Potencial de diferenciação induzido e espontâneo de células tronco mesenquimais oriundas de tecido adiposo e medula óssea de equinos (Equus caballus)
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Data
2016-07-29
Autores
Galhardo, Elaine Cristina [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A medula óssea (MO) e o tecido adiposo (TA) são fontes viáveis e amplamente estudadas de células tronco mesenquimais (CTMs), cujo alto potencial de multiplicação e diferenciação, aliado à característica imunomoduladora, permitem ampla aplicação em Medicina Veterinária com resultados satisfatórios na reconstrução de tecidos danificados. Diversos protocolos são descritos para isolamento e cultivo de CTMs, com divergência na porcentagem de suplementação de soro fetal bovino (SFB), componente xenogênico que é indicado como possível facilitador do processo de diferenciação in vitro. Este estudo analisou a proliferação e diferenciação induzida e não induzida de CTMs-TA e MO cultivadas em 10 ou 20% de SFB, e demonstrou que CTMs cultivadas em 20% de SFB apresentam maior eficiência de proliferação in vitro, e que a proliferação in vitro das CTMs-TA são superiores em comparação à CTMs-MO. O potencial de diferenciação das CTMs-TA e MO foi caracterizado por análise citológica e ultraestrutural após ensaio de diferenciação induzido por meios comerciais específicos e ensaio não induzido, onde as CTMs foram mantidas apenas nos meios basais. Esses ensaios foram também submetidos a avaliação de expressão gênica por qRT-PCR, porém estes dados não foram conclusivos. Aparente formação de matriz extracelular nas CTMs-TA cultivadas em 10% de SFB e não induzidas a diferenciação sugere possível ocorrência de diferenciação espontânea nesse grupo, o que foi evidenciado pela análise ultraestrutural. Adicionalmente, foi realizada caracterização imunofenotípica das CTMs-TA, com o objetivo de construir um banco de células aplicáveis in vivo. Em conclusão, a importância do SFB para o cultivo de CTMs foi demonstrada pelos resultados de proliferação superiores em células cultivadas em 20% de SFB, bem como a superior proliferação das CTMs-TA. A concentração de 10% de SFB no meio de cultivo pode favorecer a ocorrência de diferenciação espontânea das CTMs-TA, conforme demonstrado pela análise ultraestrutural. O isolamento, cultivo e caracterização de CTMs-TA foram eficientes e possibilitaram a formação de um banco de células viáveis para aplicação in vivo.
Bone marrow (BM) and adipose tissue (AT) are viable sources of mesenchymal stem cells (mSCs), which high potential for proliferation and differentiation, in addition to immunomodulatory property allows wide application in Veterinary Medicine, obtaining satisfactory results in reconstruction of damaged tissues. Several protocols are described for isolation and culture of mSCs, although with divergences in percentage of fetal calf serum supplementation (FCS), a xenogenic component indicated as a possible factor for in vitro differentiation process. This study examined the proliferation, induced and non-induced differentiation of mSCs from AT and BM cultured in 10 or 20% FBS, and demonstrated that mSCs cultured in 20% FBS show higher in vitro proliferation efficiency, and proliferation in vitro of MSC-AT are higher compared to mSCs-BM. The differentiation potential of mSCs-AT and BM were characterized by cytological and ultrastructural analysis after differentiation assay induced by specific commercial media and not induced assay, where mSCs were maintained only in the basal media. These assays were submitted also to gene expression analysis by qRT-PCR, but these data were not concluded. Apparent formation of extracellular matrix in mSCs-AT cultured in 10% FBS and non-induced to differentiation were observed, which suggests possible occurrence of spontaneous differentiation in this group. Additionally, immunophenotypic characterization was performed of MSCs-AT, with the aim of mantain a cell bank to in vivo application. During the process of induced differentiation into adipogenic and osteogenic lineages, the mSCs-AT and BM presented morphological and physiological changes; acquire characteristics similar to the preadipocytes and osteoblasts, which did not occur when mSCs were not exposed to the inducing differentiation media. The concentration of 10% FBS in the culture medium may induce the occurrence of spontaneous differentiation of mSCs-AT, as demonstrated by ultrastructural analysis, the concentration of 20% FBS in the culture medium promotes cell proliferation of mSCs from both sources. Isolation, culture and characterization of mSCs-AT were efficient and allowed the construction of a cell bank viable for in vivo application.
Bone marrow (BM) and adipose tissue (AT) are viable sources of mesenchymal stem cells (mSCs), which high potential for proliferation and differentiation, in addition to immunomodulatory property allows wide application in Veterinary Medicine, obtaining satisfactory results in reconstruction of damaged tissues. Several protocols are described for isolation and culture of mSCs, although with divergences in percentage of fetal calf serum supplementation (FCS), a xenogenic component indicated as a possible factor for in vitro differentiation process. This study examined the proliferation, induced and non-induced differentiation of mSCs from AT and BM cultured in 10 or 20% FBS, and demonstrated that mSCs cultured in 20% FBS show higher in vitro proliferation efficiency, and proliferation in vitro of MSC-AT are higher compared to mSCs-BM. The differentiation potential of mSCs-AT and BM were characterized by cytological and ultrastructural analysis after differentiation assay induced by specific commercial media and not induced assay, where mSCs were maintained only in the basal media. These assays were submitted also to gene expression analysis by qRT-PCR, but these data were not concluded. Apparent formation of extracellular matrix in mSCs-AT cultured in 10% FBS and non-induced to differentiation were observed, which suggests possible occurrence of spontaneous differentiation in this group. Additionally, immunophenotypic characterization was performed of MSCs-AT, with the aim of mantain a cell bank to in vivo application. During the process of induced differentiation into adipogenic and osteogenic lineages, the mSCs-AT and BM presented morphological and physiological changes; acquire characteristics similar to the preadipocytes and osteoblasts, which did not occur when mSCs were not exposed to the inducing differentiation media. The concentration of 10% FBS in the culture medium may induce the occurrence of spontaneous differentiation of mSCs-AT, as demonstrated by ultrastructural analysis, the concentration of 20% FBS in the culture medium promotes cell proliferation of mSCs from both sources. Isolation, culture and characterization of mSCs-AT were efficient and allowed the construction of a cell bank viable for in vivo application.
Descrição
Palavras-chave
Terapia celular, Diferenciação celular, Ultraestrutura, Soro fetal bovino, Cell therapy, Cell differentiation, Ultrastructure, Fetal bovine serum