Objective: A challenge in using human embryonic stem cells (hESCs) as the source of surrogate β cells is the establishment of methods that could effectively direct their differentiation into functional β cells. The aim of this study was to assess the effect of NANOG gene suppression in differentiating hESCs as a mean of increasing the efficiency with which endoderm-derived pancreatic cells could be generated.
Methods: A homogenous cell population with stable suppression of NANOG was generated in hESC ENVY line using plasmid-based siRNA approach. Pancreatic differentiation was undertaken according to the ontology-based in vitro selection protocol and followed by transplantation into immunodeficiency mice to mature in vivo.
Results: We observed up-regulation of definitive endoderm genes, which expand the role of NANOG in blocking definitive endoderm differentiation. The ontology-based differentiation protocol resulted in increased expression of markers essential for pancreatic epithelium development. Transplantation of these cells further revealed a homogenous pancreatic exocrine-like morphology that stained positively for amylase.
Conclusions: The suppression of NANOG displayed an effective differentiation toward endoderm and pancreatic progenitors. Investigation of the factors required for endocrine formation combined with a prolonged in vivo culturing could be further used to increase the ratio of endocrine-exocrine cells fate.
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