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

Abstract

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 O2 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.