Publicação
Mesenchymal stem cell-based differentiation of smooth muscle cells
| Resumo: | Valvular heart disease is a major health and socioeconomic problem worldwide with approximately 300 000 valve replacements performed annually. Tissue-engineered heart valves with repair and remodelling capabilities could overcome the limitations of today’s valvular prostheses. One major limitation to this approach has been finding a reliable source of smooth muscle cells (SMC) because biopsies of these cells can be impractical and morbid as also present limited replicative capacity. The ideal cell source should be harvested in a non-or minimally invasive way and should deliver an initial high number of cells in order to drastically reduce the time needed for cell expansion. For these reasons there are many studies endeavoured to explore whether functional SMC could be generated from various types of adult mesenchymal stem cells (MSC): Umbilical cord (UC-MSC), bone marrow (BM-MSC), adipose derived (AD-MSC) and chorionic villi (CV-MSC) as possible sources for heart valve therapy. The MSC from different sources were isolated and expanded of healthy and different donor. In this study an isolation protocol was established for the CV-MSC, because it was never performed on this research group. Since the CV-MSC are barely reported in studies, this MSC source was characterized by flow cytometry to compare with other sources that are well-characterized in literature. After that, the MSC were differentiated by culture them in a culture medium containing transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 4 (BMP4), based on a published 7-day differentiation protocol. The differentiation was analysed using 4 different smooth muscle markers (α-SMA, SM22α, Calponin and SM-MHC) by immunofluorescence (IF) and immunohistochemistry (IHC). A more extensively analysis was performed by flow cytometry using the smooth muscle markers and the other markers used on different studies to characterize the MSC population for a complete phenotype characterization before and after differentiation. The IF showed promising results as the smooth muscle markers stained positive for the differentiated MSC and negative for the undifferentiated MSC. On the other hand, the IHC and flow cytometry show some contradicting results for differentiation, since the expression is not consistent within the undifferentiated and differentiated MSC. These results highlight the concept that MSC represent an easily accessible, novel cell source for heart valve therapy, but despite of the wide experiments and results in this work, it is necessary further research in this field due to the conflicting evidence and inadequate information about several cell surface markers. |
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| Autores principais: | Amaral, Luis Manuel Fonseca |
| Ano: | 2014 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Valvular heart disease is a major health and socioeconomic problem worldwide with approximately 300 000 valve replacements performed annually. Tissue-engineered heart valves with repair and remodelling capabilities could overcome the limitations of today’s valvular prostheses. One major limitation to this approach has been finding a reliable source of smooth muscle cells (SMC) because biopsies of these cells can be impractical and morbid as also present limited replicative capacity. The ideal cell source should be harvested in a non-or minimally invasive way and should deliver an initial high number of cells in order to drastically reduce the time needed for cell expansion. For these reasons there are many studies endeavoured to explore whether functional SMC could be generated from various types of adult mesenchymal stem cells (MSC): Umbilical cord (UC-MSC), bone marrow (BM-MSC), adipose derived (AD-MSC) and chorionic villi (CV-MSC) as possible sources for heart valve therapy. The MSC from different sources were isolated and expanded of healthy and different donor. In this study an isolation protocol was established for the CV-MSC, because it was never performed on this research group. Since the CV-MSC are barely reported in studies, this MSC source was characterized by flow cytometry to compare with other sources that are well-characterized in literature. After that, the MSC were differentiated by culture them in a culture medium containing transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 4 (BMP4), based on a published 7-day differentiation protocol. The differentiation was analysed using 4 different smooth muscle markers (α-SMA, SM22α, Calponin and SM-MHC) by immunofluorescence (IF) and immunohistochemistry (IHC). A more extensively analysis was performed by flow cytometry using the smooth muscle markers and the other markers used on different studies to characterize the MSC population for a complete phenotype characterization before and after differentiation. The IF showed promising results as the smooth muscle markers stained positive for the differentiated MSC and negative for the undifferentiated MSC. On the other hand, the IHC and flow cytometry show some contradicting results for differentiation, since the expression is not consistent within the undifferentiated and differentiated MSC. These results highlight the concept that MSC represent an easily accessible, novel cell source for heart valve therapy, but despite of the wide experiments and results in this work, it is necessary further research in this field due to the conflicting evidence and inadequate information about several cell surface markers. |
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