Abstract
Synthetic human pluripotent stem cell (hPSC) culture surfaces with defined physical and chemical properties will facilitate improved research and therapeutic applications of hPSCs. In this study, synthetic surfaces for hPSC culture in E8 medium were produced for screening by modifying two polymer brush coatings [poly(acrylamide-co-acrylic acid) (PAAA) and poly(acrylamide-co-propargyl acrylamide) (PAPA)] to present single peptides. Adhesion of hPSC colonies was more consistently observed on surfaces modified with cRGDfK compared to surfaces modified with other peptide sequences tested. PAPA-coated polystyrene flasks with coupled cRGDfK (cRGDfK-PAPA) were then used for long-term studies of three hPSC lines (H9, hiPS-NHF1.3, Genea-02). Cell lines maintained for ten passages on cRGDfK-PAPA were assessed for colony morphology, proliferation rate, maintenance of OCT4 expression, cell viability at harvest, teratoma formation potential, and global gene expression as assessed by the PluriTest™ assay. cRGDfK-PAPA and control cultures maintained on Geltrex™ produced comparable results in most assays. No karyotypic abnormalities were detected in cultures maintained on cRGDfK-PAPA, while abnormalities were detected in cultures maintained on Geltrex™, StemAdhere™ or Synthemax™. This is the first report of long term maintenance of hPSC cultures on the scalable, stable, and cost-effective cRGDfK-PAPA coating.
Highlights
Human pluripotent stem cells show potential for drug discovery, studying disease mechanisms, and for clinical applications including the generation of differentiated cell types for transplantation. human pluripotent stem cell (hPSC) cultures were originally maintained in co-culture with mitotically inactivated feeder cells
An OCT4-mCherry reporter cell line was generated from the human embryonic stem cell line H9 to screen poly(acrylamide-co-acrylic acid) (PAAA) and poly(acrylamide-co-propargyl acrylamide) (PAPA) coatings modified with test peptides for adhesion and short-term maintenance of hPSCs
After identification of the lead hPSC adhesion peptide and optimisation of the substrate coating, three hPSC lines were maintained on the lead surface (PAPA-cRGDfK) for ten passages and compared to cultures maintained in parallel on the commercially available
Summary
Human pluripotent stem cells (hPSCs) show potential for drug discovery, studying disease mechanisms, and for clinical applications including the generation of differentiated cell types for transplantation. hPSC cultures were originally maintained in co-culture with mitotically inactivated feeder cells. Surfaces coated with feeder cells can provide the necessary supporting factors for hPSC maintenance, their use inherently results in variable culture conditions. Synthetic peptide-presenting polymer coatings, able to support adhesion and maintenance of hPSC cultures, were used to screen for peptide ligands that were individually capable of mediating hPSC adhesion. For the coatings presented here, a methodology was optimised which relied on multiple passes of the multiwell plates or flasks beneath a high intensity UV light in the presence of monomer solution and in the absence of oxygen. Produces a surface coating, the properties of which can be readily modified and optimised for a variety of applications, including in vitro expansion of pluripotent stem cells as presented here
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