Combination of rock inhibitor, hypoxia and melatonin improved differentiation of rabbit induced pluripotent stem cells into cardiac progenitor cells

Cardiac progenitors are a promising cell source for treating myocardial infarction. In this study, we improved the protocol for cardiac differentiation of rabbit induced pluripotent stem cells (iPSC) using Rho-associated protein kinase (ROCK) inhibitor (Y-27632), low O-2 tension (hypoxia) and melatonin treatments. In experiment 1, the rabbit iPSCs were differentiated into cardiac cell fate via embryoid body (EB) formation with or without ROCK inhibitor. EB diameters were measured on day 3 of differentiation. In experiment 2, the EBs were plated on gelatin-coated dishes and further cultured in different oxygen tensions, hypoxia (5% oxygen) and normoxia (20% oxygen). The plated EBs were examined for proliferative activity and the production of reactive oxygen species (ROS). Experiment 3 studied the effects of oxygen tensions and melatonin on the differentiation of cardiac cell fate in terms of cardiac progenitor gene expression (NKX2.5) and FLK1 positive cells. ROCK inhibitor significantly improved EB formation by mean of increased EB diameter (P<0.05) compared with the control. Hypoxia also significantly increased the numbers of newly DNA synthetic cells indicating greater proliferative activity when compared with normoxia (P<0.05). The melanin treatment during iPSCs differentiation significantly decreased ROS production only in hypoxia (P<0.05). In addition, the combination of hypoxic condition and melatonin treatment significantly upregulated a NKX2.5 cardiac progenitor gene and FLK1 positive cells compared with the controls and normoxia-melatonin treatment (P<0.05). It is concluded that an optimizing condition using a combination of ROCK inhibitor, hypoxic condition and melatonin improved differentiation of rabbit iPSCs towards cardiac progenitor cells.