Hepatic stellate cells promote the differentiation of embryonic stem cell-derived definitive endodermal cells into hepatic progenitor cells.

Aim: Hepatic non-parenchymal cells are well known to be capable of providing an important microenvironment and growth factors for hepatic regeneration, but their capacity for directing embryonic stem cells (ESC) toward hepatocytes remains to be assessed. Thus, this study aims to investigate the role of hepatic stellate cells (HSC), the major type of hepatic non-parenchymal cells, in the differentiation of ESC as well as exploring the potentiality of ESC in regeneration medicine for cell-based therapy.

Methods: A two-step differentiation procedure that utilized the capability of HSC to regulate proliferation and differentiation of hepatocytes was used to develop an approach for directing the differentiation of ESC towards hepatic progenitor cells. Mouse ESC were cultivated in a serum-free medium containing Activin A and fibroblast growth factor to generate definitive endodermal cells characterized by the CXCR4 cell-surface marker. After 6-8 days in culture, approximately 60% of the differentiated cells expressed CXCR4, and more than 90% of the CXCR4 positive cells could be recovered by cell sorting. The purified CXCR4 positive cells were co-cultured with mouse HSC as feeder cells in basal medium without additional hepatocyte growth factors. Differentiation was complete after 10-12 days of co-culture, and hepatic progenitor cell markers such as α-fetoprotein (afp) and albumin (alb) were detected in the terminally differentiated ESC.

Conclusion: These results show that HSC provide an appropriate microenvironment and pivotal growth factors for generation of hepatic progenitor cells from ESC-derived definitive endodermal cells, and suggest that this approach possibly allows for hepatic differentiation of ESC imitating the process of hepatic regeneration.