Rho-Associated Kinases and Non-muscle Myosin IIs Inhibit the Differentiation of Human iPSCs to Pancreatic Endoderm

Abstract

Summary: There has been increasing success with the generation of pancreatic cells from human induced pluripotent stem cells (hiPSCs); however, the molecular mechanisms of the differentiation remain elusive. The purpose of this study was to reveal novel molecular mechanisms for differentiation to PDX1+NKX6.1+ pancreatic endoderm cells, which are pancreatic committed progenitor cells. PDX1+ posterior foregut cells differentiated from hiPSCs failed to differentiate into pancreatic endoderm cells at low cell density, but Rho-associated kinase (ROCK) or non-muscle myosin II (NM II) inhibitors rescued the differentiation potential. Consistently, the expression of phosphorylated myosin light chain 2 and NM IIA was downregulated in aggregation culture. Notably, the soluble factors we tested were substantially effective only with ROCK-NM II inhibition. The PDX1+NKX6.1+ cells induced with NM II inhibitors were successfully engrafted and maturated in vivo. Taken together, these results suggest that NM IIs play inhibitory roles for the differentiation of hiPSCs to pancreatic endoderm cells. : Toyoda and colleagues show that ROCK or NM II inhibitors facilitate differentiation of PDX1+ posterior foregut cells into PDX1+NKX6.1+ pancreatic endoderm cells in hiPSC cultures. Consistently, NM II expression was elevated at low cell density, but upon aggregation, it was downregulated and associated with NKX6.1+ cell induction. The results provide mechanistic insight into the benefit of three-dimensional cultures for directed differentiation. Keywords: ESC, iPSC, differentiation, pancreas, pancreatic bud, pancreatic endoderm, NKX6.1, ROCK, non-muscle myosin II, cytoskeleton