ogenic differentiation of hATMSCs. The expressions of the three cardiac specific proteins were used to evaluate the cardiomyogenic differentiations of hATMSCs. The results of group D (CMCM + 1.0 μM S1P) were presented. On the 7th day, the cells began to express α-actin, connexin-43 and MYH-6, but the fluorescence intensities were weak. At the 14th day, most of the cells expressed three specific proteins and the fluorescence intensities were stronger than those on the 7th day. The expressions of cardiac specific proteins on the 28th day were similar to those on the 14th day, but most of the cells were dead and only a small number of them remained alive. The bars are 100 μm Figure 3. Influence of S1P concentration on the differentiation of hUCMSCs toward cardiomyocytes in induction media. This experiment was divided into six groups, including CMCM (group A), CMCM + 0.1 μM S1P (group B), CMCM + 0.5 μM S1P (group C), CMCM + 1.0 μM S1P (gorup D), DMEM + 5-azacytidine (group E) and DMEM + 5-azacytidine + 0.5 μM S1P (group F). The differentiations were examined by immunofluorescence staining of cardiac specific proteins, and the results were evaluated by confocal microscopy. All the differentiated cells in the six groups expressed three cardiac specific proteins (α-actin, connexin-43 and MYH-6), but the fluorescence intensities from these expressions were different. The bars are 20 μm Figure 4. Influence of S1P concentration on the differentiation of hATMSCs toward cardiomyocytes in induction media. This experiment was divided into six groups, including CMCM (group A), CMCM + 0.1 μM S1P (group B), CMCM + 0.5 μM S1P (group C), CMCM + 1.0 μM S1P (gorup D), DMEM + 5-azacytidine (group E) and DMEM + 5-azacytidine + 0.5 μM S1P (group F). The differentiations were examined by immunofluorescence staining of cardiac specific proteins, and the results were evaluated by confocal microscopy. All the differentiated cells in the six groups expressed three cardiac specific proteins (α-actin, connexin-43 and MYH-6), but the fluorescence intensities from these expressions were different. The bars are 20 μm 167 s1p stimulates cardiac differentiation to the function of S1P. Meanwhile, the expression of MYH-6 increased with the concentration of S1P and reached the peak value in CMCM + 0.5 μM S1P group (Figure 3). Addition of S1P thus made significant differences compared with group A as in the case of the other two proteins ( Figure 5C; p < 0.05). In contrast, addition of 5-azacytidine with and without S1P in DMEM produced lower expressions of MYH-6 than those in group A, a trend similar to those observed with the other two proteins, but there was no significant difference compared with group E (5-azacytidine) ( Figure 5C; p < 0.05).
For hATMSCs, whether in CMCM groups or 5azacytidine groups, the expressions of the three cardiac specific proteins were all enhanced by S1P and increased with its concentration (Figure 6), although the extent of increase varied and was different from the respective case as observed in hUCMSCs. In the CMCM groups, addition of 1 μM S1P led to substantial increases in α-actin and MYH-6. In the 5-azacytidine groups, the levels of expressions of all three proteins were similar to their respective ones in group E (5-azacytidine), but addition of 0.5 μM S1P led to increased expressions of conexion-43 and MYH-6 ( Figure 6; p < 0.05).

Influence of S1P concentration on cell activity
The influences of S1P concentrations on cell activities of hUCMSCs and hATMSCs were assessed by MTT assays and are shown in Figures 7 and 8. For the CMCM series in groups A-D, cell activities decreased with time (as assayed on the 7th and 14th days) and decreasing S1P concentration. There were significant differences compared with the control (group A) (p < 0.05). In 5-azacytidine groups (groups E and F), however, the trends are opposite as far as time and 5-azacytidine are concerned, i.e. cell activities increased with time and addition of 5-azacytidine, although the results did show the same trend of decrease of cell activity with addition of S1P (p < 0.05). In the DMEM systems (groups E and F) increase in cell viability with time because of DMEM acting as a popular cell growth medium. It was observed that cell activities cultured in DMEM were higher than those cultured in CMCM. CMCM is not a proliferation medium but a differentiation medium, hence explaining why the viabilities of the cells cultured in CMCM with or without S1P on the 14th day were lower than those on the 7th day. Figure 5. Fluorescence intensity analysis of the three cardiac specific proteins expressed in the six groups for hUCMSCs. Expressions of α-actin and connexin-43 were enhanced with increasing S1P concentration whilst that of MYH-6 showed an initial increase with the concentration of S1P and then declined, reaching the peak value in CMCM + 0.5 μM S1P group. The fluorescence intensities were analysed using the software Image J. The data were expressed as mean ± SD (n = 3). Asterisk indicates statistically significant (p < 0.05) difference of fluorescence intensity compared with CMCM group or 5-azacytidine group Figure 6. Fluorescence intensity analysis of the three cardiac specific proteins expressed in the six groups for hATMSCs. The expressions of the three cardiac specific proteins were all enhanced with increasing S1P concentration, whether in CMCM groups or in 5-azacytidine groups. The fluorescence intensity was analysed using the software Image J. The data were expressed as mean ± SD (n = 3). Asterisk indicates statistically significant (p < 0.05) difference of fluorescence intensity compared with CMCM group or 5-azacytidine group Figure 7. Cell activity assays in different groups of hUCMSCs by MTT. The experiment was divided into seven groups, including CMCM (group A), CMCM + 0.1 μM S1P (group B), CMCM + 0.5 μM S1P (group C), CMCM + 1.0 μM S1P (group D), DMEM + 5-azacytidine (group E), DMEM + 5azacytidine + 0.5 μM S1P (group F) and DMEM (control group). Cell activities of hUCMSCs decreased with increasing S1P concentration according to the results of groups A-D on both 7th and 14th days. In groups A-D, the cell viability on the 14th day was lower than that on the 7th day. In group E and group F, S1P significantly reduced the cell viability. In group A and control group, cell activity cultured in DMEM was higher than that cultured in CMCM. The data were expressed as mean ± SD (n = 3). Aterisk indicates statistically significant (p < 0.05) difference of cell activity compared with CMCM group. Number sign indicates statistically significant (p < 0.05) difference of cell activity compared with 5-azacytidine group  S1P (group B), CMCM + 0.5 μM S1P (group C), CMCM + 1.0 μM S1P (group D), DMEM + 5-azacytidine (group E), DMEM + 5azacytidine + 0.5 μM S1P (group F) and DMEM (control group). Cell activity of hATMSCs decreased with increasing S1P concentration according to the results of groups A-D on both 7th and 14th days. In groups A-D, the cell viability on the 14th day was lower than that on the 7th day. In groups E and F, S1P significantly reduced cell viability. In group A and control group, cell activity cultured in DMEM was higher than that cultured in CMCM. The data were expressed as mean ± SD (n = 3). Asterisk indicates statistically significant (p < 0.05) difference of cell activity compared with CMCM group. Number sign indicates statistically significant (p < 0.05) difference of cell activity compared with 5-azacytidine group Effect of S1P on calcium transient Figure 9 shows the electrophysiological features of the cells from the four groups including group A (CMCM), group C (CMCM + 0.5 μM S1P), group E (5-azacytidine) and group F (5-azacytidine + 0.5 μM S1P). The results showed that the calcium transient happened only in the cells from group C, while the phenomenon could not be observed in other groups (data were shown in Figures S2 and S3 of Supporting Information).

DISCUSSION
MSCs are considered as sources of seed cells in tissue engineering, as they can be obtained from a wide variety of tissues, including bone marrow, adipose tissue, cord blood, placenta and peripheral blood. [19][20][21] Recent studies have shown that umbilical cord and adipose tissue are two most attractive sources of MSCs 22-24 as they are easy to obtain. This would change the current situation of treating them as medical wastes. A further benefit is that their future applications are not subject to