Design of a Vitronectin-Based Recombinant Protein as a Defined Substrate for Differentiation of Human Pluripotent Stem Cells into Hepatocyte-Like Cells.

Masato Nagaoka,Motohiro Kobayashi,Sunil K Mallanna,Stephen A Duncan,Chie Kawai,Atsushi Asakura

doi:10.1371/journal.pone.0136350

Masato Nagaoka, Motohiro Kobayashi + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0136350

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Journal: PloS one	Publication Date: Aug 26, 2015
Citations: 16	License type: CC BY 4.0

Affiliation: University of Fukui, Medical College of Wisconsin

Abstract

Maintenance and differentiation of human pluripotent stem cells (hPSCs) usually requires culture on a substrate for cell adhesion. A commonly used substratum is Matrigel purified from Engelbreth—Holm—Swarm sarcoma cells, and consists of a complex mixture of extracellular matrix proteins, proteoglycans, and growth factors. Several studies have successfully induced differentiation of hepatocyte-like cells from hPSCs. However, most of these studies have used Matrigel as a cell adhesion substrate, which is not a defined culture condition. In an attempt to generate a substratum that supports undifferentiated properties and differentiation into hepatic lineage cells, we designed novel substrates consisting of vitronectin fragments fused to the IgG Fc domain. hPSCs adhered to these substrates via interactions between integrins and the RGD (Arg-Gly-Asp) motif, and the cells maintained their undifferentiated phenotypes. Using a previously established differentiation protocol, hPSCs were efficiently differentiated into mesendodermal and hepatic lineage cells on a vitronectin fragment-containing substrate. We found that full-length vitronectin did not support stable cell adhesion during the specification stage. Furthermore, the vitronectin fragment with the minimal RGD-containing domain was sufficient for differentiation of human induced pluripotent stem cells into hepatic lineage cells under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs.

Highlights

The generation of mature hepatocytes from human pluripotent stem cells (hPSCs) is a useful approach for therapeutic applications, researching drug metabolism, and the study of genetic diseases using patient-derived induced pluripotent stem cells
Because the N-terminal somatomedin B domain is not necessary for adhesion and maintenance of hPSCs [24], the somatomedin B domain was removed (R-Fc contained the RGD motif; NC-Fc consisted of RGD, hemopexin and heparin-binding domains, which is similar to VTN-NC reported by Chen et al [24])
The adhesion of hPSCs to vitronectin variants was inhibited by pre-incubation with the RGDfV peptide, which is a blocking peptide for the RGD sequence, whereas no effect was observed on cell adhesion to the E-cad-Fc-coated surface (Fig 1E)

Summary

Introduction

The generation of mature hepatocytes from hPSCs is a useful approach for therapeutic applications, researching drug metabolism, and the study of genetic diseases using patient-derived induced pluripotent stem (iPS) cells. Matrigel is used for maintenance of rodent and human primary hepatocytes in culture [8,9,10,11] It is a viable substitute for basement membrane, Matrigel is a relatively impure preparation with significant lot-to-lot variability in the content of growth factors and matrix proteins that can affect the reproducibility of cell differentiation. We constructed novel substrates consisting of vitronectin fragments and IgG Fc fusion proteins, and examined whether these fusion proteins could support the maintenance and differentiation of hPSCs into hepatocyte-like cells under completely defined culture conditions

Materials and Methods

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Discussion