Transient c-Src Suppression During Endodermal Commitment of Human Induced Pluripotent Stem Cells Results in Abnormal Profibrotic Cholangiocyte-Like Cells

Abstract

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 multistage 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 smooth muscle actin. Further analysis of these cells revealed colocalized expression of collagen and yes-associated protein (YAP; a marker associated with bile duct proliferation/fibrosis) and an increased production of interleukin-6 and tumor necrosis factor-α. 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.

YAP1 inhibits the induction of TNF‐α‐stimulated bone‐resorbing mediators by suppressing the NF‐κB signaling pathway in MC3T3‐E1 cells

“…The expression levels of IL-6 and RANKL were enhanced but that of OPG was reduced after YAP1 knockdown. A variation pattern of mRNA expression levels similar to that of YAP1 knockdown was observed in verteporfin, which was recently used as YAP1 inhibitor (Chaudhari et al, 2019;Lin et al, 2017). TEADs family are required for mediating YAP-dependent gene expression and biological functions.…”

A comparison of hepato-cellular in vitro platforms to study CYP3A4 induction

“…In this study, stem cells were differentiated into hepatocyte like cells accompanied by four differentiation stages: definitive endoderm (day 5), hepatic progenitor (day 10), immature hepatocyte (day 20) and mature hepatocyte (day 25) (Fig 5A) [36,37,48,51,52]. We quantified the basal CYP3A4 and PXR expression levels at each differentiation stage as shown in Fig 5. Interestingly, PXR levels did not change during the differentiation timeline whereas CYP3A4 levels increased by greater than 1500-fold at the final mature hepatocyte stage.…”

“…All human iPSCs used in this study were previously generated [34,37,[44][45][46][47][48][49][50]. For this study, six human iPSC lines including iHu71, iH34, iH14, iM9, iA3, and iH9 (either primary hepatocyte or fibroblast derived iPSCs) were used.…”

“…The medium was replaced every day until the cells reached the desired confluency for passaging or differentiation. Once the human iPSCs reached approximately 50-70% confluency, endodermal commitment and hepatic differentiation was induced as previously described [36,37,48,51]. Definitive endoderm (DE) stage cells were obtained after 3-5 days of iPSC culture in RPMI medium supplemented with B27 and 50-100 ng/mL activin A. Hepatic commitment was induced by culturing DE cells in RPMI medium supplemented with HGF and FGF4, 10 ng/ml each, for further 4-5 days to obtain hepatic progenitor (HP) stage cells.…”

“…Pluripotent stem cells can be differentiated into many different cell types, including hepatocytes. For this study, we employed a 25-day long protocol, where pluripotent stem cells differentiate into definitive endoderm-(day 3-5), hepatic progenitor-(day 8-10), immature hepatocyte-(day 20) and finally into mature hepatocyte-like cells (day 25) ( Fig 5A) [37,48,51,52]. We looked at the basal expression levels of CYP3A4 and PXR at different stages of the differentiation (on days 5, 10, 20 and 25).…”