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PLoS One. 2015 Aug 26;10(8):e0136350. doi: 10.1371/journal.pone.0136350. eCollection 2015.

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

Author information

1
Tenure-track Program for Innovative Research, University of Fukui, Yoshida-gun, Fukui, Japan; Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
2
Division of Tumor Pathology, Department of Pathological Sciences, Faculty of Medical Sciences, University of Fukui, Yoshida-gun, Fukui, Japan.
3
Tenure-track Program for Innovative Research, University of Fukui, Yoshida-gun, Fukui, Japan.
4
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.

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.

PMID:
26308339
PMCID:
PMC4550348
DOI:
10.1371/journal.pone.0136350
[Indexed for MEDLINE]
Free PMC Article

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