Objectives Inducedpluripotent stem cell (iPSC), which demonstrates the stem cell characteristics like clonogenicity, self-renewal and multipotency, could differentiate into all three germ layer cell types including cardiomyocytes. The cytokinecardiotrophin-1, a member of IL-6 family, is highly expressed in the primitive hearttube during the embryonic cardiogenesis. This study aimed to investigate the role of cardiotrophin-1 (CT-1) on cardiac differentiation of mouse iPSC.
Methods MiPSC is cultured according to Yamanaka’s protocol. The differentiation of miPSC was initiated by embryoid bodies formation with classic hanging drop method. CT-1(1 μmol/ml) and vehicle control were administrated at day 3 of the differentiation process. Cells were harvested at day 4, 7, 10 and 14 after differentiation to examine the embryonic and cardiac specific cell markers expression by real-time PCR, immunofluorescence staining, flow cytometry analysis and ultra-microstructure changes by transmission electron microscope.
Results The iPS clone was in compactly oval or round shape with strong three-dimensional sense and distinct boundaries, and was Oct-4 positive in IF staining. The gene expressions of various cardiac specific cell markers in CT-1 group were significantly increased at different time points: Flk-1was increased 1.61 ± 0.03 folds at day 7 and 2.01 ± 0.01 folds at day 10, respectively (P < 0.05). Nkx2.5 was increased 2.08 ± 0.08 folds at day 7, 2.12 ± 0.15 folds at day 10 and 1.99 ± 0.06 folds at day 14, respectively (P < 0.01). Tbx5 was increased 1.61 ± 0.05 folds at day 7 and 1.86 ± 0.04 at day 10, respectively (P < 0.05). Furthermore, cTnT was increased1.75 ± 0.04 folds at day 10 and 1.78 ± 0.05 folds at day 14, respectively (P < 0.05). Flow cytometry analysis showed that cTnI positive cells were significantly increased in the CT-1 group compared to the vehicle group (56.4% vs 28.5%, P<0.05). More organised myofibril, intactmitochondria structure and well-formed cell junction were observed in the CT-1 group as compared with control.
Conclusions In the present study, we determined the role of cardiotrophin-1 on cardiac differentiation of miPSCs and assessed its effect at both molecular and cellular levels. Up-regulation of several cardiac mesodermal/progenitor markers such as Flk-1, Nkx2.5 and Tbx5 were observed in CT-1 group, indicating its enhancement of early cardiac differentiation transcriptional activities. These findings support the role of CT-1in the early stage of embryonic cardiogenesis. In addition, earlier appearance of beating EBs and more mature ultrastructure of iPS-derived cardiomyocytes (iPS-CMs) indicate CT-1 could enhance the maturity of iPS-CMs. In conclusion, CT-1 significantly promotes the cardiac differentiation of miPSCs as well asiPS-CM maturity and these findings provide a potent evidence for iPSC-based cardiac cell therapy of myocardial infarction or congestive heart failure.