Human pluripotent stem cell (hPSC)-derived hepatocytes represent an unlimited source of cells for transplantation therapies, drug metabolism studies, and disease modeling. Current <i>in-vitro</i> hepatocyte differentiation protocols rely upon the use of undefined and animal derived cell adhesion substrates like Matrigel. To circumvent this challenge, defined human recombinant vitronectin and laminin have been used as substrates for directed-liver differentiation; however, they are susceptible to degradation, batch-to-batch variability, and high production cost. Synthetic peptides offer potential advantages due to their chemical stability and ability to be incorporated in different cell culture platforms beyond flat surfaces. In this study, we replaced Matrigel with a vitronectin-derived peptide (Ac-KGGPQVTRGDVFTMPC) as an adhesion substrate for human embryonic stem cells (hESCs) and their directed differentiation towards hepatocyte-like cells (HLCs) with simultaneous cell expansion. hESCs attached to the vitronectin (VN) peptide immobilized glass surfaces, proliferated, and expressed pluripotent markers after short term culture prior to hepatic differentiation. hESCs were successfully differentiated to endoderm cells, expanded, and further guided toward hepatocytes over 20 days following a stepwise differentiation protocol. Comparable level of expression of hepatocyte-specific transcripts, including albumin and alpha-1-antitrypsin (AAT), was observed between endoderm expanded HLCs on Matrigel and the VN peptide surface. Albumin and hepatocyte nuclear factor 4 alpha (HNF4a) protein expression was confirmed by immunofluorescence staining. Cells derived on the VN peptide surface exhibit characteristic morphological traits of hepatocytes derived in Matrigel such as polygonal shape and binucleation. In addition, functional studies revealed similar albumin secretion profile, drug-induced cytochrome P450 activity, and glycogen storage. Overall, we have demonstrated the application of vitronectin peptide modified surface as a completely synthetic and defined platform for the derivation of hepatocytes from hESC.

Functionalized vitronectin peptide surfaces for stem cell differentiation towards the hepatic lineage

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.