Abstract
The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.
Highlights
Over the past few decades, studies in vertebrate models have described several signaling pathways critical for the embryonic development of hepatocytes [7, 8]
We explored the potential of our previously established human embryonic stem cell (ESC)-derived fibroblast-like cells [41] for derivation of hepatocytes from pluripotent stem cells
We found that exposure of monkey and human ESCs and human induced pluripotent stem cells (iPSCs) to a high density of human ESC-derived fibroblast-like cells (hESdFs) induced robust hepatic endoderm differentiation in a regionally specific manner in ESC/iPSC colonies
Summary
Recent demonstrations of the generation of induced pluripotent stem cells (iPSCs) with defined transcription factors [34, 35] that allowed the derivation of patient- and disease-specific pluripotent stem cells [36, 37] without using human embryos have heightened interest in the in vitro hepatic differentiation potential of iPSCs (38 – 40). The demonstration of the hepatic differentiation capability of iPSCs suggests their possible application for in vitro personalized pharmacogenetics, toxicology, and metabolism studies and future in vivo transplantation trails. We found that exposure of monkey and human ESCs and human iPSCs to a high density of hESdFs induced robust hepatic endoderm differentiation in a regionally specific manner in ESC/iPSC colonies. We elaborate the possible mechanisms responsible for such hepatic induction
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