RRC ID 61365
Author Nagaoka M, Kobayashi M, Kawai C, Mallanna SK, Duncan SA.
Title Design of a Vitronectin-Based Recombinant Protein as a Defined Substrate for Differentiation of Human Pluripotent Stem Cells into Hepatocyte-Like Cells.
Journal PLoS One
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.
Volume 10(8)
Pages e0136350
Published 2015-1-1
DOI 10.1371/journal.pone.0136350
PII PONE-D-15-16461
PMID 26308339
PMC PMC4550348
MeSH Animals Blotting, Western Cell Adhesion Cell Culture Techniques Cell Differentiation* Cells, Cultured Collagen Drug Combinations Embryonic Stem Cells / cytology* Embryonic Stem Cells / metabolism Flow Cytometry Hepatocytes / cytology* Hepatocytes / metabolism Humans Laminin Mice Mice, SCID Pluripotent Stem Cells / cytology* Pluripotent Stem Cells / metabolism Proteoglycans RNA, Messenger / genetics Real-Time Polymerase Chain Reaction Recombinant Proteins / genetics Recombinant Proteins / metabolism* Reverse Transcriptase Polymerase Chain Reaction Teratoma / genetics Teratoma / metabolism Teratoma / pathology* Vitronectin / genetics Vitronectin / metabolism*
IF 2.74
Human and Animal Cells 253G1(HPS0002) 454E2(HPS0077)