Human pluripotent stem cells (hPSCs) have the potential to self-renew indefinitely and to differentiate into all adult cell-types. As such, hPSCs hold great promise as a source of cells for drug screening assays and potential regenerative treatments. hPSCs are commonly derived and cultured on surfaces composed of complex, xenogenic biological extracts or feeder cells; however most potential applications for hPSCs require culture surfaces that are xeno-free, well-defined, less expensive and more consistent than these options. Defined surfaces thought to specifically interact via various cell adhesion molecules (CAMs) have recently been reported to support hPSC culture, although the resultant cells are rarely well-characterized.
The aim of this project is to characterize and compare hPSCs cultured on a variety of chemically defined surfaces with well-defined cell-surface interactions. We have used a cell culture substrate composed of protein low-fouling poly(acrylamide-co-acrylic acid) modified to present small peptide ligands. A human ES reporter cell line, H9-OCT4-mCherry (kindly provided by Prof Ed Stanley, Murdoch Childrens Research Institute) is being used to test the ability of the surfaces with chemically coupled peptide ligands to support the in vitro maintenance of hPSCs. Cultures are monitored with a Nikon-MetaMorph live-cell imaging system, enabling cell-counting and quantification of mCherry reporter protein expression correlating with endogenous expression of the pluripotency associated transcription factor OCT-4.
H9-OCT4-mCherry cells have been demonstrated to attach, proliferate and maintain expression of mCherry on poly(acrylamide-co-acrylic acid) surfaces presenting an cRGDfK peptide and screening to identify additional lead ligands is in progress. Identifying ligands capable of supporting hPSC culture by interacting with different CAMs will allow us to perform long-term culture experiments with multiple embryo-derived and reprogrammed hPSC lines. Using a suite of molecular tools to assess gene expression and epigenetic changes will then allow investigation of the role(s) of CAMs in maintaining pluripotency. This work is expected to contribute to the development of defined culture surfaces appropriate for large scale, therapeutically relevant hPSC culture.