrito por Kajikawa et al. (2007).
Assim sendo, considera-se que o modelo proposto no Capítulo 3 não se encontra em
oposição com o modelo proposto por Ratajczak et al. (2004), mas sim em harmonia.
Propõe-se, então, uma extensão do primeiro, através da incorporação do último (Figura 2).
100
Figura 2 – Modelo proposto de contribuição de MSCs para manutenção
tecidual. Nesta representação esquemática de uma secção transversal de um vaso simples,
as MSCs situam-se sobre a membrana basal (linha vermelha), opostas às células
endoteliais. Sinais dados pelo micro-ambiente tecido-específico coordenam uma transição
gradual (representada por um gradiente de cor verde) de células indiferenciadas para
fenótipos celulares progenitores e maduros. Esse processo pode ocorrer naturalmente
conforme representado pela seta pontilhada (A). Em caso de lesão tecidual, e após a
colonização da área da lesão por progenitores circulantes (B), MSCs indiferenciadas
podem ser mobilizadas diretamente para dentro do tecido sem a transição por células
progenitoras, conforme representado pela seta curva (C).
101
Capítulo 8
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Capítulo 9
Anexos
Esta seção contém produção bibliográfica em que houve participação do autor durante o
período de seu Doutorado, e que estão relacionados com o tema desta tese.
1. Mesenchymal stem cells: isolation, in vitro expansion and characterization é uma
revisão publicada no Handbook of Experimental Pharmacology, abordando aspectos gerais
do isolamento, cultivo e caracterização de MSCs. Esta foi baseada no projeto de Doutorado
apresentado pelo autor desta tese
2. Functional characterization of cell hybrids generated by induced fusion of primary
porcine mesenchymal stem cells with an immortal murine cell line é resultante da interação
com grupo sueco que tinha como objetivo investigar os efeitos da fusão de células-tronco
mesenquimais porcinas com fibroblastos de camundongo sobre o potencial de
diferenciação dos híbridos resultantes. Cultivo, diferenciação e caracterização de
marcadores de superfície foram realizados pelo autor desta tese no Brasil.
3. Polyethylene glycol-mediated fusion between primary mouse mesenchymal stem cells
and mouse fibroblasts generates hybrid cells with increased proliferation and altered
differentiation também é resultante da interação com o grupo sueco mencionado
anteriormente, e analisou as conseqüências da fusão de células-tronco mesenquimais
murinas, isoladas e caracterizadas em nosso laboratório, com fibroblastos de camundongo,
em termos de potencial de diferenciação e marcadores de superfície. Cultivo, diferenciação
e caracterização de marcadores de superfície foram realizados pelo autor desta tese no
Brasil.
109
Mesenchymal stem cells: isolation, in vitro expansion and
characterization.
Nance Beyer Nardi & Lindolfo da Silva Meirelles
Handb Exp Pharmacol. 2006;(174):249-82.
110
HEP (2006) 174:249–282
© Springer-Verlag Berlin Heidelberg 2006
Mesenchymal Stem Cells: Isolation, In Vitro Expansion
and Characterization
N. Beyer Nardi (✉)·L.daSilvaMeirelles
Genetics Department, Universidade Federal do Rio Grande do Sul, Av Bento Gonçalves
9500, Porto Alegre RS, CEP 91540–000, Brazil
1Introduction.................................... 250
2 The I dentity of the Mesench ymal Stem Cell ................... 251
2.1 TheColony-FormingUnit-Fibroblast....................... 251
2.2 TheBoneMarrowStroma............................. 252
2.3 TheMesenchymalStemCell ........................... 253
2.4 OtherCellPopulationsRelatedtotheMesenchymalStemCell ......... 254
3 Distribution of the M esenchymal Stem Cell ................... 255
4 Isolation and Culture of Mesenchymal Stem Cells ................ 256
5 Homing and Engraftment of Transplanted Mesenchymal Stem Cells ..... 259
6 Characterization of Mesenchymal Stem Cells .................. 260
7 Differentiation of Mesenchymal Stem Cells ................... 262
8 Applications of Mesenchymal Stem Cells in Cell and Gene Therapy ...... 263
8.1 StudyofCancerBiology.............................. 264
8.2 CellTherapy.................................... 264
8.2.1 Fibrosis....................................... 265
8.2.2 Cardiovasculogenesis ............................... 265
8.2.3 ArteriogenicEffects ................................ 266
8.2.4 ImmunosuppressiveEffects............................ 267
8.3 MesenchymalStemCellsandTissueEngineering................ 267
8.4 GeneticTherapy.................................. 268
8.4.1 CorrectionofGeneticDisorders ......................... 269
8.4.2 CancerSuppression ................................ 270
9 Pharmacologic Aspects of Mesenchymal Stem Cell Biology .......... 271
10 Conclusions .................................... 272
References ........................................ 274
111
250 N. Beyer Nardi · L. da Silva Meirelles
Abstract Me senchymal stem cells (MSC), one type of adult stem cell, are easy to isolate,
culture, and manipulate in ex vivo culture. These cells have great plasticity and the potential
for therapeutic applications, but their properties are poorly understood. MSCs can be
found in bone marrow and in many other tissues, and these cells are generally identified
through a combination of poorly defined physical, phenotypic, and functional properties;
consequently, multiple names have been given to these cell populations. Murine MSCs have
been directly applied to a wide range of murine models of diseases, where they can act as
therapeutic agents per se, or as vehicles for the delivery of therapeutic genes. In addition
to their systemic engraftment capabilities, MSCs show great potential for the replacement
of damaged tissues such as bone, cartilage, tendon, and ligament. Their pharmacological
importance is related to fo ur points: MSCs secrete biologically important molecules, express
specific receptors, can be genetically manipulated, and are susceptible to molecules that
modify their natural behavior. Due to their low frequency and the lack of knowledge on
cell surface markers and their location of origin, most in formation concerning MSCs is
derived from in vitro studies. The search for the identity of the mesenchymal stem cell has
depended mainly on three culture systems: the CFU-F assay, the analysis of bone marrow
stroma, and the cultivation of mesenchymal stem cell lines. Other cell populations, more
or less related to the MSC, have also been described. Isolation and culture conditions
used to expand these cells rely on the ability of MSCs, although variable, to adhere to plastic
surfaces. Whether these conditions selectively favor the expansion of different bone marrow
precursors or cause similar cell populations to acquire different phenotypes is not clear.
The cell populations could also represent different points of a hierarchy or a continuum of
differentiation. These issues reinforce the urgent need for a more comprehensive view of
the mesenchymal stem cell identity and characteristics.
Keywords Mesenchymal stem cell · Bone marrow stroma · Differentiation ·
Stem cell niche · Cell therapy · Genetic therapy
1
Introduction
Stem cells present in early embryonic stages are pluripotent and can generate
all of the cell types found in adult organisms, whereas, adul t stem cells exhibit
a continuum of plasticity or multipotency. In adult humans, the first and
one of the best-known stem cells to be described is the hematopoietic stem
cell (HSC). A great variety of other stem/precursor cell types have also been
described, but much less is known about their origin and maintenance in vivo
as organ-specific stem cell pools (Nardi 2005).
The mesenchymal stem cell (MSC) is one of the most interesting of the adult
stem cell types. These cells are easily isolated, cultured, and manipulated ex
vivo. MSCs exhibit great plasticity and harbor the potential for therapeutic
applications, but these cells are poorly defined. This has led to a heterogeneity
of names and phenotypes ascribed by different groups to this cell population.
MSCs are present in the bone marrow and in many other tissues, and these
cells are presently identified through a combination of poorly defined physi-
cal, phenotypic, and functional properties. A number of recent reviews have
adequately described the nature of MSCs (Short et al. 2003; Zipori 2004; Barry
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Me senchymal Stem Cells: Isolation, In Vitro Expansion and Characterization 251
and Murphy 2004; Kassem et al. 2004; Baksh et al. 2004; Javazon et al. 2004),
and the focus of this review will be to describe experimental approaches for
their isolation, in vitro expansion, and characterization. We will also discuss
the cellular therapeutic potentials of MSCs and place a special emphasis on the
pharmacological prospects of these cells in vitro and in vivo.
2
The Identity of the Mesenchymal Stem Cell
For most cells present in adult organisms, mitosis is accompanied by differ-
entiation. Stem cells are defined as those cells with the ability to proliferate
without differentiating. At the moment, the very existence of a mesenchymal
stem cell in vivo is not completely understood, since it is based on indirect
evidence derived mainly from the in vitro cultivation of bone marrow and
other tissues. This is true, but only to a point, because most types of adult
stem cells can only be identified after isolation, which can then be examined
through in vitro or in vivo assays to determine if they have the two main
characteristics of stem cells: the ability to proliferate and to differentiate into
mature cell types. Most of the information for MSCs, which are present at a low
frequency, are derived from in vitro studies, due to a lack of information with
respect to specific surface markers and their location in vivo. In vitro studies,
by their very nature, may, however, introduce experimental artifacts (Javazon
et al. 2004). This possibility is clearly described in several studies, including
one reported by Rombouts and Ploemacher (2003), who compared the homing
abilities of primary and culture-expanded MSCs in a syngeneic mouse model.
Uncultured MSCs demonstrated highly efficient homing to bone marrow, but
the infusion of immortalized multipotent syngeneic stromal cells, or even pri-
mary MSCs that had been cultured for only 24 h, were rarely if ever seen in
the lymphohematopoietic organs. Murine MSCs were also reported to have
a deficient capacity to home to bone marrow by Anjos-Afonso et al. (2004).
The identification of the mesenchymal stem cell has thus far depended on
in vitro culture systems, which have provided very heterogeneous information
and made the characterization of MSCs even more difficult. Three in vitro
systems are generally employed to examine these cells: the CFU-F assay, the
analysis of the bone marrow stroma, and the cultivation of mesenchymal stem
cell lines. Other cell populations, more or less related to the MSC, have also
been described.
2.1
The Colony-Forming Unit-Fibroblast
The first direct evidence tha t nonhematopoietic, mesenchymal precursor cells
were present in the bone marrow originated from the work conducted in
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252 N. Beyer Nardi · L. da Silva Meirelles
M oscow during the 1960s and 1970s ofFriedenstein and colleagues (reviewed in
Phinney 2002). These pioneering experiments involved the incubation of bone
marrow samples in tissue culture flasks. The presence of an adherent fraction
could be seen within a few days, which proved highly heterogeneous. Around
the 3rd–5th days, individualfoci oftwo tofourfibroblastswere observed among
the histiocytes and mononuclear cells, which could differentiate into cells that
co uld form small deposits of bone or cartilage (Friedenstein et al. 1976). These
cells were termed colony forming unit-fibroblasts or CFU-F.
During the 1980s, several studies showed that cells isolated by the Frieden-
stein method were multipotent and could differentiate into osteoblasts, chon-
droblasts, adipocytes, and even myoblasts (reviewed in Prockop 1997). The
frequency of CFU-F in bone marrow suspensions is very different among
species, and the results are influenced by the cultur e conditions (reviewed in
Short et al. 2003). Growth factors stimulating the proliferation of CFU-F in-
clude platelet-derived growth factor (PDGF), epidermal growth factor (EGF),
basic fibroblast growth factor , transforming growth factor-
β, and insulin-like
gro wth factor-1 (Gronthos and Simmons 1995; Kuznetsov et al. 1997a; Baddoo
et al. 2003; Bianchi et al. 2003). In contrast, cytokines such as interleukin 4
(IL-4) and interferon-alpha can inhibit the establishment of CFU-F (Wang
et al. 1990; Gronthos and Simmon s 1995). The formation of CFU-F has been
considered indicative of mesenchymal stem cells, but a direct relationship be-
tween the two has not been clearly established, probably because of the great
heterogeneity in morphology, cell size and differentiation potential observed
among species and between colonies (Javazon et al. 2004).
2.2
The Bone Marrow Stroma
Stromal cells, along with extracellular matrix (ECM) compo nents and soluble
regulatory factors, have until recently been thought of as secondary compo-
nen ts of a microenvironment required for sustained hematopoiesis (Nardi and
Alfonso 1999). The stroma, studied both in vitro and in vivo, is composed
of a very heterogeneous population of cells, which includes macrophages, fi-
broblasts, adipocytes, and endothelial cells (Dexter et al. 1976; Ogawa 1993).
Adventitial reticular cells branch through the medullary cavity and pro vide
a reticular network that supports hematopoietic cells. Marrow adipocytes con-
trol hematopoietic volume, such that impaired hematopoiesis is associated
with increased accumulation of fat inclusions, and accelerated hematopoiesis
is associated with loss of fat vacuoles. These processes determine the space
available for hematopoietic cells (Tavassoli 1984). Adipocytes may also act as
a reservoir for lipids needed during proliferation. Macrophages are important
in the clean-up of ineffective erythropoiesis and in the removal of the nuclear
pole that is produced during this process.
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Me senchymal Stem Cells: Isolation, In Vitro Expansion and Characterization 253
Stromal cells produce ECM components and both soluble and membrane-
associated growth factors to form a dynamic structure that plays an active
role in hematopoiesis, i.e., the hematopoietic stem cell niche. Matrix pro-
teins in this microenvironment include fibronectin, collagen, vitronectin, and
tenascin, and some of the most relevant soluble factors include stem cell fac-
tor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and
the granulocyte colony-stimulating factor (G-CSF). Representative adhesion
molecules include members oftheintegrin superfamily (VLA-1, VLA-2,VLA-3,
VLA-4, VLA-5, VLA-6) (reviewed in Whetton and Grahan 1999). Physical con-
tact among the stromal cells is important for the regulation of this microen-
vironment. Although it has been shown that contact is not fundamental for
the hematopoietic process to occur (Verfaillie 1992), it seems to be related
to the quality of the hematopoietic cells produced (Breems et al. 1998). The
stromal compartment has implications fo r human health, since abnormalities
in the stromal compartment may represent a possible mechanism implicated
in aplastic anemia (Kojima 1998) and in the abnormal behavior of Ph
+
cells in
chronic myeloid leukemia (Cordero et al. 2004).
Based on the well-established generation of multiple types of mesenchymal
cell from bone marrow, stromal stem cells were additionally proposed to exist
by Owen (1985). Analogous to the hematopoietic system, they proposed that
stromal stem cells reside in the bone marrow in their own niche, where the
cells were able to self-renew and generate mature conjunctive/stromal cell
types. The identity of this stem cell—which is now almost universally termed
mesenchymal stem cell—is still, as stated above, poorly understood.
2.3
The Mesenchymal Stem Cell
The lack of consensus about the proper nomenclature needed to describe these
cells has resulted in an incorrect, but synonymous use of the terms “marrow
stromal cell” and “mesenchymal stem cell.” Actually, stromal cells encompass
all cells present in the bone marrow that are not part of the hematopoietic
system. MSCs, on the other hand, correspond to that rare cell population
that can form other MSCs and generate mature cells of mesenchymal tissues.
Pro tocols involving the isolation of bone marrow cells based on their adherence
toplastic surfacesresult intheimmediateestablishment ofstromalcellcultures,
and not of MSC cultures. A more adequate term for the large number of cell
types with the potential to differentiate into mesenchymal tissues would be
“mesenchymal progenitor cell” (MPC), which would include cell types from
a hierarch y immediately above the pluripotent MSC but intermediate to that
represented b y mature mesenchymal cell types.
Another point of debate is the fact that the HSC is itself of mesodermic
origin, hence a type of MSC. For this reason, some authors prefer the term
“nonhematopoietic mesenchymal stem cell.” The fact that these cells, which are
115
254 N. Beyer Nardi · L. da Silva Meirelles
described below, may have alternative differentiation pathways that go beyond
the normal limits of mesoderm and ectoderm formation renders the term
“mesenchymal” inadequate. Probab ly, the best nomenclature to define this
cell type would be “adul t nonhematopoietic stem cell,” followed by “plastic-
adherent, bone-marrow derived stem cells”. All these concepts, however, are
alread y included when the term “mesenchymal stem cell” is used, and there is
a tendency to accept this terminology, even though it is inadequate.
2.4
Other Cell Populations Related to the Mesenchymal Stem Cell
In addition to the heterogeneity, which characterizes MSC cultures established
from various species or in different laboratories, some groups have described
cell populations that are very similar to MSCs, but which have a different
nomenclature. Bone marrow stromal (stem) cells (BMSSCs), stromal precur-
sor cells (SPCs), and recycling stem cells (RS-1, RS-2) are some of these varia-
tions (Baksh et al. 2004 and references therein). More recently, D’Ippolito et al.
(2004) described the marrow-isolated adult multilineage inducible (MIAMI)
cells which, although isolated from humans, can proliferate extensively with-
out showing signs of senescence or loss of differentiation potential (which, as
described below, is not usual for human cells). These cells may represent a more
primitive subset of bone marrow stem cells. Higher proliferative and differen-
tiation potential has also been reported for the multipotential adult progenitor
cell (MAPC) described by Catherine Verfaillie’s group (Reyes et al. 2001; Reye s
and Verfaillie 2001). Young et al. (2001) described human reserve pluripotent
mesenchymal stem cells, present in the connective tissues of skeletal muscle
and dermis.
Isolation and culture conditions used by the different groups are also vari-
able, and probably represent the main factor responsible for the phenotype
and function of the resulting cell populations. Whether these conditions se-
lectively favor the expansion of different bone marrow precursors or cause
similar cell populations to acquire different phenotypes is not clear. The cell
populations could also represent different points in a hierarchy (Caplan 1994),
and some studies suggest that this second alternative might be more realistic.
Lodie et al. (2002), for instance, systematically compared different pro tocols
used to isolate/expand human bone marrow adherent cells and concluded
that the cell populations isolated by these various techniques are virtually
indistinguishable. We have recently observed that the maintenance of MSC
lines generated according to established protocols (M eirelles and Nardi 2003)
but grown under MAPC conditions for 4 weeks induced changes in the im-
munophenotypic profile of the cells to become more similar to that of MAPCs
(N. Nardi and L. Meirelles, unpublished results). In either case and as reported
for hematopoietic stem cells (Pranke et al. 2001), these results demonstrate that
the mesenchymal stem cell compartment is heterogeneous and that cultivation
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Me senchymal Stem Cells: Isolation, In Vitro Expansion and Characterization 255
conditions can alter some of their basic properties. These points reinforce the
urgent need for a more comprehensive view of the mesenchymal stem cell
iden tity and its characteristics.
3
Distribution of the Mesench ymal Stem Cell
Although very poorly understood, the interaction of MSCs with their niche
is as essential for their existence and function as it is for any other of the
adult stem cells (Watt and Hogan 2000; F uchs et al. 2004). The primary source
of MSCs in adult individuals is the bone marrow, where they are immersed
in the stroma (Pittenger et al. 1999). They are present at a low frequency in
bone marrow, and recent studies employing the CFU-F assay suggest that in
humans there is one MSC per 34,000 nucleated cells (Wexler et al. 2003). In
mice, the frequency was estimated to be one for 11,300–27,000 nucleated cells
(M eirelles and Nardi 2003). Once again, the heterogeneity of this microen-
vironment hampers the unraveling of its components and their relationship,
and basic questions remain unanswered. Wha t is the niche for the MSC? Do
hematopoietic and mesenchymal stem cells share the same niche and exchange
signals to drive proliferation and differentiation?
MSCs have been found in several other tissues and in ontogeny (Table 1). In
mice, they were isolated from the brain, thymus, liver, spleen, kidney, muscle,
and lungs of adult mice (L. Meirelles and N. Nardi, unpublished results), and
other MSC-related populations such as MAPCs have been observed in different
organs as well (Jiang et al. 2002). This distribution could be explained by
different scenarios: (a) adult tissues contain independent reservoirs of similar
Table 1 Distribution of MSCs in different organs/tissues and ontogeny stages
Site Species Ontogeny stage Reference
Adipose tissue Human Post-natal Zuk et al. 2001, 2002
Adipose tissue Mouse Post-natal Safford et al. 2002
Pancreas Human Fetal Hu et al. 2003
Bone marro w Human Fetal Campagnoli et al. 2001
Liver Human Fetal Campagnoli et al. 2001
Blood Human Fetal Campagnoli et al. 2001
Tendon Mouse Postnatal Salingcarnboriboon et al. 2003
Synovial membrane Mouse Postnatal de Bari et al. 2003
Amniotic liquid Human Fetal in’t Anker et al. 2003
Peripheral blood Human Postnatal Zvaifler et al. 2000; Kuwana et al. 2003
Umbilical cord blood Human Fetal/postnatal Alfonso et al. 2000
117
256 N. Beyer Nardi · L. da Silva Meirelles
stem cells, whose characteristic traits are determined by signals released by
each niche; (b) MSCs exist as a reservoir in one specific location, from which
they circulate through the organism to colonize different tissues/organs; or
(c) MSCs originate from cell populations belonging to blood vessels, and are,
as a consequence, pr esent through the whole organism. Since Bianco and
Cossu (1999) suggested that MSCs originate from marrow pericytes, this third
possibility has received experimental support (reviewed in Short et al. 2003).
The issue is, however, still unclear.
4
Isolation and Culture of Mesenchymal Stem Cells
Few adult stem cell populations can be unequivocally identified, and isolation
of these cells requires in vitro or in vivo experimentation and characterization
based on immunophenotypic or functional traits. Hematopoietic stem cells,
for instance, can be enriched through the selection of cells expressing surface
markers such as CD34 in humans and Sca-1 in mice, or by their ability to ex-
clude the DNA-binding dye Hoechst 33342 (Goodell et al. 1996). Mesenchymal
stem cells lack clearly defined surface markers, so that the most widely used
approach to isolate them relies on their ability to adhere to plastic surfaces
(Wakitani et al. 1995; MacKay et al. 1998; Makino et al. 1999; Muraglia et al.
2000).
For the selective isolation of bone marrow MSCs, total cells are washed,
co unted, resuspended in culture medium, and plated in six-well tissue culture
dishes at approximately 1.94 × 10
6
cells/cm
2
. Nonadherent cells are removed
24–72 h later by changing the medium. After 1 week, a heterogeneous culture
develops, which is generally ref erred to as bone marro w stroma (Fig. 1a).
Maintenanc e of the culture with a twice-weekly medium change and remo val
of nonadherent cells results, after 2 or 3 weeks, in a relatively homogeneous
culture of morphologically and immunophenotypically similar mesench ymal
stem cells (Fig. 1b,c). Our experience shows that the identification of MSCs
depends on the availability of a good inverted microscope with phase contrast,
since they are difficult to visualize otherwise.
Cultures can be maintained for variable periods, depending on the species
and organ of origin, by passaging and subculturing the adherent cells which
are detached by trypsinization. Although Dulbecco’s modified Eagle’s medium
(DMEM) is frequently employed for the culture of MSCs, other media have also
been shown to be appropriate (reviewed by Otto and Rao 2004). The presence
ofHEPESbufferisalsoimportantinourexperience.
In an attempt to further enrich the frequency of MSCs in the initial cell
population, other methods have been developed, such as the immunodepletion
of hematopoietic con taminants identified, for instance, by the molecules CD34,
CD45, and CD11b (Kopen et al. 1999; Badoo et al. 2003; Ortiz et al. 2003).
118
Me senchymal Stem Cells: Isolation, In Vitro Expansion and Characterization 257
Fig. 1 a BALB/c bone marrow cells cultured for 1 week in DMEM with 10% FCS generate het-
erogeneous cell populations, referred to as bone marrow stromal cells (×100). Maintenance
of the adherent cell population results in a homogeneous culture of mesenchymal stem cells
ofaflat-typemorphology(b, ×400; c, ×100)
Other techniques involved cell size-based enrichment, involving the filtration
of bone marrow cells through a 3-μm seive (Hung et al. 2002; Tuli et al. 2003),
or changing plating densities (Colter et al. 2000; Sekiya et al. 2002). Since none
of these app roaches results in the establishmen t of homogeneous cell cultures,
the development of efficient—and particularly reproducible—methods for the
119
258 N. Beyer Nardi · L. da Silva Meirelles
isolation and expansion of MSCs remains an important goal of this research
field. It is possible that only when the true origin and nature of MSCs is better
understood will we be able to confidently work with them in vitro.
Following removal of nonadherent cells 1–4 days after the establishment
of the culture, cells are maintained with periodic passages until a relatively
homogeneous population is established. Culture media may vary, but the
most frequently used are Dulbecco’s modified Eagle’s medium (DMEM) and
α-minimum essential medium (reviewed in Otto and Rao 2004). The batch of
fetal calf serum employed to cultivate these cells may introduce phenotypic
variations, which show that unknown factors influence the selection and ex-
pansion of these cells. The addition of specific growth factors is also important
in defining the final characteristics of MSC cultures, and these are probably
the main reasons for the heterogeneity observed in the mesenchymal stem
cell types described in the literature. The growth of murine MAPCs, for in-
stance, depends on the supplementation of leukemia inhibitory factor (LIF)
and the use of fibronectin-coated surfaces (Jiang et al. 2002). The persistence
of hematopoietic contaminants, shown by the presence of CD45
+
and CD11b
+
cells in the cultures, has also been reported (Phinney et al. 1999).
I deal culture conditions would maintain mesenchymal stem cells with (a)
phenotypic and functional characteristics similar to those exhibited in their
original niche, (b) indefinite proliferation, and (c) a capacity to differentiate
in to multiple lineages. Since the in situ characteristics of MSCs are not known,
efforts have concentrated in the last two objectives. The self-renewal potential
of MSCs is not definitely established and can vary greatly according to the
methodology used and the species (Bianco et al. 2001), but cells can be expected
to expand for at least 40 population doublings (PDs) before their growth
rate decreases significantly, as seen with human MSCs (Bruder et al. 1997).
Supplementation of growth factors can also modify these results. Fibroblast
gro wth factor-2 (FGF-2), for instance, was shown to increase the lifespan of
human MSCs to more than 70 PDs (Bianchi et al. 2003). Murine MSCs, on
the other hand, show apparently unlimited in vitro growth capacity (Meirelles
and Nar di 2003 and unpublished observations). The high self-renewal capacity
shownbymurineMSCswithoutevidenceofreplicativesenescenceisprobably
related to that of rat oligodendrocyte precursor cells (Tang et al. 2001).
Cell seeding density may also influence the expansion capacity of mes-
enchymal stem cells. Human MSCs, for instance, expand to much higher PDs
when plated at low density than at high density, with an increase of total cells
from 60- to 2,000-fold (Colter et al. 2000). On the other hand, the establish-
men t of long-term cultures of murine mesenchymal stem cells is dependent
on a minimal cell density of 2 × 10
6
bone marrow cells/cm
2
(Meirelles and
Nardi 2003). Long-term culture and high cell density are also determinants of
loss of differentiation potential for human cells, another indication that the
conditions for the in vitro main tenance of MSCs differ from those provided by
their natural microenvironment.
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Me senchymal Stem Cells: Isolation, In Vitro Expansion and Characterization 259
5
Homing and Engraftment of Transplanted Mesenchymal Stem Cells
In vivo tracking of implanted MSCs is very important, because the success of
cell and gene therapy protocols with these cells depends on their engraftment
abilities. The analysis of MSC engraftment is also related to the exact nature
of the grafted cells. Heterogeneous populations of adherent cells derived from
mouse bone marrow engraft in multiple organs after systemic infusion (Pereira
et al. 1995, 1998), and although thereisstrong evidence showing that the grafted
cells are comprised of mainly MSCs, engraftment of other cell types can not
be excluded.
M ore recently, studies using a well characterized and relatively homoge-
neous cell population comprised of murine bone marrow devoid of hematopoi-
etic cells, were consistent with a successful in vivo engraftment of candidate
MSCs in the central nervous system (Kopen et al. 1999; McBride et al. 2003).
This cell population, in contrast to other murine MSCs, was not expandable in
vitro (Baddoo et al. 2003; Meirelles and Nardi 2003; Gojo et al. 2003; Fang et al.
2004), and interspecies experiments were used to study MSC systemic engraft-
men t in mice. For instance, when human MSCs were injected intraperitoneally
in to 13-day-old mouse embryos in uterus, multiorgan engraftment was de-
tected 8 weeks after birth by real-time PCR (McBride et al. 2003). The sites
analyzed included femur, heart, brain, liver, kidney, spleen, and lungs, where
the highest level of human DNA was detected.
When cells obtained from bleomycin-resistant BALB/c mice, essentially as
described by Kopen et al. (1999), were injected systemically into bleomy cin-
sensitive C57BL/6 mice, engraf tment in the lungs was considerably higher
in animals with lung injury than in animals without injury. Tissue damage,
therefore, enhances MSC engraftment (Ortiz et al. 2003); however, murine
MSC engraftment has also been demonstrated in noninjured animals. Gojo
et al. (2003) reported the engraftmen t of in vitro-proliferative murine MSCs in
the heart, lung, spleen, stomach, small intestine, and skeletal muscle of non-
injured mice, where they differentiated locally into cardiomyocytes, vascular
endothelial cells, and possibly vascular luminal cells. Selective sorting of an
adherent fraction of passage two or three bone marrow cultures yielded non-
hematopoietic cells that engrafted into several organs after systemic inf usion
(Anjos-Afonso et al. 2004). Engraftment in some organs was infrequent (brain,
bone marrow), but in others (liver, lung, kidney), it exhibited higher levels of
engrafted MSCs, and the presence of donor cells in circulating blood was also
observed.
ThedepositionofMSCsinthelungsmayrepresentasignificanthurdlefor
engraftment therapies that employ systemic delivery of MSCs. This approach
caused some animals to develop fibrosis and subsequent breathing difficulties.
Gao et al. (2001) observed this phenomenon following systemic infusion of
MSCs in rats, perhaps because the MSC diameter was larger than that of lung
121
260 N. Beyer Nardi · L. da Silva Meirelles
capillaries (20–24 μm vs 10–15 μm, respectively). The use of the vasodilator
sodium nitroprusside at the time of injection, however, reduced entrapment
of MSCs in the lungs. Murine MSCs, when detached from the dish, are also
20–25
μm wide (L. Meirelles, unpublished results), and if administered in large
doses are likely to be trapped in lung capillaries before reaching important
organs such as the brain and bone marrow.
In 2002, two groups (Ying et al. 2002; Terada et al. 2002) showed that stem
cells can fuse with other cells in vitro and can acquire the characteristics of
these cells, thus raising the possibility that the stem cell contribution to target
tissues might be due to cell fusion rather than to (trans) differentiation. Fusion
was also demonstrated between MSCs and epithelial cells in vitro (Spees et al.
2002); however, chromosomal analysis of xenografts indicate that fusion is
not the principle mechanism responsible for the MSC contribution to multiple
tissues in vivo (Pochampally et al. 2004; Sato et al. 2005). In a study describing
the role of MSCs in the generation of gastric cancer in a mouse model, for
example, it was observed that the percentage of tetraploid cells in affected
and unaffected animals remained at the same levels (Houghton et al. 2004; see
Sect. 8.1 for further information).
While the experimental study of MSC engraftment in humans is elusive,
a recent paper describing microchimerism, possibly due to circulating fetal
mesenchymal stem cells in pregnancy, in bone marrow and bone of women
decades after giving birth to male fetuses has provided some insights into
human MSC engraftment properties in vivo (O’Donoghue et al. 2004). A com-
bined approach including immunocytochemistry, FISH, and PCR using rib
sections and cultured MSCs derived from rib bone marrow was used. The
res ults strongly suggest that circulating MSCs present in fetal blood (Campag-
noli et al. 2001) crossed the placenta during pregnancy, entered the maternal
circulation (O’Donoghue et al. 2003) and grafted with maternal bone and bone
marrow. Khosrotehrani et al. (2004) also reported the detection of grafted male
fetal cells in thyroid, cervix, int estine, liver, and lymph nodes when analyzing
biopsy material of women who have had male pregnancies. The male cells
were shown to express liver, hematopoietic, or epithelial markers, indicating
tissue-specific incorporation.
Although fetal stem cells entering the maternal circulation include other
cell populations, the results of experiments in animals suggest that MSCs are
the main cell type that can engraft in maternal tissues. Human adult MSCs,
therefore, can be expected to have multisite engraftment capabilities as well.
6
Characterization of Mesenchymal Stem Cells
Cultured MSCs have been extensively analyzed both morphologically and with
respect to surface and molecular markers. None of these characteristics, how-
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Me senchymal Stem Cells: Isolation, In Vitro Expansion and Characterization 261
ever, is specific enough to adequately define this cell type, and mesenchymal
stem cells are still operationally defined by the ultimate criteria used for iden-
tifying stem cells: prolonged proliferation and the potential to originate differ-
en tiated cell types. To abandon the term “operational MSC,” it is necessary to
show that these cells contribute to the formation of mesenchymal tissues after
in vivo infusion (Verfaillie 2002).
A great number of surface markers have been described for committed
mesenchymal progenitors (Otto and Rao 2004), and Deans and Moseley (2000)
have compiled a long list of candidate markers, including CD44, CD29, and
CD90, to define human MSCs. The expr ession of CD34 is not clearly defined
in murine MSCs, but the marker is known to be absent from human and
rat cells. More specific antigens such as Stro-1, SH2, SH3, and SH4 are also
important markers for MSCs (reviewed in Barry and Murphy 2004), which are
also positive for MHC-1 and Sca-1. None of these markers, however, seems
to be a reliable parameter for the analysis of culture purity, since on the one
hand even long-term cultures may exhibit some heterogeneity (maybe due to
cell cycle-related marker expression) and, on the other, functionally different
cultures may have similar immunophenotypic profiles.
Relatively little attention has been given to the morphology of the MSCs
originating in culture. Two types of morphology can be observed—large, flat
cells or elongated, fibroblastoid cells. The derivation of two types of adherent
cell cultures from cord blood has already been pointed out by our group
(Alfo nso et al. 2000). The functional significance of these differences remains
to be established.
In an earlier report (Meirelles and Nardi 2003), we described the isolation
and long-term culture of murine MSCs without the need for any other medium
supplementation than fetal calf serum. The cells exhibited a constant flat-type
morphology (see Fig. 1), even when originating from other tissues such as
spleen, lungs and brain (not published). In most publications, it is difficult to
adequately assess cell morphology, but a review of the literature shows that in
many cases the cells maintained a flattened shape, while others exhibited an
elongated, fibroblastic phenotype (Table 2). The morphology of MAPCs also
seems to be relatively flat (Reyes et al. 2001).
Stilllittleisknownabouttheprofileofgeneexpressioninmesenchymalstem
cells. Tremain et al. (2001), in a study that also emphasized the heterogeneity
of MSC cultures, reported over 2,000 expressed transcripts in a clone that
originated from a stromal cell culture. In two recent studies, gene expression
of bone marrow (Silva et al. 2003) and cord blood-derived MSCs (Panepucci
et al. 2004; Jeong et al. 2005) was analyzed by serial analysis of gene expression
(SAGE). A great number of genes were identified in the cultured cells, and an
important contribution of extracellular protein products, adhesion molecules,
cell motility, TGF-beta signaling, growth factor receptors, DN A repair, protein
folding, and ubiquination as part of their transcriptome was observed.
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262 N. Beyer Nardi · L. da Silva Meirelles
Table 2 Reports of flat or elongated/fibroblastic morphology of cultured mesenchymal stem
cells from different ori gins
Morphology Species Referenc e
Flat Mouse Meirelles and Nardi 2003 (Fig. 2)
Human Azizi et al. 1998 (Fig. 2a)
Human D’Ippolito et al. 2004 (Fig. 1)
Human Gronthos et al. 2003 (Fig. 2G)
Human Stute et al. 2004 (Fig. 6B)
Human Hung et al. 2004 (Fig. 1)
Rat Azizi et al. 1998 (Fig. 2c)
Rat Davani et al. 2003 (Fig. 1A)
Rat Kobayashi et al. 2004 (Fig. 1)
Fibroblastic Human Azizi et al. 1998 (Fig. 2b)
Human Koç et al. 2000 (Fig. 1)
Human Campagnoli et al. 2001 (Fig. 1)
Human Pittenger et al. 1999 (Fig. 1)
Human Pittenger and Martin 2004 (Fig. 1)
Other Human Seshi et al. 2000 (Fig. 1)
7
Differentia tion of Mesenchymal Stem Cells
Although considered nondifferentiated cells, MSCs are nevertheless capable
of performing at least one specialized function: they support hematopoiesis.
This function is generally attributed to the bone marrow stroma, which is
frequently confused with MSCs. Apparently homogeneous cultures of MSCs
support hematopoietic stem cells with greater efficiency than conventionally
established bone marrow stroma (Meirelles and Nardi 2003).
The in vitro differentiation of MSCs into several lineages is easily achieved.
Representative examples of the protocols employed in a number of studies
are shown in Table 3. Determination of the phenotype of differentiated cells
depends on morphological, immunophenotypic, and functional criteria. The
differentiation of osteoblasts, for instance, is determined by upregulation of
alkaline phospha tase activity and deposition of a mineralized extracellular
matrix in the culture plates that can be detected with Alizarin Red or other
stains. Adipocytes are easily identified by their morphology and staining with
Oil Red O. For identification of my ocytes or neuronal cells, immunocytochem-
istry is performed with antibodies specific for antigens such as myosin and
dystrophin, or Tau and GFAP, respectively.
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Me senchymal Stem Cells: Isolation, In Vitro Expansion and Characterization 263
Table 3 Examples of the culture protocols used for inducing in vitro differentiation of MSCs
Tissue Species Culture medium complement Reference
Bone Mouse 10
–8
M dexamethasone, 5 μg/ml ascorbic
acid 2-phosphate and 10 mM β-glycero-
phosphate
Meirelles and Nardi
2003
Cartilage Human Transforming growth factor-
β3inserum-
free medium, added to three-dimensional
cultures
Pittenger et al. 1999
Fat Human 1-methyl-3-isobutylxanthine, dexam-
ethasone, insulin, and indomethacin
Pittenger et al. 1999
Mouse 10
8
M dexamethasone and 5 μg/ml insulin Meirelles and Nardi
2003
Neuron-like Human Isobutylmethylxanthine and dibutyryl
cyclic AMP
Deng et al. 2001
Mouse 50 ng/ml of basic fibroblast growth factor
(bFGF) and 20 ng/ml of epidermal growth
factor (EGF)
Anjos-Afonso et al.
2004
Muscle Mouse Amphotericin B Phinney et al. 1999
Rat 5-azacytidine Wakitani et al. 1995
Pig 5-azacytidine Moscoso et al. 2005
In vitro cultured MSCs show great heterogeneity in their differentiation
potential. Although the analysis of established MSC cultures show them to be
pluripotent, with a tri-lineage (osteo/chondro/adipo, De Ugarte et al. 2003) or
even higher (Anjos-Afonso et al. 2004) differentiation potential, clonal assays
have shown that only one-third of the MSC clones derived from established
cultures are pluripotent (Pittenger et al. 1999; Muraglia et al. 2000). Within es-
tablished cultures, thus, a minority of cells seem to be pluripotent, with most of
them having bi- or only uni-lineage differentiation capacity (Digirolamo et al.
1999). Models have been proposed to explain these results (Baksh et al. 2004),
and it is possible that cultures are composed of a mixture of cells with different
differentiation potentials. A smal l portion may correspond to authentic stem
cells, whereas most may be committed to more differentiated phenotypes.
8
Applications of Mesenchymal Stem Cells in Cell and Gene Therapy
Although human MSCs can be immortalized through genetic modification
using expression vectors carrying the catalytic subunit of human telomerase
(Mihara et al. 2003), the study of murine MSCs in vitro is more attractive since
they represent an unmodified natural population. They can also be promptly
125
264 N. Beyer Nardi · L. da Silva Meirelles
obtained by researchers who do not have access to a source of human cells
or do not have adequate facilities for their manipulation. Mor e importantly,
murine MSCs can be directly applied to a wide range of murine models of
diseases, where they can act as therapeutic agents per se or as vehicles for the
delivery of therapeutic genes. Finally, MSCs obtained from rats, rabbits, pigs,
and sheep will also be useful for the development of engineered tissues using
autologous cells.
8.1
Study of Cancer Biology
The sustained proliferation of murine MSCs provides an interesting model for
the evaluation of genetic and epigenetic factors involved in the maintenance of
stemness, as well as the components responsible for the generation of tumors.
Murine MSC self-renewal is not linked to neoplasia: experiments in which mice
received intravenous or intraperitoneal MSC infusion do not develop donor-
derived tumors (L. Meirelles and N. Nardi, unpublished results). Unraveling
the genetic determinants of self-renewal may lead to the identification of can-
didate genes involved in tumorigenesis and to the develop men t of drugs that
can act specifically on their products. The fusion of murine MSCs and nonpro-
liferative cells, for instance, would help with the mapping of genes in volv ed in
proliferation to specific chromosomes and chromosomal regions.
While studying the behavior of marrow-derived cells in a mouse model
of gastric cancer induced by Helicobacter pylori, cells bearing the marker
TFF2 could be associated with the predominant cell type present in this can-
cer (Houghton et al. 2004). In vitro studies using purified marrow-derived
hematopoietic stem cells or MSCs exposed to cancerous tissue extracts showed
that MSCs, but not HSCs, acquired expression of TFF2. The contribution of
MSCs to tumor formation by cell fusion was ruled out by comparing the ploidy
of stomach cells from infected and noninfected mice: the number of tetraploid
cells in both groups did not differ significantly. Another recent study described
the inv olvement of stem cells in human brain tumors (Singh et al. 2004). These
results are consistent with an emerging view of cancer as a stem cell disorder,
rather than a disease confined to fully or partially differentiated cells.
Besides their potential use for the study of basic cancer biology, modified
MSCs may prove to be efficient antitumoral agents. Human MSCs have been
shown to incorporate into tumor stroma (Studeny et al. 2002), a potentially
useful tool for the delivery of gene products directly to tumors (see below).
8.2
Cell Therapy
Mesenchymal stem cells may particip ate in cell therapy protocols through
two mechanisms. First, MSCs may contribute ph ysically to injured sites when
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Me senchymal Stem Cells: Isolation, In Vitro Expansion and Characterization 265
administered locally or systemically. Second, MSCs may have a sup portive role
through means of secreted factors. Examples of these applications are given
below.
8.2.1
Fibrosis
As mentioned earlier, murine MSCs administered systemically to mice sub-
jected to lung injury show superior lung engraftment rates relative to unin-
jured animals. Furthermore, MSC treatments performed immedia tely after
an tibiotic challenge reduces the fibrotic and inflammatory effects of the lesion
significantly more than that in animals receiving the cells 7 days after the chal-
lenge (Ortiz et al. 2003). An earlier work, using a poorly characterized murine
MSC population, showed that infusion of the cells in mice subjected to lung
injury by bleomycin showed enhanced reproducibility of engraftment (Kotton
et al. 2001). The injected cells were found to engraft as type I pneumocytes,
but not type II pneumocytes. Similar results were reported by Ortiz et al.
(2003). In the case of bleomycin-induced lung injury, this indicated that the
main cellular contribution from the plastic-adherent fraction of bone marrow
can be attributed to MSCs. This information may be valuable for future ther-
apies aiming to reduce lung fibrosis in humans by autologous bone marrow
transplantation.
MSCs have also been used to treat liver fibrosis. Fang et al. (2004) depleted
murine bone marrow from CD45
+
,GlyA
+
,andCD34
+
cells to obtain adher-
ent Flk
+
cells that are expandable in vitro for more than 30 passages. Using
a murine model of tetrachloride-induced liver injury, they showed in the an-
imals receiving MSCs systemically immediately after the challenge, but not
1 week later, that the fibrotic effects caused by the lesion were reduced. The
presence of albumin-producing cells that exhibit donor-derived markers was
also detected, although at a low frequency.
8.2.2
Cardiovasculogenesis
In a study originally designed to assess the contribution of murine MSCs to
the cardiac tissue, Gojo et al. (2003) showed that a 5-azacytidine-responsive,
CD34
low/–
c-kit
+
CD140a
+
Sca-1
high
clone transduced with an EGFP con struct
contributed to several sites when implanted in vivo. After injection into the
ventricular m yocardium, EGFP
+
cardiomyocytes were detected, along with
EGFP
+
CD31
+
cells lining the vessels surrounding the site of injection. The
number of endothelial cells and cardiomyocytes grafted in the ventricle was
estimated to be, respectively, 1,625 and 75 1 week after injection, and 275
and 25 3 months later. When MSCs were infused systemically through the
inferior vena cava, the cells engrafted predominantly in the lungs 1 week after
127
266 N. Beyer Nardi · L. da Silva Meirelles
administration. Four weeks after the injection, the number of EGFP
+
cells
in the lungs declined considerably. The grafted cells lacked CD31 expression,
indicating that they had formed pericytes or smooth muscle cells. In addition
to these results, EGFP
+
cells were found in the brain, thymus, uterus, and
kidney. Engraftment in the stomach and small intestine was observed, and
thenumberofdonor-derivedcellsseemedtoincreaseovertime.Whenhigh
cell numbers were implanted in the muscle, liver, or spleen, ectopic bone
formation was observed, in contrast to the muscular and vascular fates adopted
by cells delivered in low quantities. This finding cautions against experimental
pr otocols involving the injection of large doses of MSCs directly in heart
muscle, since they might differentiate into tissues other than those expected.
The use of purified MSCs to treat human heart diseases has not yet come
into practice, despite the reported successes of blood or bone marro w-derived
mononuclear cells (Assmus et al. 2002; Perin et al. 2003). Although studies
demonstrating that bone marrow-derived c-kit
+
lin
–
cells, a cellular fraction
putatively enriched in HSCs, regenerate mouse infarcted myocardium (Orlic
et al. 2001) these findings been challenged by others (Murry et al. 2004; Baslam
et al. 2004). The plastic-adherent fraction of bone marrow has also improved
cardiac performance in a rat model of heart infarction(Olivares et al. 2004). The
CD34
–
CD45
–
cells collected for these latter experiments most likely comprised
MSCs. Histological analyses indicated that the main contribution of the cells
to the infarcted zone occurred through the formation of new m yocardium and
blood ve ssels.
Taken together, these studies indicate that the cell type most likely involved
in cardiac regeneration is the MSC, possibly due more to its arteriogenic effects
(see the next section) than to its cardiomyogenic properties.
8.2.3
Arteriogenic Effects
Kinnaird et al. (2004) induced unilateral hind limb ischemia in mice to demon-
strate that murine bone marrow, devoid of CD34
+
CD45
+
cells and delivered
in situ 24 h after lesion, improved limb function despite little long-term cell
engraftment. They found that medium conditioned by these cells contained
several potent arteriogenic cytokines, such as basic fibroblast growth fac-
tor (bFGF), vascular endothelial growth factor (VEGF), placental growth fac-
tor (PlGF), and monocyte chemoattractant protein-1 (MCP-1). Animals that
received the cells after injury contained donor-marked cells surrounded by
bFGF and VEGF positive cells, indicating that the transplanted MSCs ex-
pressed these c ytokines in situ. These findings support the hypothesis that
factors secreted by MSCs have a significant role in limb recovery. Interest-
ingly, MCP-1, which is secreted by cells present in the vascular wall, has been
shown to recruit circula ting monocytes that can differentiate into endothelial
cells (Fujiyama et al. 2003). The arteriogenic effects of MSCs may thus involve
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Me senchymal Stem Cells: Isolation, In Vitro Expansion and Characterization 267
the recruitment of circulating cells through the secretion of chemoattractant
factors.
8.2.4
Immunosuppressive Effects
The subcutaneous co-injection of primary murine MSCs or of an embry-
onic mouse mesenchymal stem cell line (CH310T1/2) with a melanoma cell
line (B16) was shown to favor tumor growth (Djouad et al. 2003). In vitro
experiments in which activated murine splenocytes were co-cultured with
CH310T1/2 cells in a transwell culture system indicated that soluble factors
secreted by MSCs inhibit CD8
+
T cell proliferation. Even though the primary
murine MSCs were not well characterized, the use of CH310T1/2 validated
the hypothesis that MCSs favored tumor growth, since the results observed
in vivo were similar for both cell types. These results indicated that the im-
munosuppressive effects should be considered whenever MSC transplantation
takes place. Krampera et al. (2003) demonstrated even more clearly the im-
munosuppressive effects of MSCs by showing that culture-expanded murine
MSCs are capable of inhibiting both naïve and memory antigen-specific T cell
activation. The authors used a mixed lymphocyte reaction system in dose-
dependent experiments, and determined that T cell inhibition was transient
and independent of MHC antigen-presenting cells or CD4
+
CD25
+
regulatory
T cells. In contrast, the study by Djouad et al. (2003) showed that the MSC
immunosuppressive effect required cell contact. Whether or not cell contact
is required, it is clear that MSCs have immunomodulatory capabilities. Treat-
ment of acute graft-versus-host disease in a human subject using third-party
haploidentical MSCs can be taken as proof of this concept (Le Blanc et al. 2004).
The mechanisms involved in MSC-mediated immunosuppression are cur-
rently being investigated. Glennie et al. (2004) reported that MSCs suppress T
cell effector function transiently, but the cells do not block activ ation. On the
other hand, they induce an irreversible proliferation arrest not only in CD4
+
and CD8
+
T cells, but also in B cells, by downregulating cyclin-D2 expression.
The interactions of MSCs and immune cells may have future implications not
only for the knowledge of MSC biology, but also for the understanding of
immune system homeostasis.
8.3
Mesenchymal Stem Cells and Tissue Engineering
In addition to systemic engraftment capabilities, MSCs show great potential
for the replacement of damaged tissues such as bone, cartilage, tendon, and lig-
ament. Although bone is capable of regeneration, the three other tissues often
develop fibrous scar tissues when injured, which usually renders them unable
to function properly. Large bone defects, however, do not heal spontaneously,
129
268 N. Beyer Nardi · L. da Silva Meirelles
Table 4 Examples of tissues engineered with the use of MSCs
Engineered tissue Methods Reference
Respiratory m ucosa Human MSCs co-cultured with normal human
bronchial epithelial cells, in vitro
Le Vi sage et al.
2004
Cartilage Human MSCs cultured under chondrogenic con-
ditions on 3D scaffold, in vitro
Chen et al.
2004a; Li et al.
2005
Bone Critical-size cranial defect crea ted in rabbit, re-
paired with BMP-2-expressing rabbit MSCs em-
bedded in alginate
Chang et al.
2004
Full-thickness mandibular defects createdin pigs,
and repaired with autologous MSCs previously
seeded in poly-DL-lactic-coglycolic acid scaffolds
and kept in osteo-inductive medium
Abukawa et al.
2004
Cardiac pacemakers Human MSCs transfected with a cardiac pace-
maker gene (mHCN2). Functional results ob-
served in vitro by co-culture with neonatal rat
ventricular myocytes, and in vivo by subepicar-
dial injection into the canine left ventricular wall
Potapova et al.
2004
suggesting that MSC-based reconstitution may be feasible. The application of
MSCs in the engineering of new tissue is dependent on the use of an appro-
priate scaffold to maintain an adequat e three-dimensional distribution and on
the use of specific molecules to drive their differentiation into cells that can
rest ore the tissue-specific matrix (Huang et al. 2004).
The use of murine MSCs for tissue engineering is limited, due to the small
size of the mouse. However, murine MSCs along with MSCs from other species
are interesting candidates for the study of cell interactions with novel bioma-
terials, and the study of new molecules on differentiation. Examples of the use
of MSCs for tissue engineering in larger animal models can be found in the
literature, some of which are listed in Table 4.
8.4
Genetic Therapy
Genetic diseases can be generally classified into two categories: those caused by
genetic alterations leading to loss of protein/gene function and those caused by
a gain of function mutation. Other factors are involved such as the restriction
of a disease phenotype to a specific organ as opposed to the whole body. When
loss of function is the cause, introduction of genetic constructs expressing the
missing product may be sufficient to revert or suppress the disease phenotype.
When the disease involves gain of function, however, the insertion of vectors
130
Me senchymal Stem Cells: Isolation, In Vitro Expansion and Characterization 269
expressing healthy transcripts is not enough to correct the disorder, and some
sort of genome or transcriptome editing is necessary.
The molecular tools currently used to address these issues include vectors
derived f rom plasmids, virus, and even transposons (reviewed by Selkirk 2004
and Nathwani et al. 2004). Anti-sense RNA and interference RNA can also be
used. Plasmid-derived vectors do not integrate into the host genome, and so
do not last for the lifetime of the individual. On the other hand, virus-derived
vectors can integrate into the genome of proliferating or nonproliferating cells,
depending on the type of viral-based expression system. Sustained expression
following integration makes them the vector system of choice. The systemic
in vivo administration of viral vectors represents the main hurdle to their
direct utilization, since they may elicit a strong host immune response that
co uld lead to death (Kaiser 2004) or cause insertional mutagenesis resulting
in cancer as reported recently in the X-SCID clinical trial (Hacein-Bey-Abina
et al. 2003). The use of stem cells to deliver genetic material represents the best
way to circumv ent the first obstacle. Stem cells can be manipulated ex vivo
and receive the genetic modifications necessary fo r correction of the disease,
avoiding the need to expose the patient directly to vectors. When proliferative
stem cells such as MSCs are used, there is also the possibility of selecting
successfully altered clones for reinfusion, which might suffice to minimize
insertional mutagenesis risks. This is in contrast to the use of HSCs, the stem
cell type altered in the X-SCID trial, which are largely nonproliferative in vitro.
Systemic delivery of genetic constructs mediated by stem cells is feasible:
stem cells engraft in vivo, and particularly in the case of MSCs, they engraft to
multiple sites. When auto logous cells are genetically corrected, they are likely
to acquire a proliferative advantage over the patient’s cells, increasing the like-
lihood of engraftment and providing continued expression of the therapeutic
construct. Moreover, if HLA-matched allogeneic stem cells are used there may
be no need to use genetic manipulation tools, as the cells themselves may exert
therapeutic effects through the expression of donor genes.
8.4.1
Correction of Genetic Disorders
The availability of homogeneous populations of murine MSCs has profound
implications for the treatment of genetic diseases in mouse models, and by
analogy to humans. The use of murine models is appropriate when trying
to develop new therapeutic strategies. The cost of main taining mice is not
excessively high, and mouse genetics are well described. Moreover, knock-out
mice can be generated by site-directed mutagenesis in embryonic stem cells,
which means that many loss-of-function diseases, excluding those that lead
to embryonic lethality, can be simulated in mice. The results obtained using
a small animal model should, nevertheless, be validat ed using larger animals
to avoid any unexpected effects when the therapy is applied to humans.
131
270 N. Beyer Nardi · L. da Silva Meirelles
Table 5 Selected candidate mouse models of genetic disease for MSC-mediat ed therapy
Model Expected role of transplanted MSCs See also
Mucopolysaccharidosis
type I
Production of α-L-iduronidase, par-
ticularly in the brain
Koç et al. 2002
Hemophilia A Production and release into the blood
of coagula tion factor VIII
Van Damme et al. 2003
Niemann-Pick disease Sphingomyelinase production, partic-
ularly in the brain
Jin et al. 2002
Osteogenesis
imperfecta
Production of healthy collagen type I
fibers
Pereira et al. 1995;
Chamberlain et al. 2004
Reference: http://jaxmice.jax.org/jaxmice-cgi/jaxmicedb.cgi as of 03/28/2005 (mouse
models database). See also complementary references for further information
In a search at The Jackson Laboratory mouse database (http://jaxmice.jax.
org/jaxmice-cgi/jaxmicedb.cgi) using the term “genetic disease,” 140 results
were retrieved. Some of them were selected (Table 5) as examples of experi-
men tal models that could be used to test a MSC-based therapeutic approach,
including the mouse model of mucopolysaccharidosis type I (Ohmi et al. 2003)
that we currently study.
The application of MSC-mediated gene therapy in humans is still in its in-
fancy, with no clinical trials reported so far. In vitro studies, however, show
promise. Baxter et al. (2002) have successfully restored
α-L-iduronidase ex-
pression by retroviral transduction of the human IDUA cDNA into MSCs
obtained from patients affected by mucopolysaccharidosis type I. While this
work demonstrates the possibility of reversing loss-of-function genetic disor-
ders in humans, another study (Chamberlain et al. 2004) went even further.
In this latter study, the correction of a gain-of-function genetic disease, os-
teogenesis imperfecta, was addressed. MSCs obtained from affected patients
were genetically modified to disrupt the dominant-negative mutant allele of
the COL1A1 gene. Because the site-directed mutagenesis method could affect
both normal and mutant alleles and lead to inappropriate integration events in
the genome, screening for correctly altered clones was performed. The investi-
gators specifically screened several clones to identify those cells that had been
appropria tely modified. Once selected, these clones co uld generate cells tha t
accumula ted limited amounts of intracellular procollagen and could produce
relatively normal collagen extracellular matrix.
8.4.2
Cancer Suppression
As mentioned earlier, the ability of MSCs to incorporate into tumor stroma
could be used to design strategies to fight cancer. Studeny et al. (2002) trans-
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Me senchymal Stem Cells: Isolation, In Vitro Expansion and Characterization 271
duced human MSCs with a con struct expressing human IFN-β, which has im-
munomodulatory properties and antiproliferative effects over melanoma cell
lines. The transduced MSCs were administered subcutaneously to nude mice
together with the A375SM human melanoma cell line. The results showed that
the tumor area was strikingly reduced even when only 10% IFN-
β-expressing
MSCs were co-injected with the melanoma cells, as compared to control ani-
mals that received the melanoma cells alone. The same effect was not achieved
when nontransduced MSCs were used, indicating that IFN-
β was the mediator
of tumor suppression. Moreover, the tumor area was not reduced by IFN-
β
injection alone, indicating that MSCs were required. The survival period of
treated animals, compared to controls, also significantly increased (i.e., 41–
110 days for animals receiving different proportions of IFN-
β-expressing MSCs
vs 21–27 days for animals that received melanoma cells alone). A study exam-
ining tumor metastasis in lungs yielded similar results (Studeny et al. 2004).
The results obtained using xenograft tumor models in immunoincompetent
mice also show pro mise.
Equi valent studies with syngeneic or allogeneic murine MSCs in immuno-
competent mice are required to evaluate the efficacy of this treatment in in-
dividuals with normal immune activity, since the development of anti-cancer
protocols without the need for immunosuppression are highly desirable for
application in humans.
9
Pharmacologic Aspects of Mesenchymal Stem Cell Biology
The pharmacological relevance of MSCs can be divided into four categories.
First, the molecules secreted by MSCs may be employed as therapeutic agents
or adjuvan ts in animal models. A long list of biologically important molecules
secreted by MSCs (Majumdar et al. 1998; Kinnaird et al. 2004) include inter-
leukins 6, 7, 8, 11, 12, 14, and 15, M-CSF, Flt-3 ligand, SCF, LIF, bFGF, VEGF,
PlGF, and MCP-1. Second, specific receptors expressed by MSCs (Table 6) may
be used as targets for drugs aimed at MSCs in vivo. These studies may pro-
vide information on homing mechanisms when systemically infused. Third,
genetic constructs can be made that are preferentially expressed in MSCs by
the incorporation of cell-specific regulatory regions, similar to that described
earlier. Fourth, natural or artificial molecules may be used modify the natural
behavior of MSCs and alter the MSC com partment in vivo.
While a set of natural and synthetic compounds have been shown to ex-
ert many biological effects, such as differentiation induction (Table 7), many
other compounds remain to be discovered and/or fully characterized. For
instance, a small molecule termed reversine (a 2-(4-morpholinoanilino)-6-
cyclohexylaminopurine analog) has the ability to reprogram myogenesis-
committed precursor cells (the murine cell line C2C12) into a less differentiat ed
133
272 N. Beyer Nardi · L. da Silva Meirelles
Table 6 Receptors expressed by MSCs render them responsive to specific molecules
Category Expressed Nonexpressed
Cytokine receptors IL-1R (CD121a) IL-2R (CD25)
IL-3Ra (CD123)
IL-4R (CDw124)
IL-6R (CD126)
IL-7R (CD127)
Chemokine receptors CXCR4
Factor receptors EGFR EGFR-3
IGF1 R (CD221) Fas ligand (CD178)
NGFR
IFN
γR (CDw119)
TNFIR (CD120a)
TNFIIR (CD120b)
TGF
βIR
TGF
βIIR
bFGFR
PDGFR (CD140a)
Transferrin (CD71)
Matrix receptors ICAM-1 (CD54) ICAM-3 (CD50)
ICAM-2 (CD102) E-selectin (CD62E)
VCAM-1 (CD106) P-selectin (CD62P)
L-Selectin (CD62L) PECAM-1 (CD31)
LFA-3 (CD58) vW factor
ALCAM (CD166) Cadherin 5 (CD144)
Hyal uronate (CD44) Lewis
x
(CD15)
Endoglin (CD105)
References: Pittenger et al. 1999; Gronthos et al. 1998; Wynn et al. 2004
state equivalent to that of MSCs (Chen et al. 2004b). The resulting MSCs were
shown to differentiate into osteoblastic and adipocytic cells upon appropriate
stimulation.
10
Conclusions
M esenchymal stem cells are finally attracting the attention of the scientific
community, some 30 years after the first insights on the existence of non-
hematopoietic stem cells in bone marrow. Their ease of derivation and manip-
134
Me senchymal Stem Cells: Isolation, In Vitro Expansion and Characterization 273
Table 7 Specific molecules can direct MSC differentiation or modulate their expansion
capacity
Molecule Main effect Reference
5-azacytidine Myogenesis Wakitani et al. 1995
All-trans-retinoic acid Neurogenesis Sanchez-Ramos
et al. 2000
Amphotericin B Myogenesis Phinney et al. 1999
Ascorbic acid Osteogenesis; chondrogenesis Pittenger et al. 1999
Beta-glycerophosphate Osteogenesis; chondrogenesis Pittenger et al. 1999
Beta-mercaptoethanol Ne urogenesis Woodbury et al. 2000
bFGF Proliferation Kuznetsov et al. 1997;
Bianchi et al. 2003
BHA Neurogenesis Woodbury et al. 2000
Dexamethasone Adipogenesis; osteogenesis; Pittenger et al. 1999
chondr ogenesis
EGF proliferation Kuznetsov et al. 1997
ETYA Adipogenesis Kopen et al. 1999
Hydrocortisone Myogenesis Zuk et al. 2001
IBMX Adipogenesis Pittenger et al. 1999
Indomethacin Adipogenesis Pittenger et al. 1999
Insulin Adipogenesis Pittenger et al. 1999;
Zuk et al. 2001
PDGF Proliferation Kuznetsov et al. 1997
TGF-beta family members Proliferation; differentiation Kuznetsov et al. 1997;
(incl. BMPs) Roelen and Dijke 2003
ulation ex vivo, together with the growing body of information provided by
studies on their characterization and differentiation potential, have generated
excitement in the field of stem cell based therapies. Clinical applications with
MSCs are also relatively close to realization, when compared with most other
stem cells.
Most of the knowledge generated so far, however, concerns their behavior in
vitro,becauselittleisknownabouttheirpropertiesinvivo.Althoughthisdoes
not necessarily hinder their application for the treatment of severe diseases or
for the replacement of damaged tissues, it is clear that a comprehensive under-
standing of their biology is required to achieve maximal benefits. The task will
not be easy, because at present, tracking the fates of ex vivo manipulated MSCs
after their systemic delivery in animal models may lead to skewed results. In
vitro manipulation seems to alter the original properties of the cells. The lack
of definitive markers also does not allow the direct observation of MSCs in
situ, so that putativ e MSCs should be functionally characterized in vitro prior
135
274 N. Beyer Nardi · L. da Silva Meirelles
to their use in the experiments. Ultimately, concrete data indicating that MSCs
are present in multiple adult tissues together with findings suggesting the exis-
tence of a perivascular niche for mesenchymal precursors will help us to better
understand the role of the mesenchymal stem cell in vivo and conseq uently
will help in the development of more efficient strategies to treat a wide range
of diseases.
Acknowledgements The a uthors are indebted to Conselho Nacional de Desenvolvimento
Cientifico e Tecnologico (CNPq) and Fundação de Amparo a Pesquisa do Estado do Rio
Grande do Sul (FAPERGS) for funding.
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144
Functional characterization of cell hybrids generated by
induced fusion of primary porcine mesenchymal stem cells
with an immortal murine cell line.
Islam MQ, Ringe J, Reichmann E, Migotti R, Sittinger M, da S Meirelles L, Nardi NB,
Magnusson P, Islam K.
Cell Tissue Res. 2006 Oct;326(1):123-37.
145
REGULAR ARTICLE
Functional characterization of cell hybrids generated
by induced fusion of primary porcine mesenchymal
stem cells with an immortal murine cell line
M. Q. Islam & J. Ringe & E. Reichmann & R. Migotti &
M. Sittinger & L. da S. Meirelles & N. B. Nardi &
P. Magnusson & K. Islam
Received: 17 January 2006 / Accepted: 11 April 2006
#
Springer-Verlag 2006
Abstract Bone marrow mesenchymal stem cells (MSC)
integrate into various organs and contribute to the regener-
ation of diverse tissues. However, the mechanistic basis of
the plasticity of MSC is not fully understood. The change
of cell fate has been suggested to occur through cell fusion.
We have generated hy brid cell lines by polyethylene-
glycol-mediated cell fusion of primary porcine MSC with
the immortal murine fibroblast cell line F7, a derivative of
the GM05267 cell line. The hybrid cell lines disp lay
fibroblastic morphology and proliferate like immortal cells.
They contain tetraploid to hexaploid porcine chromosomes
accompanied by hypo-diploid murine chromosomes. Inter-
estingly, many hybrid cell lines also express high levels of
tissue-nonspecific alkaline phospha tase, which is consid-
ered to be a marker of undifferentiated embryonic stem
cells. All tested hybrid cell lines retain osteo genic differen-
tiation, a few of them also retain adipogenic potential, but
none retain chondrogenic differentiation. Conditioned me-
dia from hybrid cells enhance the proliferation of both
early-passage and late-passage porcine MSC, indicating
that the hybrid cells secrete diffusible growth stimulatory
factors. Muri ne F7 cells thus have the unique property of
generating immortal cell hybrids containing unusually high
numbers of chromosomes derived from norm al cells. These
hybrid cells can be employed in various studies to improve
our understanding of regenerative biology. This is the first
report, to our knowledge, describing the generation of
experimentally induced ce ll hybrids by using normal
primary MSC.
Keywords Mesenchymal stem cell
.
Cell hybrid
.
Reprogramming
.
Immortality
.
Growth factor
.
Mouse
.
Pig
Introduction
Mammalian somatic cells in vitro divide for a limited
number of times before entering a n on-dividing state called
replicative senescence (Hayflick 1965). Cellular senesc ence
is a complex phenotype characterized by irreversible G1
growth arrest. There are at least three major types of
senescence: (1) replicative senescence, though t to be caused
Cell Tissue Res
DOI 10.1007/s00441-006-0224-2
M. Q. Islam
:
K. Islam
Laboratory of Cancer Genetics, Laboratory Medicine Center (LMC),
University Hospital Linkoping,
SE-581 85 Linkoping, Sweden
M. Q. Islam
:
K. Islam
Department of Biomedicine and Surgery,
Faculty of Health Sciences, Linkoping University,
SE-581 85 Linkoping, Sweden
J. Ringe
:
E. Reichmann
:
R. Migotti
:
M. Sittinger
Tissue Engineering Laboratory, Department of Rheumatology,
Charité University of Medicine Berlin,
Tucholskystr 2,
10117 Berlin, Germany
L. da S. Meirelles
:
N. B. Nardi
Laboratorio de Imunogenetica, Departamento de Genetica,
Universidade Federal do Rio Grande do Sul,
Av. Bento Goncalves,
Porto Alegre, RS, Brazil
P. Magnusson
Division of Clinical Chemistry, Department of Biomedicine
and Surgery, Faculty of Health Sciences, Linkoping University,
SE-581 85 Linkoping, Sweden
M. Q. Islam (*)
Laboratory of Cancer Genetics, Main Building, Floor 11,
Division of Clinical Chemistry, Department of Biomedicine
and Surgery, Faculty of Health Sciences, Linkoping University,
SE-581 85 Linkoping, Sweden
e-mail: [email protected]
146
by shortening of telomeres with successive cell divisions;
(2) induced senescence, caused by forced expres sion of
activated oncogenes in normal cells; (3) stre ss-induced
senescence, resultin g from cells cultured u nder sub-optimal
conditions (Ben-Po rath and Weinberg 2005).
In contrast to embryonic stem cell s, somatic stem cells
have a limited capacity for in vitro proliferation (Czyz et al.
2003). Somatic stem cells have been found to reside in a
variety of tissues, e.g., bone marrow, muscle, heart, skin,
intestine, liver, lung, prostate, central nervous system, and
mammary gland (Poulsom et al. 2002; Young and Black
2004). Bone marrow is a major source of mesenchymal
stem cells (MSC), which represent a population of plastic
adherent non-hematopoietic cells that develop into mesen-
chymal tissues such as bone, cartilage, muscle, ligament,
tendon, adipose, and stroma in vitro and in vivo (Baksh et
al. 2004; Barry and Murphy 2004). Transplantation of MSC
in humans and in animal models has demon strated that they
integrate into various organs and contribute to the regener-
ation of many cell lineages. Numerous studi es have recently
been undertaken to understand the mechanistic basis of
somatic stem cell plasticity, i.e., the capacity of cells
derived from one tissue type to form cells of other tissue
types, but no clear result has emerged yet. Recently, cell
fusion has received much attention as one explanatio n of
stem cell plasticity in vivo (Vassilopoulos and Russell
2003; Camargo et al. 2004). However, convincing results
are still required before accepting this hypothesis
(Eisenberg and Eisenberg 2003; Dahlke et al. 2004). More
research in this field is therefore desirable to uncover the
role of cell fusion in stem cell plasticity. In this respect, new
findings might be useful for develo ping cell-based therapies
(Barry and Murphy 2004; Camargo et al. 2004).
By fusing cells of embryonic origins (embryonic stem
cells, ES; embryonic germ cells, EG; embryonal carcinoma
cells, EC) with unlimited proliferation and somatic cells of
limited proliferation, many investigators have demonstrated
that hybrid cells can be generated with indefinite prolifer-
ation through the reprogramming of somatic cell nuclei
(Takagi 1997; Pells et al. 2002; Flasza et al. 2003 ; Ambrosi
and Rasmussen 2005; Cowan et al. 2005). To ascertain the
reprogramming of the somatic cell genome, various
transcription factors and other traits of pluripotent embry-
onic cells have been used as markers (Tada et al. 2003;Do
and Scholer 2004, 2005; Ambrosi and Rasmussen 2005).
These studies have in esse nce demonstrate d tha t the
inactive X chromosome derived from female somatic cell
becomes activated (Takagi 1997; Tada and Tada 2001; Tada
et al. 2003), and a range of epigenetic changes takes place
during the reprogramming of the two genomes of the
hybrid cells through various modifications of histone
proteins and alterations of the status of DNA methylation
(Forejt et al. 1999; Tada and Tada 2001; Flasza et al. 2003;
Kimura et al. 2004). One major difference between the
reprogramming potentials of ES and EG cells is that
the fusion of EG cells with somatic cells often erases the
epigenetic marks of both imprinted and non-imprinted
genes of the two genomes, resulting in a switching of
monoallelically expressed imprinted ge nes into biallelic
forms, unlike the ES-derived hybrid cells where epigenetic
markers of both genomes remain intact (Tada et al.
1997;
Surani 2001). However, hybrid cells derived from both ES
and EG cells retain limited development potentials (Tada
et al. 1997, 2001; Matveeva et al. 1998).
Cell fusion studies involving the generation of somatic
cell hybrids between immortal and normal cells have
suggested that the phenotype of limited proliferation of
the normal cell is dominant over the unlimited proliferation
of the immortal cell (Tominaga et al. 2002). In contrast, we
have recently found that the fusion of the immortal murine
cell line F7 with normal porcine fibroblasts produces
immortal cell hybrids containing near-tetraploid (4N)
chromosomes derived from porcine fibroblasts (M.Q.
Islam, V. Panduri, K. Islam, in preparation). Normal MSC
divide in vitro only for 30–40 population doublings before
they become senescent (Sethe et al. 2005). This study has
been designed to investigate (1) whether the fusion of the
murine F7 cell line with porcine MSC generates hybrid
cells capab le of dividing indefinitely, (2) whether the
generated hybrid cells retain the 4N genome from the
porcine MSC, (3) whether the hybrid cells express distinct
characteristics of porcine MSC, and (4) whether the
conditioned media of hybrid cells can stimulate the
proliferation of parental porcine MSC.
Materials and methods
Isolation of porcine MSC
Porcine MSC were isolated as described previously (Ringe
et al. 2002). Briefly, femur and tibia bones from 6– month-
old to 8-month-old porcine donors were sawed open, and
bone marrow was extracted under sterile conditions.
Gelatinous bone marrow was suspended in phosphate-
buffered saline (PBS) and dispersed mechanically by
passing through syringes fitted with a series of 16-, 18-,
and 20-gauge needles. Cells were centrifuged and plated in
complete Dulbecco’s Modified Eagle’s Medium (DMEM,
Biochrom) containing 10% fetal bovine serum (FBS,
Biochrom), at a density of 300,000 cells/cm
2
. The medium
was changed after 72 h and every 2–3 days thereafter. At
90% confluency, monolayer cells were detached by the
addition of a solution containing 0.5% trypsin-EDTA
(Biochrom) and replated at a density of 6,000 cells/cm
2
.
Cell Tissue Res
147
Murine parental cell line F7
The immortal murine cell line GM05267, obtained from the
National Institutes of General Medical Sciences (NIGMS),
is deficient for the enzyme hypoxanthine-phosphoribosyl-
transferase (HPRT-) and consequently is sensitive to
hypoxanthine-aminopterin-thymidine (HAT). This fibro-
blast cell line was originally isolated from a postnatal/adult
kidney of a male mouse heterozygous for the tfm (testicular
feminization locus) mutation (Migeon et al. 1981; Jabs et
al. 1984). Subsequently, these cells were transformed with
simian virus 40 and sub-clones were isolated that were
resistant to the toxic effects of 6-thioguanine (6-TG
R
)
because of the lack of HPRT. Although the original 6-TG
R
cells had the modal numb er of 64 chromosomes, the
derivative cell line available at the NIGMS, designated as
GM05267 (http://www.locus.umdnj.edu/nigms/nigms_cgi/
display.cgi?G M05267), contains the modal number of 38
chromosomes.
The GM05267 cell line was maintained in DMEM,
supplemented with 15% FBS, 1% DMEM nonessential
amino acids, and 1% penicillin-streptomycin. Tissue culture
reagents were obtained from PAA Laboratories, if not stated
otherwise. In order to selectively isolate hybrid cells and to
eliminate the two parental cell types, we introduced the
G418-resistant gene to the GM05267 cell line by a
modified calcium-phosphate co-precipitation method (Islam
and Islam 2000). Briefly, exponentially growing cells were
seeded into 60-mm Petri dishes. The following day,
calcium-phosphate-DNA solution was added and incubated
with the cells overnight. Selective medium (complete
DMEM plus 1% G418, Invitrogen) was added, and cells
were fed with fresh selective medium twice a week. After
3 weeks, a single colony of G418-resistant cells was
transferred to a culture flask, and the resulting cell line
was designated as F7 (M.Q. Islam, V. Panduri, K. Islam, in
preparation).
Cell fusion and isolation of hybrid cell lines
Cell fusion was induced in co-cultured porcine MSC and F7
cells briefly treated with polyethylene glycol (PEG) as
follows. The F7 cells (HAT-sensitive and G418-resistant)
and the porcine MSC (HAT-resistant and G418-sensitive)
were detached from culture flasks by trypsin-EDTA treat-
ment. Approximately equal numbers of cells from each
parent were mixed in a centrifuge tube and plated in 60-mm
Petri dishes to achieve nearly confluent cultures after 3–4h.
At this stage, growth medium was discarded, and fresh
serum-free medium containing phytohemagglutinin-P
(100 μg/ml, Sigma) was added for 30 min at 37°C to
increase cell-cell contact. After the medium had b een
discarded, 2 ml 45% PEG (Sigma, MW 1 500, prepared in
serum-free DMEM, w/v) was added for 1 min at room
temperature and then aspirated off. The cell layer was
washed four times with serum-free medium and then
incubated in 5 ml complete DMEM (containing 15% FBS
and 1% penicillin/streptomycin) at 37°C for 30 min. Medium
was then aspirated away and replaced with complete
DMEM, followed by incubation overnight at 37°C. The cell
layer was then dissociated by trypsinzation; half of the cells
were cryopreseved and the other half was mixed with
complete DMEM supplemented with 2% HAT (Invitrogen)
and 1% G418. The cells were plated into four 100-mm Petri
dishes and two T75 flasks. HAT medium eliminated the F7
cells, and the G418 eliminated the porcine MSC. After
7 days, macroscopic double-drug-resistant colonies of hybrid
cells were visible. Independently derived HAT and G418
double-resistant colonies (n=17) of hybrid cells were
isolated individually by using cloning rings and were
expanded in separate culture flasks as clonal cell lines.
The expanded 17 cell lines were derived from two
independent cell fusion experiments, 11 from one fusion
and six from the other. Two cell lines (Pool A and Pool B),
from each of the two fusion experiments, were also
established by mixing all hybrid cell colonies of T75 flasks.
All cell lines were cryopreserved after multiplication.
Chromosome analysis
Chromosome preparation and the chromosome banding
procedure were as described previously (Islam and Levan
1987). Metaphase cells (20–35) were captured from each
cell line by a charge-coupled device camera by using
CytoVision software program (Applied Imaging). The
chromosomes of the parental cell lines/strains were identi-
fied and counted from the metaphase images by using
CytoVison. In the hybrid cells, chromosom es belonging to
the same species were counted together, and then the total
number of chromosomes was determined by adding the
chromosomes of two species.
Induction of cellular differentiation
Osteogenic differentiation was induced by culturing cell s of
a selected hybrid cell line in DMEM (15% FBS) supple-
mented with 100 nM dexamethasone (Dex; Fortecortin
Mono 40, Merck), 0.05 mM L-ascorbicacid-2-phosphate
(AsAP; Sigma), and 10 mM β-glycerophosphate (Sigma;
Bellows et al. 1990; Jaiswal et al. 1997). Cells were fixed
with ice-cold methanol at specific time points and used for
further histochemical analysis.
For chondrogenic differentiation, cells of a selected
hybrid cell line were centrifuged to form a pelleted micro-
mass (Johnstone et al. 1998 ). Pelleted cells were cultured in
a defined medium consisting of DMEM, ITS+1 (Sigma:
Cell Tissue Res
148
10 mg/l insulin, 5 mg/l transferrin, 5 μg/l selenium, 0.5 mg/ml
bovine serum albumin, 4.7 μg/ml linoleic acid), 1 mM
sodium-pyruvate (Sigma), 100 nM Dex (Sigma), 0.35 mM
proline (Sigma), 0.17 mM AsAP, and 10 ng/ml trans-
forming growth factor-β3 (R&D Systems). For histological
staining, the pellets were cryopreserved in OCT (Sakura),
and 6- μ m-thick sections were used for further analysis.
To induce adipogenic differentiation, cells of a selected
hybrid cell line were treated with adipogenic induction
medium containing DMEM, 15% FBS, 1 μM Dex, 0.2 mM
indomethacin (Sigma), 10 μg/ml insulin (Hoechst Marion
Roussel), and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma;
Gimble et al. 1992 ; Pittenger et al. 1999). Cells were used
for further histochemical analysis or were lyzed for total
RNA isolation.
Histological methods and immunohistochemistry
Osteoblasts exhibit high levels of alkaline phosphatase
(ALP), which can be visualized by staining with SIGMA
FAST BCIP/NBT (Sigma). Von Kossa staining identifies
the deposition of mineralized bone matrix.
Proteoglycan-secreting chondrocytes were stained with
Alcian Blue 8GS (Roth) at pH 2.5. Adipocytes were
identified morphologically and by staining with Oil Red
O (Sigma). For immunohistochemistry of type II collagen,
cryosections (6 μm) were incubated for 1 h with primary
antibodies (rabbit anti-human type II collagen, DPC-
Biermann). Subsequently, sections were incubated with
biotinylated anti-rabbit antibody and peroxidase-conjugated
streptavidine (Dako). The color reaction was developed by
the AEC substrate kit (Dako), followed by counterstaining
with hematoxylin (Merck).
Real-time revers e transcription/polymerase chain reaction
To demonstrate adipogenesis on the mRNA level, total
RNA of porcine MSC and a selected set of hybrid cell lines
was isolated as described previ ously (Chomczynski 1993).
Subsequently, 5 μg total RNA was reverse-transcribed by
using the iScript cDNA Synthesis Kit (BioRad) according
to the manufacturer′s instructions. The housekeeping gene,
D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
was used to normalize marker gene expression in each
sample in various concentrations. Real-time polymerase
chain reaction (PCR) with the i-Cycler system (BioRad)
was performed with 1 μl cDNA sample by using the SYBR
Green PCR Core Kit (Applie d Biosystems) . Relative
quantitation of adipogenic marker gene expression (Table 1)
was performed in an ABI Prism 7700 as detailed by the
manufacturer and was recorded as the percentage of the
GAPDH product.
Measurement of ALP activity and protein concentration
Confluent cult ures of hybrid cell lines wer e rinsed with
PBS and homogenized in 10 mM TRIS buffer (pH 8.3)
containing 10 μM zinc acetate, 0.1 mM MgCl
2
, and 0.1%
Triton X-100. ALP activities of homogenized cells were
determined in 96-well microtiter plates. In brief, a total
volume of 300 μl solution containing 1.0 M diethanolamine
buffer (pH 9.8), 10 mM p-nitrophenyl phosphate (Sigma),
and 1.0 mM MgCl
2
was added per well. The time-
dependent increase in absorbance at 405 nm (reflecting
p-nitropheno l production) was determined on a kinetic
microplate reader (Model VMax, Molecular Devices).
In humans, tissue-nonspecific ALP (TNALP) isoforms
are expressed in many tissues including bone, liver, and
kidney. Various heat inactivation and inhibition techniques
can distinguish the ALP isoenzymes (Magnusson et al.
1992). Heating at 56°C for 15 min inactivates TNALP
activity, particularly bone ALP activity. Heat inactivation at
65°C for 10 min inactivates 100% of TNALP and intestinal
ALP activity, but placental and germ cell ALP are resistant
at 65°C. L-Phenylalanine (10 mM) inhibits intestinal ALP
activity, but other isoenzymes are resistant to this treatment.
Protein concentrations were deter mined with the bicin-
choninic acid (BCA) method (Smith et al. 1985) by using
the BCA Protein Assay Kit (Pierce, Rockford). Since ALP
genes are highly conserved across species (Narisawa et al.
2005), we used the same method established for human cell
systems to assay ALP activity in the hybrid cell lines.
Table 1 Oligonucleotide sequences
Gene Accession number Oligonucleotides (5′→3′; up/down) Product size
in base pair
GAPDH AF017079 AGG GGC TCT CCA GAA CAT CAT
TTG GCA GTA GGG ACA CGG AAG G
117
aP2 AF102872 GGC ATG GCC AAA CCC AAC CT
TGT ACC AGG GCG CCT CCA TCT A
179
PPAR-γ2 AF103946 TGG CGA TAT TTA TAG CTG TCA TTA
TGT CCG TCT CTG TCT TCT TTA TTA
237
Cell Tissue Res
149
Results
Inter-species porcine-murine hybrid (MSC-F7) cell lines
were generated by fusing porcine MSC with the F7 murine
cell line. Cell fusion was induced in co-cultured monolayers
of two parental cell types followed by PEG treatment. To
amplify the recombinant hybrid cells, the mixture of fused
cells was plated in growth medium containing HAT (to
eliminate F7 cells) and G418 (to eliminate porcine MSC).
From these cells, 17 indepe ndently derived HAT and G418
double-resistant colonies of hybrids were isolated and
expanded as clonal cell lines. Two cell lines (Pool A and
Pool B), from each of the two fusion experiments, were
also established by mixing all hybrid cell colonies that had
developed in T75 flasks.
All MSC-F7 hybrid cell lines and two parental cell lines/
strains were subjected to cytogenetic analysis by high-
quality trypsin G-ba nding. The murine F7 cell line
contained about 35 chromosomes, including single copies
of chromosomes 6, 7, 8, 9, 12, 17, and 18, three to four
copies of chromosome 15, and a single copy of the small
marker chromosome M (Fig. 1a). The parental porcine
MSC contained 38 normal chromosomes including two X
chromosomes (Fig. 1b). Cytogenetic analyses of the 17 cell
lines showed that 13 retained approximately 4N, and three
retained approximately 6N porcine chromosomes, together
with hypo-diploid murine chromosomes (Table 2, Fig. 1c).
The hybrid cell lines retained about 22 murine chromo-
somes (Table 2) indicating that, following fusion, about 13
chromosomes were lost from the murine F7 cell parent.
Retention of porcine chromosomes in the hybrid cell lines
was extremely high compa red with the murine chromo-
somes, with an average of 72 chromosomes, ranging from
56 to 119. The total number of chromosomes in the hybri d
cell lines was about 94, ranging from 85 to 128. Thus, the
ratio of murine to porcine chromosomes in the hybrid cells
was approximately 1:4. Importantly, the chromosome
number of all hybrid cell lines was highly varia ble, and
therefore both murine and porcine genomes were repre-
sented by high anuploidy.
The morphology of all hybrid cell lines was fibroblastic,
and the cells were large (Fig. 2). Closer examination
revealed that minor morphological variations existed
among the hybrid cell lines. In general, hybrid cell lines
proliferated quickly (Fig. 3), but a few were slowly growing
(e.g., C6, Fig. 2i, Table 2).
One cell line (Pool A) has been growing continuously in
culture for more than a year and has exceeded 200
population doublings. So far, this cell line has not exhibited
any sign of growth retardation. Interestingly, all these cell
lines lack contact inhibition of growth and consequently
produce multiple cell layers on plastics.
Since the in vitro expanded parental porcine MSC were
capable of differentiating into different lineages (Ringe et
al. 2002), a selected set of hybrid cell lines were
investigated by usi ng various differentiation induction
protocols. The results of these investigations demonstrated
Fig. 1 Representative karyotypes of parental cells and derived hybrid
cells. a Hypo-diploid karyotype of murine cell line F7 (M unidentified
marker chromosome). Note that many chromosomes are represented
by single copies including chromosome 9 and 17. b Diploid karyotype
of porcine MSC. c A representative metaphase of hybrid cell line C4
containing a total of 124 chromosomes: 97 porcine (hyper-tetraploid,
circles five copies of porcine chromosome 1) and 27 murine
chromosomes (hypo-diploid, arrows)
Cell Tissue Res
150
that the tested cell lines retained osteogenic differentiation
(Table 3,Figs.4, 5), a few of them also retaining
adipogenic potential (Figs. 6, 7); none of them retained
chondrogenic potential (Table 3).
Table 2 Cytogenetic characterization of cell hybrids generated by fusion of primary porcine MSC with immortal murine cell line F7. All
chromosome numbers represent mean values
Hybrid line Murine
chromosomes
Range Porcine
chromosomes
Range Total
chromosomes
Range No. of
cells
analyzed
Hybrid
type
containing
M and P
a
Relative
cell
growth
b
MSC-F7-C1 27 14–31 70 63–83 97 88–112 21 1:2=M/P/P ++
MSC-F7-C2 23 9–29 71 59–77 94 77–104 25 1:2=M/P/P ++++
MSC-F7-C3 27 20–42 79 73–119 106 97–161 24 1:2=M/P/P ++++
MSC-F7-C4 29 18–32 93 67–111 122 88–142 25 1:3=M/P/P/P +++++
MSC-F7-C5 21 8–30 71 33–91 91 48–114 29 1:2=M/P/P ++++
MSC-F7-C6 24 19–28 68 61–75 92 86–97 21 1:2=M/P/P +
MSC-F7-C7 23 10–34 105 69–138 128 93–164 23 1:3=M/P/P/P ++++
MSC-F7-C8 28 22–32 68 63–73 96 90–102 21 1:2=M/P/P ++
MSC-F7-C9 27 18–34 71 66–78 98 83–106 24 1:2=M/P/P ++
MSC-F7-C10 29 24–39 61 47–68 91 76–106 23 1:2=M/P/P ++
MSC-F7-C11 17 10–23 70 66–75 87 83–97 28 1:2=M/P/P ++
MSC-F7-C12 29 19–33 74 66–84 103 95–112 22 1:2=M/P/P ++++
MSC-F7-C13 21 16–25 67 66–73 88 90–96 24 1:2=M/P/P ++
MSC-F7-C14 25 10–31 96 82–105 121 105–131 20 1:3=M/P/P/P +++
MSC-F7-C15 13 8–19 72 65
–76 85 81–92 27 1:2=M/P/P +++
MSC-F7-C16 18 7–33 72 56–94 90 71–127 28 1:2=M/P/P ++
MSC-F7-C17 26 17–33 70 57–84 97 74–117 35 1:2=M/P/P +++
Mean 22 72 94
Murine F7 35 33–36 24
Porcine MSC 38 30
a
Each M and P represents one diploid cell
b
Qualitative measurement from slowly growing (+) to rapid growth (+++++)
Fig. 2 Phase-contrast photomi-
crographs of murine F7 parental
cells plated at low (a) and high
(b) cell densities, porcine MSC
(c), hybrid cell line C1
(d), hybrid cell line C2 (e),
hybrid cell line C4 (f), hybrid
cell line C5 (g), hybrid cell line
C6 (h), and hybrid cell line C14
(i). The porcine MSC and all
hybrid cell lines were photo-
graphed at passage 4. Bars
100 μm
Cell Tissue Res
151
We tested all hybrid cell lines for the expression of
TNALP. The results showed that all hybrid cell lines
expressed TNALP, although there was wide range variation
for the amount of expressed enzyme among the cell s lines
(Table 4). Variable expression of TNALP enzyme by the
hybrid cell lines was not unexpected because th ey
contained variable numbers of the TNALP structural gene
(ALPL), as the hybrid cell lines were highly aneuploid
(Table 2).
Since the MSC-F7 hybrid cells were highly proliferative,
we collected conditioned media from hybrid cell lines Pool
A and Pool B at different time points to determine whether
they secreted growth stimulatory factors into the media.
The early-pa ssage porcine MSC grown in continuous
culture for 10 months in the presence of 20% conditioned
medium showed the morphology of freshly isolated MSC
with high mitoti c activity (Fig. 8a), compared with the same
cells that were grown without conditioned media and that
exhibited signs of aging (Fig. 8b). Despite prolonged
passaging in conditioned media, these cells maintained
their diploid karyotype (Fig. 2b). The MSC grown without
the addition of conditioned media showed mitotic activity
within 5 days when sub-cultured and treated with hybrid-
cell-derived conditioned media and, within 20 days, these
cells acquired a morphology similar to that of freshly
isolated porcine MSC (Fig. 8a,c). On the contrary, hybrid
cells grown for more than 1 year as continuous cultures in
standard DMEM containing 15% FBS showed high mitotic
Fig. 3 Growth kinetics of the hybrid cell lines C10, C11, and C12
(scale left: 1,0E+10 1.0×10
10
, etc.). Hybrid cells were expanded for up
to 103 days in monolayer culture. Cultures starting with fewer than
2×10
6
cells expanded to more than 1×10
29
cells after 103 days. The
average specific growth rate (μ
max
) of the cell lines was about
5.1×10
−1
±2.48×10
−2
/day
Table 3 Differentiation of a selected set of hybrid cell lines generated by fusion of primary porcine MSC with immortal murine cell line F7
Hybrid cell
line
Osteogenic
differentiation
a
Chondrogenic
differentiation
b
Adipogenic differentiation
MSC-F7-C3 + ND Cells contracted and detached before they could fully differentiate into adipocytes.
MSC-F7-C4 +++ ND Cells contracted and detached before any sign of adipogenic differentiation could be
detected
MSC-F7-C5 +++ ND Cells contracted and detached before any sign of adipogenic differentiation could be
detected
MSC-F7-C7 +++ ND Cells contracted and detached before any sign of adipogenic differentiation could be
detected
MSC-F7-C10 +++ – Adipogenic differentiation of hybrid cells was weaker than the parental porcine MSC
MSC-F7-C11 +++ – Adipogenic differentiation of hybrid cells was much weaker than the parental porcine
MSC
MSC-F7-C12 +++ – Adipogenic differentiation of hybrid cells was much weaker than the parental porcine
MSC
a
Qualitative measurement from low differentiation (+) to high differentiation (+++)
b
ND not done, − no differentiation
Cell Tissue Res
152
activity with no signs of aging (Fig. 8d). These results
indicated that the MSC-F7 hybrid cells secreted diffusible
factors into the culture media; these enhanced their own
growth and that of other cells.
Discussion
We have demonstrated that the fusion of normal primary
porcine MSC with the immortal murine fibroblast cell line
F7 generates immortal hybrid cells containing 4N to 6N
porcine chromosomes. This result indicates that whatever
chromosome numbers may come from normal cell during
fusion, the immortal F7 cell is capable of erasing the
memory of the normal cell and resetting it into the immortal
cell program. This unusual species-specific combinati on of
genomes has not previously been described f or a ny
experimentally induced somatic cell hybrid. Although the
spontaneous fusion of MSC, both in vitro and in vivo, with
other cell types is known to occur (Vassilopoulos and
Fig. 4 Osteogenic differentiation of the hybrid cell line C10
documented by von Kossa staining of the mineralized bone matrix
(a, e day 7, b, f day 14, c, g day 21, d, h day 28). a–d Untreated
control cultures were negative for von Kossa staining. e–h Dexameth-
asone-stimulated hybrid cells started to form a calcified matrix and
therefore were von-Kossa-positive. In contrast to untreated controls,
induced hybrid cells were von-Kossa-positive on day 7
Cell Tissue Res
153
Russell 2003; Camargo et al. 2004; Que et al. 2004; Rodic
et al. 2004), this is the first report, to our knowledge,
describing the generat ion of experimentally induced cell
hybrids by fusion of primary porcine MSC with an
immortal cell line. The retention of an unusually high
number of normal cell chromosomes is not limited to the
MSC-F7 hybrid cell line. Similar hybrid cell lines have
been generated by fusing normal porcine fibroblasts with
F7 cells (M.Q. Islam, V. Panduri, K. Islam, in prepar ation).
The fusion of F7 cells with normal porcine fibroblasts (both
proliferating and senescent) containing the complete ge-
nome, but not with immortal cells containing a deleted
genome, consistently produces hybrid cells with activated
growth accompanied by a nearly 4N porcine genome.
Introduction of the human telomerase gene (hTERT) into
the immortal cell line and the subsequent fusion of these
cells with F7 cells does not improve the growth of the
resulting hybrid cells, indicating that a telomerase-indepen-
dent mechanism is responsible for the activated growth
phenotype.
We have previously demonstrated that porcine MSC can
be induced to differentiate into various cell lineages
Fig. 5 Osteogenic differentiation potential of the hybrid cell line C10
documented by alkaline phosphatase (ALP) activity (a, e day 7, b, f
day 14, c, g day 21, d, h day 28). a–d Controls showed low ALP
activities during the whole culture period. e–h Osteogenic stimulation
resulted in increased ALP activity that peaked at day 7
Cell Tissue Res
154
including bone, cartilage, and fat (Ringe et al. 2002). In the
present study, we have shown that the hybrid cell lines
retain full potential for osteogenic differentiation, partial
potential for adipogenic differentiation, but no potential for
chondrogenic differentiation. Several investigators have
reported that cho ndrogenic differentiation is a sensitive
phenotype that can be influenced by cell culture conditions
and by genetic factors (Muraglia et al. 2000; Baddoo et al.
2003; Gregory et al. 2005; Magne et al. 2005; Sethe et al.
2005; Vacanti et al. 2005). The reprogramming of two
genomes in hybrid cells is also known to be a general
phenomenon during which the expression of previously
silent genes and the extinction of previously expressed
genes can occur, particularly if the hybrid cells are
generated by fusing dissimilar cell types (Gourdeau and
Fournier 1990; Takagi 1997; Surani 2001; Ambrosi and
Rasmussen 2005). Whether the loss of chondrogenic
potential in the hybrid cells is attributable to the silencing
of a specific porcine gene(s) required for the differentiation
pathway or because of interspecies genome incompatibility
is currently unknown.
Although TNALP is one of the most consistent markers
of embryonic stem cells (Carpenter et al. 20 03), this
enzyme is not expressed at a significant level in adult stem
cells. A recent report, however, indicates that pluripotent
epiblastic-like adult stem cells express high levels of
TNALP. Interestingly, these pluripotent stem cells lack
contact-inhibited cell growth and consequently form mul-
tiple cell layers on plastics (Young and Black 2004). Since
the MSC-F7 hybrid cells also exhibit these features, we
have tested all hybrid cell lines for the expression of
TNALP. Interestingly, we have found that some of the
MSC-F7 hybrid cell lines, particularly the C5, C7, C10,
C11, C15, Pool A, and Pool B lines, produce high levels of
TNALP in contrast to the immortal fibroblast cell line F7
(Table 4 ). Previous somatic cell genetic studies have
revealed that the expression of this en zyme is extinguished
upon cell fusion and re-expressed when substantial num-
bers of chromosomes are ejected fr om hybrid cells
(Johnson-Pais and Leach 1995, 1996). Our limited data
indicate that prolonged culture of hybrid cells in vitro has a
positive effect on the expression of TNALP (Table 4). One
Fig. 6 Adipogenic differentiation potential of the hybrid cell line C10 documented by Oil red O staining of lipid droplets (a, d day 5, b, e day 15,
c, f day 22). a–c Induced. d–f Untreated control
Cell Tissue Res
155
possibility is that the extended culture selects cells of high
TNALP content. Alternatively, chromosome carrying the
repressor gene for ALPL is lost during cell culture resulting
in the re-expression of this gene. Although the significance
of high level expression of TNALP by embryonic stem
cells is not fully understood, it may reflect the undifferen-
tiated cell state, whereas reduced or loss of expression may
reflect the differentiated state (Anneren et al. 2004). Our
results indicate that there is ample scope for the selection of
hybrid cell lines with high TNALP content.
Normal cells have been demonstrated to produce various
growth factors and cytokines that are beneficial for cell
proliferation and cell survival (Le Pillouer-Prost 2003;Liet
al. 2005). Normal MSC are also known to produce multiple
growth factors and cytokines (Majumdar et al. 1998, 2000;
Deans and Moseley 2000; Dormady et al. 2001; Minguell
et al. 2001). We have found that conditioned media from
MSC-F7 hybrid cells can enhance the proliferation of both
early-passage and late-passage MSC indicating that the
hybrid cells secrete diffusible growth stimulatory factors
into the culture media. We have previously reported that
hybrid cells, generated by the fusion of normal porcine
fibroblasts with F7 cells, also secrete growth stimulatory
factors, which not only can enhance their own proliferation,
but also can reinitiate cell division in the late-passage
porcine fibroblasts (M.Q. Islam, V. Panduri, K. Islam, in
preparation).
Unlike the fusion between embryonic and soma tic cells
(Tada and Tada 2001; Ambrosi and Rasmussen 2005;
Cowan et al. 2005), we have generated somatic cell hybrids
by fusing two types of somatic cells, viz., the immortalized
murine fibroblast cell line F7 and primary porcine MSC, to
ascertain whether the somatic cell hy brids grow indefinite-
ly, and whether they exhibit any features of MSC. Our
results demonstrate that porcine MSC with limited prolif-
eration can be reprogrammed to give unlimited prolifera-
tion, and that they express many characteristics of MSC,
although phenotypic differences occur among the hybrid
cell lines (Tables 2 and 3). Somat ic cell genetic studies have
revealed that the phenotype of hybri d cells is determined by
the relative contribution of the two parental genomes. For
example, hybrid cells generated by fusion of two differen-
tiated cell types representing the 2N genome from both
parent (1:1 hybrid) express only housekeeping genes, with
tissue-specific genes being systematically silenced. On the
contrary, hybrid cells representing the 4N genome from one
parent and the 2N genome from the other (2:1 or 1:2)
generally maintain the phenotype of the 4N parent
(Gourdeau and Fournier 1990; Massa et al. 2000). In our
MSC-F7 hybrid cells, the porcine MSC genome is
represented at the 4N to 6N level, and unsurprisingly many
hybrid cell lines express the phenotypes of the MSC parent
(e.g., TNALP, osteogenic and adipogenic differentiation
potentials, and production of growth promoting factors).
Interestingly, the lim ited proliferation of MSC is not
evident in the hybrid cells, although the murine genome is
represented by fewer than 2N chromosomes. Of note, the
F7 is a unique cell line containing many single copy
chromosomes in the karyotype, including chromosomes 9
and 17 (Fig. 1a). Fusion of F7 cells with other types of
normal primary cells derived from various species (porcine,
bovine, equine, canine, murine, and primate) consistently
produces hybrid cells containing the 4N normal cell
genome with activated growth phenotype. On the contrary,
fusion of GM05267-derived independent cell lines, carry-
ing disomy of chromosomes 9 and 17, with normal cells
produces hybrid cells of neither activated growth nor the
4N normal cell genome (M.Q. Islam and K. Islam,
unpublished). The activated growth phenotype seems to
be mediated by epigenetic reprogramming of the normal
cell genome through a direct involvement of the F7 genome
containing a specific karyotype (Fig. 1a). Further studies
are needed to understand the mechanism of this unusual
growth phenotype of the hybrid cells resulting from the
fusion of two somatic cell types.
Fig. 7 Real-time RT-PCR performed on the hybrid cell line C12 for
adipogenic marker genes (scale left: 4,0E-05 4.0×10
−5
,etc.).a PPAR-γ2,
the early transcription factor. b aP2, the late marker. Each measure
corresponds to the mean±SD of three independent experiments
Cell Tissue Res
156
One limitation of present study is that we have not
employed the parental cell lines as controls in all of the
experiments. We have previously demonstrated the multi-
lineage potentials of bone-marrow-derived porcine MSC
(Ringe et al. 2002). The porcine MSC in the present study
have been isolated from bone marrow in the same laboratory
by identical methods and under standar d cell culture
conditions as previously. Additionally, the differentiation
studies of the hybrid cell lines have been carried out in the
same laboratory with identical protocols to those of our
previous study (Ringe et al. 2002). Since the multilinage
potential of MSC is well documented (Baksh et al. 2004;
Barry and Murphy 2004), we have reasonably assumed that
the MSC used in the present study retain similar properties to
those obtained previously. Contrary to the potentials of
MSC, postnatal mature fibroblasts do not differentiate into
other lineages (Pitteng er et al. 1999). Similarly, other
workers have also concluded that tissue-specific fibroblasts
have limited plasticity, unlike embryonic fibroblasts and
stromal cells (also termed MSC; Bayreuther et al. 1988;
Ronnov-Jessen et al. 1996). Since the F7 fibroblast cell line
is also derived from a postnatal kidney, we consider that they
are tissue-specific mature fibroblasts.
Although mammalian somatic cells under standard cell
culture conditions do not proliferate indefinitely, several
independent reports indicate that normal cells can be grown
for extended periods under improved cell culture conditions
(Loo et al. 1987; Math on et al. 2001; Ramirez et al. 2001;
Romanov et al. 2001; Tang et al. 2001). However, these
cells are not expected to fulfill the strict criteria for
immortal cells. The emergence of true immortal cells with
unlimited proliferative capacity seems sometimes to occur
spontaneously; however, this requires both structural and
numerical chromosomal aberrations and multiple gene
mutations (Romanov et al. 2001; Yaswen and Stampfer
2002; M.Q. Islam, V. Panduri, K. Isla m, in preparation). In
the present study, we have demonstrated that the prolifer-
ation of both early-passage and late-passage porcine MSC
can be improved by the addition of conditioned media
derived from hybrid cells (Fig. 8). Of note, the improved
proliferation of porcine MSC following this treatment is a
conditional phenotype; these cells are not truly immortal
because porcine MSC grown for extended periods maintain
their diploid karyotype (Fig. 1b). However, our present
results and the findings of other investigators mentioned
above support the notion that cellular senescence may not
Table 4 Quantitative kinetic assay of ALP in the hybrid cell lines generated by fusion of porcine MSC with immortal murine cell line F7
Cell line Total protein
(mg/l)
Total
ALP
(U/l)
ALP/protein
(mU/mg)
Remaining
ALP
activity after
56°C,
15 min (%)
Remaining
ALP
activity after
65°C,
10 min (%)
Remaining ALP
activity after
10 mM
L-phenylalanine
(%)
Passage
number at
which ALP
assayed
MSC-F7-C1 4837 25 5 35 0 78 4
MSC-F7-C2 2977 47 16 33 0 83 4
MSC-F7-C3 3587 338 94 19 0 76 4
MSC-F7-C4 3347 133 40 30 0 76 4
MSC-F7-C5 3017 543 180 23 0 71 4
MSC-F7-C6 1357 57 42 26 0 72 4
MSC-F7-C7 3027 506 167 35 0 76 4
MSC-F7-C8 4427 346 78 26 0 77 4
MSC-F7-C9 3217 362 113 24 0 76 4
MSC-F7-C10 3147 2185 694 24 0 76 20
MSC-F7-C11 2747 490 178 32 0 78 20
MSC-F7-C12 3077 101 33 19 0 77 4
MSC-F7-C13 3917 13 3 39 0 81 4
MSC-F7-C14 2747 24 9 34 0 79 4
MSC-F7-C15 3437 971 282 18 0 78 4
MSC-F7-C16 3167 61 19 28 0 71 4
MSC-F7-C17 3497 469 134 24 0 79 4
MSC-F7-Pool
A
4052 1281 316 18 0 59 20
MSC-F7-Pool
B
5422 515 95 34 0 83 20
Murine F7 11163 100 9 28 4 92 Not known
Porcine MSC 1031 272 264 20 0 77 4
Cell Tissue Res
157
be a genetically programm ed event, but rather the pheno-
type may be caused by the lack of availability of
appropriate growth factors in the culture media.
With some exceptions, cell fusion is not a common
phenomenon in vivo, particularly in mammals (Ogle et al.
2005). Interestingly, an increasing number of reports
indicate that transplanted MSC frequently undergo cell
fusio n with native cells, and clonal expansion of th e
resulting hybrid cells is required for effective tissue
regeneration. This is apparent in the fumarylacetoacetate-
hydrolase-deficient mouse model in which regenerated
livers contain mostly hybrid cells (Wang et al. 2003 ). The
hybrid cells may have a selective growth advantage in vivo.
Since the fusion of cell types with only limited proliferation
produces hybri d cells of limited proliferation (Hoehn et al.
1978), methods must be developed in order to obtain
proliferating hybrid cells in vitro before testing their ability
in vivo.
Somatic stem cells have potentials for therapeutic
applications, but their limited prolifer ation is a major
barrier to achieving desirable therapeutic effects (Baxter et
al. 2004; Javazon e t al. 2004; Sethe et al. 2005). Although
large numbers of cells are a requisite for in vivo use, cells
with unlimited proliferation may not be essential for tissue
regeneration. We have demonstrated here that our two-step
cell culture protocol, viz., (1) immortalization of primary
somatic stem cells of limited proliferation by fusi on with F7
cells and (2) improvement of proliferation of primary cells
through their treatment with hybrid-cell-derived condi-
tioned media , may provide a means to produce large
number of somatic stem cells of limited proliferation and
of the hybrid cells derived from them. Thus, the application
of our improved cell culture protocol should allow an
evaluation of the regenerative capacity of hybrid and
normal adult stem cells of limited proliferation.
Acknowledgement The authors thank Cecilia Linder for excellent
technical assistance.
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Polyethylene glycol-mediated fusion between primary
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161
STEM CELLS AND DEVELOPMENT 15:905–919 (2006)
© Mary Ann Liebert, Inc.
Biotechnology
Polyethylene Glycol-Mediated Fusion between Primary
Mouse Mesenchymal Stem Cells and Mouse Fibroblasts
Generates Hybrid Cells with Increased Proliferation
and Altered Differentiation
M.Q. ISLAM,
1,2
L. DA S. MEIRELLES,
4
N.B. NARDI,
4
P. MAGNUSSON,
3
and K. ISLAM
1,2
ABSTRACT
Bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into different cell lineages
with the appropriate stimulation in vitro. Transplantation of MSCs in human and other animal
models was found to repair tissues through the fusion of transplanted MSCs with indigenous cells.
We have generated mouse–mouse hybrid cell lines in vitro by polyethylene glycol-mediated fusion
of primary mouse MSCs with mouse fibroblasts to investigate the characteristics of hybrid cells, in-
cluding their potentials for proliferation and differentiation. Similar to the parental MSCs, hybrid
cells are positive for the cell-surface markers CD29, CD44, CD49e, and Sca-1, and negative for Gr-
1, CD11b, CD13, CD18, CD31, CD43, CD45, CD49d, CD90.2, CD445R/B220, and CD117 markers.
The hybrid cells also produce a high level of tissue nonspecific alkaline phosphatase compared to
the parental cells. Conditioned medium of hybrid cells contain biologically active factors that are
capable of stimulating proliferation of other cells. Although the parental MSCs can differentiate
into adipogenic and osteogenic lineages, hybrid cells held disparate differentiation capacity. Hybrid
cell lines in general have increased proliferative capacity than the primary MSCs. Our study dem-
onstrates that proliferative hybrid cell lines can be generated in vitro by induced fusion of both im-
mortal and primary somatic cells with primary MSCs.
905
INTRODUCTION
A
DULT TISSUES CONTAIN STEM CELLS
that contribute to
the repair and regeneration of organs (1–3). Stem
cell populations derived from variety of tissues offer great
promise for cell-based therapies (4,5). Mesenchymal
stem cells (MSCs) isolated from bone marrow (BM) are
multipotent and capable of differentiating into many cell
lineages when cultured under defined in vitro conditions
(6–9). The MSCs also contribute to the regeneration of
tissues such as bone, cartilage, muscle, ligament, tendon,
adipose, and stroma in vivo (10–14). Transplantation of
MSCs in vivo so far have demonstrated very limited re-
generative capacity for using this procedure for mean-
ingful therapies (12,15,16). However, delivery of MSCs
in injury models shows promise for cell-based therapies,
1
Laboratory of Cancer Genetics, Laboratory Medicine Center (LMC), University Hospital Linkoping, Sweden.
2
Department of Biomedicine and Surgery and
3
Divison of Clinical Chemistry, Faculty of Health Sciences, Linkoping Univer-
sity, SE-581 85 Linkoping, Sweden.
4
Laboratorio de Imunogenetica, Departamento de Genetica, Universidade Federal do Rio Grande do Sul, Porto Alegre RS,
Brazil.
162
particularly in the fumarylacetoacetate hydroxylase-defi-
cient mice where cell fusion plays a major role in regen-
erating new liver tissues (17–20). This observation may
indicate that hybrid cells have better proliferative capacity
than the unmodified natural MSCs. Because the frequency
of spontaneous hybrid cell formation in vivo was expected
to be low due to random chances of spontaneous cell fu-
sion, optimum regeneration of organs may not be possible
through the delivery of natural MSCs and relying on their
unplanned fusion. In this respect, in vitro generation of hy-
brid cells by induced fusion of MSCs with organ-specific
somatic cells and their proper characterization would per-
mit selection of hybrid cell lines with improved regenera-
tive capacity suitable for in vivo transplantation. This hy-
pothesis has prompted us to generate hybrid cells by fusing
mouse MSCs with mouse fibroblasts to investigate the pro-
liferative capacity of in vitro-generated hybrid cells and
their potentials for differentiation.
MATERIALS AND METHODS
Mouse MSCs
The isolation of mouse (m) MSC line LTC-4D (here-
after 4D) was described previously (21). Briefly, the 4D
cell line was established from the BM of the mouse strain
C57BL/6 with a targeted deletion of the gene Idua
(IDUA-KO) introduced through a neomycin-resistant
gene cassette (22). Animals were killed by cervical dis-
location, and BM was flushed out of tibias and femurs.
To initiate the mMSC culture, BM cells were plated in
tissue culture dishes and kept in a humidified incubator
with 5% CO2 at 37°C for 72 h, when nonadherent cells
were removed by changing the medium. Confluent pri-
mary culture was trypsinized and passaged to a new cul-
ture dish with a split ratio of 1:2. Subsequent passages
were done when the culture approached confluence, and
split ratios were increased as needed to permit two sub-
cultures a week. Long-term growing 4D cells at passage
22 were used for the present investigation. They were
grown in complete Dulbecco’s modified Eagle medium
(DMEM) supplemented with 15% fetal bovine serum
(FBS), 1% minimal essential medium (MEM) nonessen-
tial amino acids, and 1% penicillin and streptomycin. Tis-
sue culture reagents were purchased from PAA Labora-
tories GmbH (Pasching, Austria), if not stated otherwise.
Primary mouse fibroblast cells
Primary mouse fibroblast cells were isolated from an
adult female mouse of the strain T37H (kindly provided by
M.F. Lyon) carrying reciprocal translocations involving the
X chromosome and chromosome 4. The T37H fibroblasts
(hereafter T37) were cultured in complete DMEM supple-
mented with 15% FBS, 1% MEM nonessential amino acids,
and 1% penicillin and streptomycin. The T37 cells at pas-
sage 8 were used for the present study.
Immortal mouse fibroblast cells
The neomycin-resistant mouse cell line F7, a deriva-
tive of GM05267, has been described recently (23). The
original immortal mouse fibroblast cell line GM05267,
obtained from the National Institutes of General Medical
Sciences, was deficient for the enzyme hypoxanthine
phosphoribosyl transferase (HPRT
Ϫ
) and consequently
sensitive to hypoxanthine aminopterin thymidine (HAT).
This fibroblast cell line was isolated from a postnatal/
adult kidney of a heterozygous male for the testicular
feminization (tfm) locus mutation (24,25). The F7 cell
line was maintained in DMEM, supplemented with 15%
FBS, 1% MEM nonessential amino acids, and 1% peni-
cillin-streptomycin.
Introduction of hygromycin-resistant gene into
T37 fibroblasts
A hygromycin-resistant gene was introduced into the
T37 fibroblasts through retroviral infection using a pub-
lished protocol (26). Briefly, proliferating cells were
plated in a T75 culture flask with DMEM containing 15%
FBS supplemented with 0.03 g/ml tunicamycin (Sigma)
and incubated overnight. Conditioned medium contain-
ing the HyTk retrovirus (kindly provided by R.F. New-
bold) was collected from confluent culture of the pro-
ducer cells and filtered through a 0.45-m filter to
remove any intact cell. The filtered supernatant was
placed in the T75 flask after adding 4 g/ml Polybrene
(Sigma) and allowed to stay 5 h. The medium was then
changed several times with serum-free DMEM and fi-
nally replaced with complete DMEM. Next day, com-
plete DMEM with hygromycin (0.1 mg/ml; Calbiochem)
was added to the flask. The medium was renewed every
3–4 days for 2 weeks. A pool of hygromycin-resistant
cells was used for the present study.
Cell fusion
Cell fusion was induced following a protocol reported
elsewhere (23). Briefly, a monolayer of 4D cells (HAT-
resistant) and F7 cells (HAT-sensitive) were detached by
trypsin treatment. Trypsinized cells were suspended in
complete DMEM and 1 ϫ 10
6
cells were taken from each
parent and mixed in a centrifuge tube. Supernatant was
discarded after centrifugation, and the pelleted cells were
suspended in 5 ml of complete DMEM. The mixed
parental cells were then seeded on a 60-mm Petri dish
and incubated at 37°C with 5% CO
2
for 3–4 h. After as-
pirating medium, serum-free DMEM containing phyto-
ISLAM ET AL.
906
163
hemagglutinin-P (100 g/ml; Sigma) was added to in-
crease the cell–cell contact and incubated at 37
o
C with
5% CO
2
for 30 min. After aspirating the medium, 2 ml
of 45% polyethylene glycol (PEG; Sigma, MW 1,500),
prepared by mixing with serum-free DMEM (wt/vol) and
filter sterilized (MediaKap-2, 0.2 m, Microgon, Inc.,
Laguna Hills, CA), was added at room temperature and
allowed to cover the entire cell layer. One minute later,
the PEG was aspirated and washed four times with serum-
free medium. Complete DMEM was added to the dish
after the final washing and then incubated at 37°C with
5% CO
2
. The next day, the fused cell layer was
trypsinized, and the dissociated cells were suspended in
DMEM. The cells were divided equally into two cen-
trifuge tubes. Cells from one tube were cryopreserved
and the cells of the other tube were resuspended in com-
plete DMEM supplemented with 2% HAT and plated into
a 75-cm
2
tissue culture flask. Cells were grown in HAT
medium for 7 days to eliminate F7 cells leaving the pro-
liferating parental 4D cells and hybrid cells between 4D
and F7 (half-selection). After propagating the mixed pop-
ulations of HAT-resistant cells in DMEM for additional
14 days, 20 cells were plated per 100-mm Petri dish and
allowed to form macroscopic colonies. Larger-size cell
colonies were circled under the Petri dishes with a marker
pen, and morphologically distinct colonies were isolated
using cloning rings for establishing cell lines.
To generate the second series of hybrid cell lines, 4D
cells (G418-resistant and hydromycin-sensitive) were
fused with T37 cells (hygromycin-resistant and G418-
sensitive) in monolayer culture similar to the first fusion
experiment as described above. Hybrid cells were se-
lected in DMEM containing hygromycin (0.1 mg/ml;
Calbiochem) and G418 (1.5%, Invitrogen) to eliminate
both 4D and T37 parental cells (complete selection). They
were grown for 14 days in the presence of selective
medium, and then 20 cells were plated per 100-mm cell
culture dish to allow the formation of macroscopic
colonies. After 7 days, hybrid cell colonies were isolated
and then cryopreserved after multiplication.
Chromosome analysis
Chromosome preparation and the chromosome banding
procedure were described previously (27). Metaphase cells
(20–43) were captured from each cell line by a charge-
coupled device camera by using CytoVision software pro-
gram (Applied Imaging). Chromosomes of the parental and
hybrid cell lines were identified and counted from the
metaphase images using the CytoVision program.
Flow cytometry
Immunophenotyping of a selected set of hybrid cell
lines and the MSC parental cell line 4D was performed
by flow cytometric analysis following a published pro-
tocol (21,28). Briefly, cells were collected after
trypsinization and incubated for 30 min at 4°C with phy-
coerythrin (PE)- or fluorescein isothiocyanate (FITC)-
conjugated antibodies against murine Sca-1, Gr-1,
CD11b, CD13, CD18, CD29, CD31, CD34, CD43,
CD44, CD45, CD49d, CD49e, CD90.2, CD445R/B220,
and CD117 (Pharmingen). Excess antibodies were re-
moved by washing. Detection of PE and FITC labeling
was accomplished on a FACScalibur cytometer equipped
with 488-nm argon laser (Becton Dickinson) using Cell-
Quest software. At least 10,000 events were collected.
WinMDI 2.8 software was used to create histograms.
Induction of osteogenic differentiation
Osteogenic differentiation was induced by culturing
cells of selected hybrid cell lines and the MSC parental
cell line 4D for up to 4 weeks in DMEM containing 10%
FBS and 15 mM HEPES supplemented with 10
Ϫ8
M
dexamethasone, 5 g/ml ascorbic acid 2-phosphate, and
10 mM -glycerophosphate (9,21,28). To observe cal-
cium deposition, cultured cells were fixed with 4% para-
formaldehyde in phosphate-buffered saline (PBS) for 1
min at room temperature (RT), washed once with PBS,
and stained for 5 min at RT with Alizarin Red S stain,
pH 4.2. Excess stain was removed by several washes with
distilled water.
Induction of adipogenic differentation
To induce adipogenic differentiation, selected hybrid
cell lines and the MSC parental cell line 4D were kept in
culture for up to 4 weeks in DMEM containing 10% FBS
and 15 mM HEPES supplemented with 10
Ϫ8
M dexa-
methasone and 5 g/ml insulin. Adipocytes were easily
distinguished from the undifferentiated cells by phase-con-
trast microscopy. To further confirm their adipogenic dif-
ferentiation, cells were fixed with 4% paraformaldehyde
in PBS for 1 h at RT, and stained with Oil Red O (Sigma)
solution (three volumes of 3.75% Oil Red O in isopropanol
plus two volumes of distilled water). In some experiments,
CoCl
2
was added to the differentiation medium to a final
concentration of 100 M to mimic hypoxic conditions, be-
cause hypoxia has been previously shown to induce lipid
vacuole formation in human MSCs (29).
Measurement of ALP activity and
protein concentration
Parental and hybrid cells were rinsed with PBS and
homogenized in 10 mM Tris buffer, pH 8.3, containing
10 M zinc acetate, 0.1 mM MgCl
2
, and 0.1% Triton X-
100. Alkaline phosphatase (ALP) activities of homoge-
nized cells were determined in 96-well microtiter plates.
MOUSE MSC HYBRIDS
907
164
In brief, a total volume of 300 l of solution was added
per well, containing 1.0 M diethanolamine buffer, pH 9.8,
10 mM p-nitrophenyl phosphate (Sigma), and 1.0 mM
MgCl
2
. The time-dependent increase in absorbance at
405 nm (reflecting p-nitrophenol production) was deter-
mined on a kinetic microplate reader (Model VMax, Mo-
lecular Devices Corp.).
Tissue-nonspecific ALP (TNALP) isoforms are ex-
pressed in many tissues. Different heat inactivation and in-
hibition techniques can distinguish the ALP isoenzymes
(23,30). Heating at 56°C for 15 min inactivates the TNALP
activity, particularly bone ALP activity. Heat inactivation
at 65°C for 10 min inactivates 100% of TNALP and in-
testinal ALP activity, but placental and germ cell ALP are
resistant at 65°C.
L
-Phenylalanine (10 mM) inhibits the in-
testinal ALP activity, but other isoenzymes are resistant to
this treatment. Protein concentrations were determined by
the bicinchoninic acid (BCA) method (31), using the BCA
Protein Assay Kit (Pierce, Rockford). Because ALP genes
are highly conserved across species (32), we used the same
method established for human cell systems to assay ALP
activity in the mouse cells.
RESULTS
Generation of hybrid cells
As the fusion of cells of limited proliferation usually
generates hybrid cells of limited proliferation (33), we
fused the long-term growing mouse MSC line 4D with
the immortal mouse fibroblast cell line F7 to overcome
the potential proliferative limitations in the resulting hy-
brid cells. Cell fusion was induced by PEG in co-cultured
monolayer of F7 (HAT-sensitive) and 4D cells (HAT-re-
sistant). Subsequently, HAT selection was applied for the
initial enrichment of hybrid cells. The HAT medium pre-
vented the proliferation of parental F7 cells but not the
parental 4D cells and hybrid cells between 4D and F7
(half-selection, see Materials and Methods). We isolated
clonal cell lines by dilution plating of mixed populations
of the parental 4D cells and 4D–F7 hybrid cells at pas-
sage three. On the basis of distinct morphologies, we iso-
lated five independently derived cell colonies using
cloning rings and expanded separately as cell lines for
further characterization.
Karyological evidence of generation
of hybrid cells
Previous cytogenetic analyses revealed that the
parental F7 fibroblast cell line contained approximately
35 chromosomes with monosomy of at least seven chro-
mosomes. This cell line contained many copies of chro-
mosome 15, one copy of X, one copy of a small marker
chromosome, and no Y chromosome (23). Cytogenetic
analyses of the parental cell line 4D showed nearly 80
chromosomes (Table 1). Initially, it appeared that this cell
line was euploid and contained tetraploid chromosome
complements. However, detailed karyotype analyses re-
vealed that this was an aneuploid cell line containing vari-
able numbers of copies of individual chromosome. This
cell line also contained two copies of X, one to two copies
of Y, and four marker chromosomes (Fig. 1A). Theoret-
ically, fusion of one F7 cell with one 4D cell expected
to produce a hybrid cell of 115 chromosomes. Cytoge-
netic analysis of HAT-resistant cell lines derived from
the fusion of F7 and 4D cells revealed that four cell lines
ISLAM ET AL.
908
T
ABLE
1. C
YTOGENETIC
A
NALYSIS OF
C
ELL
H
YBRIDS
G
ENERATED BY
F
USING
M
OUSE
MSC
C
ELL
L
INE
LTC-4D
WITH THE
I
MMORTAL
F
IBROBLAST
C
ELL
L
INE
F7
F7-derived Presumable
Total Normal X 4D-derived Y small Number of cells fusion type
Cell line chromosome
a
chromosome
a
chromosome
a
marker
a
analyzed 4D/F7
b
Hybrid
4D-F7-C1 106.96 (99–110) 2.81 (1–3) 2.50 (1–4) 1.04 (1–2) 26 2Ϻ1
4D-F7-C2 107.70 (84–140) 3.10 (2–4) 1.40 (0–2) 1.20 (1–3) 20 2Ϻ1
4D-F7-C3 116.64 (63–139) 3.24 (2–4) 1.48 (0–2) 1.68 (1–2) 25 2Ϻ1
4D-F7-C4 107.91 (74–126) 2.78 (2–4) 0.56 (0–2) 1.66 (0–2) 32 2Ϻ1
4D-F7-C5 87.96 (67–116) 2.23 (1–3) 1.33 (0–3) 0.89 (0–1) 27 2Ϻ1
Parent
F7 34.70 (33–36) 1.00 (1–1) NA 1.04 (1–2) 24 NA
4D 79.12 (74–82) 1.98 (1–1) 1.07 (0–2) NA 43 NA
NA, Not applicable.
a
All numbers represent the mean value, and the number in parentheses represents the range.
b
The number “1” represents a diploid cell and “2” represents a tetraploid cell.
165
contained chromosome numbers exceeding 100 ranged
from 88 and 117 chromosomes (Table 1), and this num-
ber is close to the expected chromosome number of a hy-
brid cell. However, one cell line (4D–F7–C5) contained
less than 90 chromosomes, which may resulted from the
loss of several chromosomes. To confirm whether the
HAT-selected cell lines were actually hybrid cells and
carrying marker chromosomes of both 4D and F7 parental
cells, we performed cytogenetic analysis of all cell lines.
The results of these analyses revealed that they contained
two copies of each of X and Y chromosomes, presum-
ably derived from the 4D cell line, and one copy each of
X and a small marker chromosome, derived from the F7
cell line (Table 1, Fig. 1B). The presence of marker chro-
mosomes of both 4D and F7 parents in the metaphases
of all cell lines confirmed that they were truly hybrid
cells. Absence of any pure parental karyotype of 4D cells
in the isolated cell line indicates that the hybrid cells had
a proliferative advantage compared to the parental 4D
cells.
Next, we generated a second series of cell hybrids by
fusing the 4D cell line with primary T37 fibroblasts. The
4D cell line was constitutively carrying a neomycin-re-
sistant gene (G418-resistant and hygromycin-sensitive),
and a hygromycin-resistant gene was introduced into the
T37 fibroblasts (hygromycin-resistant and G418-sensi-
tive) before inducing cell fusion. After fusion, hybrid
cells were selected in DMEM containing G418 (to kill
T37 cells) and hygromycin (to kill 4D cells). Recombi-
nant hybrid cells between 4D and T37 cells were prolif-
erated in the medium containing two drugs because of
genetic complementation. Cytogenetics is a powerful tool
to identify hybrid cells generated by fusion of two cell
types belonging to different species. However, it is dif-
MOUSE MSC HYBRIDS
909
FIG. 1. Cytogenetic evidence of generation of hybrid cells be-
tween mouse 4D cells and mouse fibroblast cells. (A) Repre-
sentative karyotype of the 4D cell line containing a total of 81
chromosomes. Besides the presence of four marker chromosomes
(M), the karyotype showed variable numbers of copies of auto-
somes represented by three to seven copies, two copies of X chro-
mosomes, and a single-copy Y chromosome. (B) Representative
metaphase cell of the hybrid cell line 4D–F7–C1, containing a
total of 105 chromosomes including two copies of X and one
copy of Y, presumably derived from the 4D cell, and one copy
each of X and a small marker chromosome derived from the F7
cell. (C) Representative karyotype of a diploid cell of primary
mouse fibroblast strain T37 showing 40 chromosomes, includ-
ing two translocated X;4 chromosomes, generated by reciprocal
translocations of 4 and X, and one normal X chromosome. (D)
Represenetative metaphase of the hybrid cell line 4D–T37–H
containing a total of 114 chromosomes including two normal X
chromosomes and two Y chromosomes, presumably derived
from a 4D cell, and one normal X and two translocated X;4 chro-
mosomes, presumably derived from a T37 cell.
166
ficult to determine unambiguously the parental origins of
chromosomes of hybrid cells generated by fusion of cells
of same species. To discriminate the parental origin of
chromosomes in the mouse–mouse hybrid cells, we used
mouse fibroblast cells T37 carrying reciprocal transloca-
tions between chromosome 4 and X chromosome (Fig.
1C). Cytogenetic analysis of the T37 parental cells
showed that 56% of the metaphase cells contained
diploid, 12% tetraploid, and 32% heteroploid chromo-
somes (Table 2). The fusion of 4D cells with 80 chro-
mosomes with T37 cells having 40 chromosomes was ex-
pected to produce a hybrid cell of 120 chromosomes.
Double drug-resistant eight hybrid cell lines (4D–T37 hy-
brid) of independent origins were subjected to cytoge-
netic analysis using the G-banding technique. These
analyses showed that they contained chromosome num-
bers ranging from 111 to 145 (Table 2). It should be noted
that two hybrid cell lines contained chromosome num-
bers slightly less than 120 (hybrid A and hybrid H), two
contained slightly more than 120, and four contained be-
tween 134 and 145 chromosomes. These results suggest
that hybrid cells were generated by the fusion of near-
tetraploid 4D cells with three possible types of T37 cells
(diploid, tetraploid, and heteroploid) (Table 2). Detailed
karyotype analyses of 4D–T37 hybrid cells confirmed
that that they contained marker chromosomes of both 4D
and T37 parental cells (Table 2, Fig. 1D). The use of the
T37 cells in the fusion experiments allowed us to deter-
mine indirectly the genomic contribution of each parent
(2:1 or 2:2 combination) in the generation of hybrid cells
by counting the copy numbers of t(4;X) markers, normal
X, and Y chromosomes derived from two parents (Table
2).
Cellular morphology and growth properties
Morphologically, the F7 parental cells were smaller in
size with high proliferation (Fig. 2A) and the 4D cells
were flat with slow proliferation (Fig. 2B). The 4D–F7
hybrid cells had flattened morphology with high prolif-
eration rates. These hybrid cells grew even at low cell
densities and proliferated like immortal cells (Fig. 2C–F).
The morphology of the parental T37 fibroblas was thin,
elongated, and tightly packed (Fig. 3A). The morphology
of 4D–T37 hybrid cells was large and flat (Fig. 3C–F).
The 4D–T37 hybrid cells have been grown for at least
60 population doublings, and they showed superior pro-
liferative capacity compared to the parental cells (Fig.
3A–H). In general, the growth of 4D–F7 hybrid cell lines
(Fig. 2C–F) was better compared to the growth of
4D–T37 hybrid cell lines (Fig. 3C–F). It should be noted
that the 4D–T37 hybrid cells required moderately high
cell density for optimum proliferation.
Immunophenotyping
The MSC parental cell line 4D found to express a dis-
tinct set of cell-surface antigens (21). We randomly se-
lected cell lines from two series of hybrids and performed
immunophenotyping by flow cytometry. Similar to the
cell-surface antigens of the parental 4D cell line, all tested
hybrid cell lines were found to be positive for the mark-
ISLAM ET AL.
910
T
ABLE
2. C
YTOGENETIC
A
NALYSIS OF
C
ELL
H
YBRIDS
G
ENERATED BY
F
USING
M
OUSE
MSC C
ELL
L
INE
LTC-4D
WITH
A
DULT
F
IBROBLASTS
D
ERIVED FROM THE
M
OUSE
S
TRAIN
T37H
T37-derived Presumable
Hybrid Total Normal X 4D-derived Y long X;4 Number of cells fusion type
cell line chromosome
a
chromosome
a
chromosome
a
marker
a
analyzed 4D/T37
b
Hybrids
4D-T37-A 111.1 (104–142) 3.2 (3–4) 2.2 (1–4) 0.9 (0–1) 22 2Ϻ1
4D-T37-B 126.8 (120–131) 3.8 (3–4) 2.2 (2–3) 1.0 (1–1) 20 2Ϻ1
4D-T37-C 143.1 (118–146) 3.9 (3–4) 2.0 (1–3) 1.7 (1.2) 20 2Ϻ2
4D-T37-D 140.4 (108–150) 3.1 (3–4) 1.6 (1–2) 1.0 (1–1) 20 2Ϻ2
4D-T37-E 145.4 (133–158) 3.6 (3–4) 1.3 (1–2) 1.0 (1–1) 20 2Ϻ2
4D-T37-G 134.5 (106–146) 3.3 (2–4) 2.1 (1–3) 1.2 (1–2) 20 2Ϻ2
4D-T37-H 114.0 (110–146) 3.2 (3–4) 2.6 (2–4) 1.0 (1–1) 20 2Ϻ1
4D-T37-I 126.5 (107–131) 3.7 (3.4) 1.7 (1–2) 1.0 (1–1) 20 2Ϻ1
4D-T37-J 125.3 (113–130) 3.4 (2–4) 2.5 (2–3) 1.0 (1–1) 20 2Ϻ1
Parent
4D 79.1 (74–82)0 1.9 (1–2) 1.1 (0–2) NA 43 NA
T37
c
52.4 (60–84)0 1.2 (1–2) NA 1.2 (1–2) 25
NA, Not applicable.
a
All numbers represent mean value, and the number in parentheses represents the range.
b
The number “1” represents a diploid cell, and “2” represents a tetraploid cell.
c
A total of 56% of the cells were diploid, 12% cells tetraploid, and 32% cells aneuploid.
167
ers CD29 (integrin 1 chain), CD44 (hyaluronan recep-
tor), CD49e (integrin ␣5 chain), and Sca-1 (stem cell anti-
gen-1). There was no expression or barely detectable ex-
pression of following markers: CD49d (integrin ␣4
chain), CD11b and CD45 (hematopoetic markers), CD13
(a monocyte marker), CD18 (a leukocyte marker), CD31
(an endothelial marker), CD34 (a hematopoetic progen-
itor marker), CD117 (stem cell factor receptor), GR-1 (a
granulocyte marker), CD41 (a megakaryocyte marker),
CD90.2 (a thymus cell antigen), and CD445R/B220 (B
lymphocyte marker) (Fig. 4). These results indicate that
the fusion of mouse MSCs with mouse fibroblasts does
not alter the cell-surface antigen expression of MSCs, ir-
respective of the mortality status of fibroblast parents.
TNALP analysis
In contrast to embryonic stem (ES) cells, adult stem
cells express limited amounts of TNALP. Recently, we
have demonstrated that fusion of pig MSCs with mouse
F7 fibroblast cells produces hybrid cells where the ex-
pression of TNALP was elevated (23). To know whether
the fusion of mouse MSCs with mouse fibroblasts pro-
duces hybrid cells of similar characteristics, we assayed
the total TNALP content of all cell lines of 4D–T37 and
4D–F7 hybrids. The results of these analyses showed that
the expression of TNALP was elevated in most of the
hybrid cell lines compared to the parents (Table 3). It
should be noted that the expression level of TNALP in
the 4D–T37 hybrids was much higher than that of 4D–F7
hybrids.
Differentiation studies
The parental 4D cells were found to differentiate into
osteoblasts and adipocytes under induced conditions. To
find out whether the fusion of these cells with fibroblasts
maintained similar differentiation as MSCs, we tested se-
MOUSE MSC HYBRIDS
911
FIG. 2. Phase-contrast photomicrographs of the parental mouse immortal fibroblast F7 cells (A), mouse MSC 4D cells (B), hy-
brid cell lines 4D–F7–C1 (C), 4D–F7–C2 (D), 4D–F7–C3 (E), and 4D–F7–C4 (F). Note the increased proliferation of hybrid
cells, as evident from the presence of numerous dividing rounded cells, compared to the parental 4D cells. Bars, 100 m.
168
lected hybrid cell lines for osteogenic and adipogenic dif-
ferentiation. In vitro differentiation assays revealed that
hybrid cell lines generated by fusion of 4D cells with nor-
mal fibroblasts (4D–T37 hybrids) retained osteogenic dif-
ferentiation, although with variable degrees in different
lines (Table 4, Fig. 5). On the contrary, hybrid cell lines
generated by the fusion of 4D cells with immortal fibro-
blasts (4D–F7 hybrids) failed to differentiate into os-
teogenic lineage except one cell line (4D–T37–C1).
Most of the 4D–T37 hybrid cell lines retained adi-
pogenic differentiation but the degree of fat accumula-
tion was variable among the cell lines (Table 4, Fig. 5).
Adipogenic studies of the 4D–F7 hybrid cell lines could
not be completed because of detachment of cells from
the plastic surface before cellular fat deposition could
be observed.
Unusual cellular proliferation and bioactivity of
conditioned medium
It should be noted that the confluent monolayer of
4D–F7 hybrid cells could be kept in culture for weeks
with a regular change of medium. However, when con-
fluent cells were maintained in culture for prolonged pe-
riods, the renewal of medium was difficult because of de-
tachment of cells from the sides of culture flasks. This
ISLAM ET AL.
912
FIG. 3. Phase-contrast photomicrographs of the parental
mouse normal fibroblast T37 cells (A), cells of mouse MSC 4D
cell line (B), hybrid cell lines 4D–T37–A (C), 4D–F7–B (D),
4D–T37–C (E), and 4D–F7–E (F). Note the increased number
of mitotic cells in hybrid cells compared to the parental 4D
cells. Bars, 100 m.
FIG. 4. Immunophenotypic profile of mouse MSC 4D cell
line and 4D–T37 and 4D–F7 hybrid cells. (Left panel)
4D–hTERT (an hTERT introduced cell line); (middle panel)
4D–T37–C; and (right panel) 4D–F7–5. Flow cytometry his-
tograms demonstrate the expression of Sca-1, CD29, CD44, and
CD49e and lack of expression or weak expression of CD34,
CD45, CD49d, CD90.2, and CD117 (see text for detail de-
scription of markers). Note the consistency of retention of cell-
surface antigen expression of the 4D cells after fusion of these
cells with both normal and immortal fibroblast cells.
169
happened particularly when the acidic old medium was
replaced by alkaline new medium (Fig. 6A). The de-
tached cell sheets frequently folded into multiple cell lay-
ers, showing brownish to blackish colors (Fig. 6B), and
eventually the empty space created on the plastic surface
because of cell detachment was covered by new cell di-
vision from the side of the folded cells (Fig. 6C). Occa-
sionally, the detached cells never reattached on plastics
and they eventually died. On the contrary, the confluent
culture of 4D–T37 hybrid cells never detached, even if
the medium was not changed regularly. On one occasion,
we found a culture flask of the hybrid cell line 4D–T37–J
where medium was not renewed at least for 115 days.
Although the cells were in the nondividing state, they still
MOUSE MSC HYBRIDS
913
T
ABLE
3. Q
UANTITATIVE
K
INETIC
A
SSAY OF
ALP
IN THE
H
YBRID
C
ELL
L
INES
G
ENERATED
BY
F
USION OF
M
OUSE
MSC
S WITH
P
RIMARY AND
I
MMORTAL
M
OUSE
F
IBROBLASTS
Remaining Remaining Remaining ALP
Total Total ALP/ ALP after ALP after after 10 mM
protein ALP protein 56°C, 15 min 65°C, 10 min
L
-phenylalanine
Cell strains/lines (mg/L) (U/L) (mU/mg) (%) (%) (%)
Parents
F7
a
6431 78 12 18 2 80
LTC-4D
b
611 5 8 28 0 76
T37
c
3301 5 2 34 10 85
Hybrids (4DXF7)
4D-F7-C1 3001 119 39 15 0 93
4D-F7-C2 6791 85 12 14 2 77
4D-F7-C3 5031 53 10 18 2 77
4D-F7-C4 4931 10 2 33 9 92
4D-F7-C5 3654 205 56 15 1 76
Hybrids (4DXT37)
4D-T37-A 1171 441 377 11 0 80
4D-T37-B 2101 295 140 10 0 76
4D-T37-C 1371 4 3 27 1 76
4D-T37-D 791 327 413 11 0 77
4D-T37-E 1781 31 17 11 0 81
4D-T37-G 1321 6 4 27 1 79
4D-T37-H 1271 373 293 11 0 76
4D-T37-I 761 19 25 18 0 82
4D-T37-J 1557 56 36 14 0 76
a
F7, Immortal mouse cell line derived from GM05267.
b
LTC-4D, Long-term growing mouse MSC line.
c
T37, Long-term growing adult fibroblasts derived from the mouse strain T37H.
T
ABLE
4. D
IFFERENTIATION
P
OTENTIALS OF THE
M
OUSE
MSC H
YBRID
C
ELL
L
INES
Quantitative score Quantitative score
Cell line of adipogenesis of osteogenesis Remarks
mMSC parent
LTC-4D ϩϩϩ ϩրϪ
4DXF7 hybrids
4D-F7-C1 ϩϩϩPartial adipogenic differentiation
4D-F7-C2 Cell detached during differentiation
4D-F7-C5 Cell detached during differentiation
4DXT37 Hybrids
4D-T37-C ϩϩϩ ϩրϪ
4D-T37-E ϩ ϩրϪ
4D-T37-H Ϫ ϩրϪ
4D-T37-J ϩϩϩ ϩրϪ
170
looked healthy (Fig. 7A). These post-mitotic hybrid cells
when subcultured in standard DMEM containing 15%
FBS did not show any mitotic activity (Fig. 7B). How-
ever, subculturing of these cells at low cell densities with
DMEM containing 15% FBS supplemented with 20%
conditioned medium derived from the hybrid cell line
4D–F7–C5 reinitiated mitotic activities (Fig. 7C,D). In
contrast, the conditioned medium of 4D–T37 hybrid cells
failed to show same effect (not shown).
DISCUSSION
We have generated two series of intraspecies somatic
cell hybrids by fusing the mouse MSC cell line 4D with
the immortal mouse fibroblast cell line F7 and primary
fibroblast strain T37. Hybrid cell lines derived from the
fusion of immortal F7 cell line were rapidly proliferating
with immortal growth potential, and the T37-derived hy-
brid cell lines were moderately proliferating with an ex-
tended life span. Previously, we generated interspecies
pig–mouse hybrid cell lines with indefinite growth by
fusing the F7 cell line with primary pig MSCs (23) and
primary pig fibroblasts (M.Q. Islam, V. Panduri, and K.
Islam, in preparation). These results demonstrate that the
fusion of the immortal F7 cell line with various primary
cells consistently produces hybrid cells with immortal
growth. Significantly, there are reports indicating that fu-
sion of immortal cells of unlimited proliferation and nor-
mal cells of limited proliferation often produce hybrid
cells of limited proliferation (34,35). In this respect, the
F7 line is a unique immortal cell line that not only can
produce immortal hybrid cells but also contains an ex-
cessive number of chromosomes derived from normal
ISLAM ET AL.
914
FIG. 5. Differentiation of mouse MSC 4D cells and derived hybrid cell lines. Induced adipogenic differentiation of parental
4D cell line (A), hybrid cell line 4D–T37–J (B), and hybrid cell line 4D–T37–E (C). Induced osteogenic differentiation of parental
4D–hTERT cell line (D), hybrid cell line 4D–T37–J (E), and hybrid cell line 4D–T37–E (F).
171
cell. It is interesting to note that the karyotype of the F7
cell line contains as many as seven single-copy auto-
somes (23) and at least six of these chromosomes are
known to carry imprinted genes that are normally ex-
pressed from one of the two alleles (36). We suggested
earlier that the F7 cells might have achieved the rapid
proliferative capacity by accumulating imprinted gene(s)
with growth-promoting effects, located on one or more
single-copy chromosomes, through the loss of antago-
nistically acting growth suppressive imprinted gene(s),
located on the missing chromosome(s) (23). Several re-
ports indicate that fusion between two normal cell types
of limited proliferation commonly produces hybrid cells
of only limited proliferation (33,37,38). Although the 4D
cell line has been growing in culture for an extended pe-
riod and carrying polyploid chromosome complements,
this cell line may not be truly immortal. This is probably
why the fusion of 4D cells with primary T37 cells pro-
duces hybrid cells of extended proliferation instead of in-
definite proliferation (33).
Cytogenetic analysis of the hybrid cell lines revealed
that they contained variable chromosome contents. Al-
though the chromosome numbers of the F7-derived hy-
brid cell lines ranged from 88 to 117, the chromosome
numbers of T37-derived hybrid cell lines ranged from
111 to 145. The presence of two sets of marker chromo-
somes from the 4D cell line and one set of marker chro-
mosomes from the F7 cell line in the metaphases of hy-
brid cell lines indicates that they were produced by the
fusion of tetraploid 4D cells with hypo-diploid F7 cells.
Cytogenetic analyses of 4D–T37 hybrid cell lines re-
vealed that most of them were generated either by the fu-
sion of tetraploid 4D cells with diploid of T37 cells or
by the fusion of tetraploid 4D and tetraploid T37 cells.
To discriminate the parental origin of chromosomes in
4D–T37 hybrid cell lines, we fused primary mouse fi-
broblasts derived from a female mouse of strain T37, car-
rying reciprocal translocations involving chromosome 4
and X, with 4D cells carrying an XXYY sex chromo-
some composition. The use of the T37 fibroblast cells al-
lowed us to determine indirectly the genomic contribu-
tion of parental cells in the resultant hybrids (2:1 or 2:2
hybrids) by counting the copy numbers of the t(4;X)
marker chromosome and normal X and Y chromosomes
(Table 2). Taken together, our cytogenetic data indicate
that the 4D–T37-derived hybrid cell lines were chromo-
somally more diverse than the 4D–F7-derived hybrid cell
lines. It should be noted that the fusion of mouse ES cells
and MSCs with various somatic cell types frequently pro-
duces hybrid cells with near-diploid chromosome num-
bers (17,18,39–41). This observation has led to the sug-
gestion that the hybrid cells undergo a reduction division
resulting the loss of nearly a diploid set of chromosomes
(18,42). Interestingly, hybrid cells containing tetraploid
chromosomes, without the loss of chromosomes from any
parent, have also been generated by fusing embryonic
and adult stem cells with somatic cells (43–51).
Because the normal MSCs produce diffusible growth
stimulatory factors in the culture medium (10,52–54), we
saved the conditioned medium of mouse 4D-derived hy-
brid cells and tested their ability to promote the prolifer-
ation of other cells. Although the conditioned medium of
4D–F7 hybrids retained full capacity to promote prolif-
eration of other cells, the conditioned medium derived
MOUSE MSC HYBRIDS
915
FIG. 6. Phase-contrast photomicrographs of abnormal growth
phenotype of hybrid cell line 4D–F7–C5. Note that cells reach-
ing the confluent state tended to detach from the plastic and
eventually formed a multilayered cell sheet (A), reinitiating cell
growth from the side of multilayered cells (B), and reaching
confluency upon renewal of medium (C). Bars, 100 m.
172
from the 4D–T37 hybrid cells was less effective for this
capacity. This may indicate that the immortal F7 cell line
has a special competence to convert the polyploid normal
genome into an immortal state capable of producing
growth-promoting factors in the conditioned medium. This
conclusion is consistent with our recent demonstration that
the fusion of F7 cells with both pig fibroblasts and pig
MSCs generates hybrid cells capable of producing dif-
fusible factors in the conditioned media that can enhance
their own growth as well as growth of other cells (23)
(M.Q. Islam, V. Panduri, and K. Islam, in preparation).
This unique property of the F7 cell line can be applied for
the generation of hybrid cells by fusing this cell line with
various types of normal cells to produce cell type-specific
growth-factor(s) to improve the proliferation of other cells.
By immunophenotyping the hybrid cell lines, we have
demonstrated here that they express common cell-surface
antigens as the parental 4D cells. For example, the
parental 4D cell line and hybrid cell lines were positive
for the makers CD29, CD44, CD49e, and Sca-1 and neg-
ative for the markers CD11b, CD13, CD18, CD31, CD41,
CD45, CD90.2, CD117, CD445R/B220, and GR-1.
These data indicate that the fusion of mouse MSCs with
mouse fibroblast cells does not change the expression pat-
tern of cell-surface antigens of MSCs in the resulting hy-
brid cells. One possible explanation of dominant expres-
sion of cell-surface markers of MSCs by the hybrid cells
is that the parental 4D cells contained more chromosomes
than the fibroblastic parents. Alternatively, hybrid cells
between MSCs and fibroblasts maintained mesenchymal
ISLAM ET AL.
916
FIG. 7. Phase-contrast photomicrographs of hybrid cell line 4D–T37–J grown under nonpermissive conditions for normal cells
and their subsequent recovery with addition of conditioned media derived from the hybrid cell line 4D–F7–C5. (A) 4D–T37–J
cells kept in the same culture flask for 115 days without change of medium remained alive, but without proliferation. (B) Pas-
saging of these cells into a new flask containing DMEM with 15% FBS show no sign of proliferation, at day 4. (C) Cells pas-
saged in a new flask containing DMEM with 15% FBS (80%) and conditioned medium (20%) derived from the hybrid cell line
4D–F7–C5 show no mitotic cells at day 0. (D) The same cells show both mitotic and nonmitotic cells at day 4. (E) The same
cells at day 8 show complete recovery and normal growth. (F) A culture of 4D–T37–J cells maintained in DMEM containing
15% FBS with regular change of medium served as control. Bars, 100 m.
173
cell-surface markers, because both cell types have a com-
mon mesenchymal origin. To our knowledge, this is the
first report demonstrating the expression pattern of cell-
surface antigens by intraspecies mouse hybrid cells us-
ing flow cytometry.
Although the parental mouse cell line 4D is capable of
differentiating into adipogenic and osteogenic lineages
(21), the differentiation capacity is largely altered fol-
lowing the fusion of 4D cells with fibroblasts. For ex-
ample, most of the hybrid cell lines generated by fusion
of 4D cells with immortal fibroblasts (4D–F7 hybrids)
failed to differentiate into either adipogenic or osteogenic
cell lineages. On the other hand, the osteo- and adi-
pogenic differentiation potentials of hybrid cell lines gen-
erated by the fusion of 4D cells with primary fibroblasts
(4D–T37 hybrids) were mostly maintained, but quantita-
tive differences exist among the cell lines. Because most
of the 4D–F7 hybrid cell lines can proliferate ceaselessly,
the failure of differentiation in these hybrid cells may be
caused by lack of cell cycle arrest. Alternatively, this fail-
ure may be due to resetting of the genetic program of
parental genomes during cell fusion through the modifi-
cation of gene expression pattern. The second possibility
is unlikely in light of the observation that one of the hy-
brid cell lines maintained both adipo- and osteogenic dif-
ferentiation, although all hybrid cell lines were derived
from the same cell fusion experiment. These results dem-
onstrate that the fusion between MSCs and fibroblasts
generates hybrid cells with dissimilar differentiation po-
tentials. The phenotypic diversity in our hybrid cell lines
is not unexpected because they were genetically differ-
ent as they contained disparate chromosome content.
Previously, we produced interspecies hybrid cells by
fusing pig MSCs with mouse F7 fibroblast cells where
osteogenic differentiation largely maintained, adipogenic
differentiation diminished, and chondrogenic differenti-
ation was completely lost (23). Collectively, these results
clearly indicate that generation of hybrid cells by fusing
MSCs with other somatic cells does not necessarily mean
that the MSC phenotypes are maintained by the hybrid
cells. In this respect, hybrid cells with contrasting char-
acters could be useful to select cell lines with desirable
traits by screening a large number of cell lines to use
them as therapeutic agents.
Recently, it has been reported that systemic transplan-
tation of various adult stem cells in human and other an-
imal models resulted in spontaneous hybridization with
endogenous cells generating hybrid cells in vivo
(20,55–64). Spontaneously formed hybrid cells through
the fusion of exogenous MSCs with endogenous somatic
cells were found to repair experimentally damaged or-
gans more effectively than the MSCs alone (17–20). In
case of the fumarylacetoacetate hydroxylase-deficient
mouse model, the regenerated livers contained mainly hy-
brid cells (17,18). This may indicate that the hybrid cells
have potentials for improved proliferation compared to
nonfused natural MSCs.
In the present study, we generated intraspecies hybrid
cells through experimentally induced fusion of mouse
MSCs with mouse fibroblasts to discover whether the re-
sulting hybrid cells gain a superior capacity for prolifer-
ation and whether they retain the phenotypes of normal
MSCs, including multilineage differentiation. Our results
demonstrate that the hybrid cells in general maintain bet-
ter proliferative capacity than the parental cells, and they
also express many phenotypes of MSCs. Although the
differentiation phenotypes of parental MSCs were dras-
tically altered following the fusion of these cells with fi-
broblasts, some hybrid cell lines maintained phenotypes
of parental cell differentiation. The nonuniform differen-
tiation capacities of our experimentally induced hybrid
cell lines indicate that the in vivo fusion of transplanted
MSCs with indigenous cells may not produce hybrid cells
with a consistent cell differentiation phenotype.
In the present study, we deliberately used at least one
parental cell line with the capacity of either indefinite cell
growth or extended cell growth to generate proliferating
hybrid cells. In this context, in vitro generation of hybrid
cells by fusing diploid MSCs with diploid primary so-
matic cells would be more similar with the spontaneously
formed hybrid cells in vivo following the fusion of sys-
temically transplanted MSCs with indigenous cells. Ap-
plication of our two-step cell culture protocol, immortal-
ization of adult stem cells by fusing with F7 cells, and
improvement of proliferation of other cells by treatment
of the conditioned medium of F7-derived hybrid cells
would allow the production of a large number of
diploid–diploid hybrid cells, derived from the fusion of
normal somatic cells with normal adult stem cells to eval-
uate the differentiation potential of hybrid cells in vitro
and their regenerative competence in vivo.
ACKNOWLEDGMENT
The authors thank Cecilia Linder for excellent techni-
cal assistance.
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Address reprint requests to:
Dr. M. Quamrul Islam
Laboratory of Cancer Genetics
Department of Biomedicine and Surgery
Division of Clinical Chemistry
Main Building, Floor 11
Faculty of Health Sciences
Linkoping University
S-581 85 Linkoping, Sweden
E-mail: [email protected]
Received September 7, 2006; accepted September 14,
2006.
MOUSE MSC HYBRIDS
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Errata
As referências Jin et al., 2002, e Van Damme et al., 2003, citadas na Tabela 5,
página 270 do artigo Mesenchymal stem cells: isolation, in vitro expansion and
characterization, não aparecem listadas na bibliografia do mesmo. Estas são, em sua forma
completa:
Jin HK, Carter JE, Huntley GW and Schuchman EH (2002) Intracerebral transplantation of
mesenchymal stem cells into acid sphingomyelinase-deficient mice delays the onset of
neurological abnormalities and extends their life span. J Clin Invest 109:1183-1191.
Van Damme A, Chuah MK, Dell'accio F, De Bari C, Luyten F, Collen D and
VandenDriessche T (2003) Bone marrow mesenchymal cells for haemophilia A gene
therapy using retroviral vectors with modified long-terminal repeats. Haemophilia. 2003
Jan;9(1):94-103.
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