Overexpression of growth factors to improve cardiac differentiation of human mesenchymal stem cells derived from the amniotic membrane

Purpose: Mesenchymal stem cells (MSC) derived form human amniotic membrane (A-MSC) differentiate into cardiomyocyte (CMC) more promptly than bone marrow-derived MSC (BM-MSC). Still, the differentiation efficiency is not very high. We found that BM-MSC, particularly from elderly patients, express lower amount of several proteins potentially involved in cardiac differentiation compared with A-MSC. We hypothesized that by overexpressing these factors, cardiac differentiation efficiency of A-MSC may further increase. Methods: Human A-MSC were transduced with a lentiviral vector encoding for either GFP alone (GFP-A-MSC control cells), or TGFβ1 (TGFβ1-A-MSC), or BMP2 (BMP2-A-MSC), or IGF-I (IGF-I-A-MSC), or FGF2 (FGF2-A-MSC). To confirm transduction efficiency, after 48 hours the cells were analyzed by FACS, Western Blot (WB), ELISA and immunocytochemistry (ICC). The cells were then maintained in culture for 5 weeks and analyzed by qPCR, WB and ICC at two different time points: 2 weeks, to search for expression of early cardiac genes (GATA4, Nkx2.5 and Tbx5), and 5 weeks, to verify acquisition of cardiac-specific proteins (Cx43, cTnT and α–MHC). Results: The transduction efficiency with all four lentiviral vectors was higher than 90%. WB, ELISA and ICC confirmed the over-expression of the protein of interest. Compared with GFP-A-MSC, the over-expression of each factor triggered the expression of cardiac markers. qPCR and ICC showed the over-expression of GATA4 and Nkx2.5 in IGF-I-A-MSC and of Tbx5 in TGFβ1-A-MSC and FGF2-A-MSC. qPCR also documented that IGF-I and FGF2 significantly increased the expression levels of Cx43 (+15.8 and +9.5 vs GFP-A-MSC respectively, p<0.05). Moreover, compared with GFP-A-MSC, both IGF-I and BMP2 significantly increased the expression level of cTnT (+6.02 and +12.3 respectively, p<0.05) and αMHC (+10.6 and +5.9 respectively, p<0.05). WB confirmed the acquisition of late cardiac genes also at protein level. Finally, ICC showed that IGF-I promoted the localization of Cx43 on the membrane of the IGF-I-A-MSC, while BMP2, TGFβ1 and FGF2 increased Cx43, but the distribution in the cells resulted perinuclear. Conclusions: We have shown that the overexpression of either IGF-I, or BMP2, or TGFβ1 or BMP2, significantly increases the differentiation efficiency of A-MSC into CMC. In particular, IGF-1 and BMP2 seem to be the most relevant cardiac differentiation triggers. Their combined use may result in further synergistic effect and improve cardiac regeneration capacity of MSC.