Transient c-Src suppression during endodermal commitment of human iPSCs results in abnormal profibrotic cholangiocyte-like cells.

Directed differentiation of human induced pluripotent stem cells (iPSCs) toward hepatobiliary lineages has been increasingly used as models of human liver development/diseases. As protein kinases are important components of signaling pathways regulating cell fate changes, we sought to define the key molecular mediators regulating human liver development using inhibitors targeting tyrosine kinases during hepatic differentiation of human iPSCs. A library of tyrosine kinase inhibitors was used for initial screening during the multi-stage differentiation of human iPSCs to hepatic lineage. Among the 80 kinase inhibitors tested, only Src inhibitors suppressed endoderm formation while none had significant effect on later stages of hepatic differentiation. Transient inhibition of c-Src during endodermal induction of human iPSCs reduced endodermal commitment and expression of endodermal markers including SOX17 and FOXA2 in a dose dependent manner. Interestingly, the transiently treated cells later developed into profibrogenic cholangiocyte-like cells expressing both cholangiocyte markers such as CK7 and CK19 and fibrosis markers including Collagen1 and SMA. Further analysis of these cells revealed co-localized expression of collagen and YAP (a marker associated with bile duct proliferation/fibrosis), and an increased production of IL-6 and TNF-alpha. Moreover, treatment with verteporfin, a YAP inhibitor, significantly reduced expression of fibrosis markers. In summary, these results suggest that c-Src has a critical role in cell fate determination during endodermal commitment of human iPSCs, and its alteration in early liver development in human may lead to increased production of abnormal YAP expressing profibrogenic proinflammatory cholangiocytes, similar to those seen in livers of patients with biliary fibrosis. SIGNIFICANCE STATEMENT: Using human iPSCs, we discovered that a protein kinase c-Src is important for endoderm formation and that a transient alteration of its activity in the early stage of liver development results in abnormal fibrotic-biliary cells resembling those observed in patients with biliary fibrosis. Since it is not feasible to study early endoderm formation in human fetus, this study provides a basis for establishing human-specific experimental systems for studying abnormal liver development which may potentially associated with liver fibrosis and cirrhosis. © AlphaMed Press 2018.