Abstract
SummaryThere 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.
Highlights
Pancreatic cells generated from pluripotent stem cells, such as human embryonic stem cells and induced pluripotent stem cells, are considered a promising cell source for regenerative therapies
Rho-associated kinase (ROCK)-non-muscle myosin II (NM II) Inhibitors Facilitate the Differentiation of Posterior Foregut to Pancreatic Endoderm Cells PDX1+ posterior foregut cells re-seeded at low cell densities (1.6 3 105 cells/cm2) were treated with three soluble factors: KGF, NOGGIN, and EGF (Figure 1A)
These results suggest that the signaling of ROCKs and downstream NM IIs regulates differentiation into NKX6.1+ cells, we cannot exclude the possibility that the inhibitors used in this study may target molecules other than ROCK-NM II (Figure S2A)
Summary
Pancreatic cells generated from pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are considered a promising cell source for regenerative therapies. It is possible that these physical changes lead to modifications in intracellular signaling, resulting in modulation of the propensity for differentiation into pancreatic lineages (Cortijo et al, 2012; Kesavan et al, 2009) Supporting this idea, we previously showed that high cell density or aggregation promotes the differentiation of PDX1+ posterior foregut cells to the earliest stage of PDX1+NKX6.1+ pancreatic endoderm cells in hESC/iPSC differentiation cultures (Toyoda et al, 2015). Proper cytoskeletal regulation is required for normal pancreatic organogenesis; the roles of the cytoskeleton in the formation of pancreatic endoderm remain unclear
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