Modeling hepatobiliary development using human induced pluripotent stem cells

Due to the limitations of research using human embryos and the lack of a biological model of human liver development, the liver developmental stages, the underlying molecular mediators governing them and roles of various markers associated with these stages in humans are largely speculative, and based on studies utilizing animal models and certain patient tissues. Human pluripotent stem cell-based in vitro directed and multistage hepatic differentiation systems may thus serve as good surrogate models for mimicking normal human liver development, pathogenesis and injury/regeneration studies, and is now been increasingly utilized as in vitro models of human liver development and diseases. In my thesis here, I describe the implications of various liver stem/progenitor cell markers and their bipotency (i.e. hepatocytic- and biliary- epithelial cell differentiation), based on the pluripotent stem cell derived model of human liver development which includes distinct endoderm, hepatic progenitor (HP) and mature hepatocyte (MH) differentiation stages. To elucidate the underlying key molecular mediators regulating multiple stages of human liver development, I utilized various inhibitors targeting specific tyrosine kinases during directed hepatic differentiation of human induced pluripotent stem cells (iPSCs). Among the 80 inhibitors tested in each of the stages during endoderm, early and late hepatic differentiation of human iPSCs, only Src inhibitors significantly suppressed the formation of endoderm cells. In contrast, Src inhibition during HP commitment of endodermal cells results in a transient increase in expression of HP markers, which of further maturation gives rise to hepatocytes with compromised functionality, while inhibition of Src Kinase activity during hepatocyte commitment of HP cells or in MH cells showed no significant cell fate changes. Transient inhibition of c-Src, a non-receptor tyrosine kinase, during endodermal induction of human iPSCs clearly reduced endodermal commitment and inhibited expression of endodermal markers including SOX17 and FOXA2 in a dose dependent manner. Surprisingly, the treated cells gradually became profibrogenic cholangiocyte-like cells by expressing both cholangiocyte markers such as CK7 and fibrosis markers such as Collagen1 and SMA during prolonged culture in normal hepatic differentiation condition. Interestingly, collagen expression in the abnormal profibrogenic cholangiocytes was highly co-localized with expression of YAP (a marker of biliary proliferation and fibrosis), and inflammatory markers IL-6 and TNF-alpha also increased in these cells. A YAP inhibitor, verteporfin significantly reduced expression of collagen and other fibrosis markers in these cells. Collectively, these results suggest that c-Src has critical roles in cell fate determination during endodermal commitment of human pluripotent stem cells, and its alteration in early liver development in human may lead to increased production of YAP expressing profibrogenic proinflammatory cholangiocytes, similar to those seen in livers of patients with biliary fibrosis including biliary atresia (BA). Co-expression of YAP1 and COL1 in BA patient biliary remnant tissue samples was increased significantly, compared to the expression seen in normal liver tissue. Additionally, co-expression of COL1 and CK7 also increased significantly in BA patient biliary remnant tissue samples was increased significantly, compared to the expression seen in normal liver tissue. Using multistage hepatic differentiation culture of human iPSCs, I 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 previously observed in patients with biliary fibrosis. Since it is not feasible to study early endoderm formation in human fetus, this study provides new insights on endoderm development in human and a basis for establishing human-specific experimental systems for modeling abnormal liver development which may potentially associated with hepatobiliary cirrhosis