| Due to their multipotential, available in large numbers and easily accessible, human adipose-derived mesenchymal stem cells (AD-MSCs) and human umbilical cord mesenchymal stem cells (UC-MSCs) have become an attractive stem cell source for cell therapy and tissue regeneration. More than one month is needed for cell therapy, and time plays an important role in cell therapy. Further more, cell viability of MSCs in transplanted area is another key factor for improving cell therapy. However, bad transplanted environment (such as ischemia, oxidative stress, inflammatory reaction) and contact inhibition, which cause abundant death or apoptosis of MSCs, are unfavorable for application of cell therapy. Therefore, it is needed to find a suitable source of MSCs possessing more tolerance to the contact inhibition for improving the cell therapy. In addition, UC-MSCs and AD-MSCs have the potential to differentiate into cardiomyocytes and can express cardiomyocytes specific proteins, making them promising therapeutic candidates for treating damaged cardiac tissues. However, the differentiated cells induced from UC-MSCs or AD-MSCs by5-azacytidine or cardiomyocytes culture medium can hardly display functional characteristics similar to cardiomyocytes. Therefore, it is important to find a bioactive factor combining with cardiac differentiation medium for UC-MSCs or AD-MSCs to improve their functional cardiomyogenic differentiation.In this study, the growth and cell viability of AD-MSCs and UC-MSCs were investigated by CCK-8kits and hemocytometer everyday in30days of culture. Live/Dead staining and flow cytometry (FCM) were used to evaluate the cell viability of AD-MSCs and UC-MSCs during one month of culture. The results showed that AD-MSCs could be cultured up to the30th day in one passage while maintaining high level cell viability, and were seemed to be more tolerance to the contact inhibition. In addition, the cells displayed two plateau phases and three logarithmic phases during one month of culture. While UC-MSCs were not tolerance,to the contact inhibition, they couldn’t maintain high level cell viability in whole culture. Their cell viability significantly declined in later period of one month of culture. And the grow curves of UC-MSCs showed that they only displayed one logarithmic phase then went into the plateau phase. The cell numbers and cell viability of UC-MSCs kept mostly unchanged in the late of one month of culture. As a cell source of transplanting for clinical implication, the cells are required to not only maintain high cell viability, but also maintain their stem cell characteristics and normal chromosome type. The stem cell characteristics of AD-MSCs at the beginning and the end of culture were detected by flow cytometry (FCM) and histochemical staining. In addition, G banding karyotype analysis was used to evaluate biosecurity of AD-MSCs after one month of culture. The results showed that AD-MSCs could maintain their stem cell characteristics up to the end of one month of culture, including the capacity of mutilineage differentiation and the expression of hADSCs relative surface marker proteins. The results of G banding karyotype analysis showed that AD-MSCs after one month of culure still possessed a normal female chromosome type with no chromosome abnormalities being observed. This study demonstrates that AD-MSCs can be used as seed cells for application of cell therapy.In adition, to investigate the control factors of hADSCs growth during one month of culture, we detected the cell cycle distribution profiles by FCM using propidium iodide (PI) staining of DNA content, and the expression of cell cycle regulators including cyclin D1and cyclin A by western blotting. The results showed that increasing expression of cyclin A at protein level resulted in an increase in the percentage of AD-MSCs in the S and G2/M phases, promoting cells to go into logarithmic phases. Meanwhile, increasing or decreasing expression of cyclin D1at protein level caused a rise or decline in the proportion of AD-MSCs in the S phase, regulating cells to move into rapid proliferation or plateau phases. In conclusion, cyclin A and cyclin D1affect the distribution of cell cycle and regulate the growth of AD-MSCs.In this study, we have investigated the effects of sphingosine-1-phosphate (SIP) on cardiomyogenic differentiations of UC-MSCs and AD-MSCs in cardiomyocyte culture medium (CMCM) and5-azacytidine in vitro. Cardiomyogenic differentiations were identified through immunofluorescence staining, and the results were observed with fluorescence microscopy and confocal microscopy. The effects of respective combinations of SlP-5-azacytidine and S1P-CMCM on cell viability were evaluated by methyl thiazolyl tetrazolium (MTT) assays. Functional characteristics similar to cardiomyocytes were evaluated through detecting calcium transient. The results showed that the differentiations towards cardiomyocytes of UC-MSCs or AD-MSCs in CMCM were enhanced with SIP concentration increasing, but cell activities declined. Therefore, our data showed that the suitable differentiation time was around14days, and the optimal concentration of SIP was0.5μmol/L in CMCM differential medium. Moreover, S1P working together with CMCM can not only enhance the differentiations of UC-MSCs or AD-MSCs into cardiaomyocytes, but also promote their differentiations towards functional cardiomyocytes, generating the calcium transients from the induced cardiomyocytes. Wherase SIP with5-azacytidine only enhanced the expressions of the three cardiac specific proteins but could not give rise to the specific electrophysiological properties (the calcium transient) in the committed UC-MSCs or AD-MSCs. This difference could be due to some cellular stimulus (e.g. transforming growth factor-beta, TGF-β) existing in CMCM but not in5-azacytidine induction medium. |
PDF Full Text Request