Synthetic surfaces for long-term maintenance of hPSC cultures
2017-03-02T02:39:58Z (GMT) by
The self-renewal and multilineage differentiation potential of human pluripotent stem cells (hPSC) has inspired a range of potential applications, the most exciting of which are cell replacement therapies using hPSC-derived cells. Many such therapies are under development, and therapies using various hPSC-derived cells to treat four diseases have reached phase one clinical trials. The widespread use of standardised, chemically-defined xeno-free hPSC culture conditions, including culture surfaces, are very important. The use of a global standard for hPSC culture conditions would simplify regulation, prevent xenogeneic contamination and improve consistency of not only the therapeutic product but also of all hPSC-based research and products. This thesis presents the optimisation of two peptide-presenting co-polymer brush coatings as chemically defined xeno-free hPSC culture surfaces, namely poly(acrylamide-co-acrylic acid) (PAAA) and poly(acrylamide-co-propargyl acrylamide) (PAPA). Both coatings were synthesised from mixed monomer solutions by repeated exposure to ultraviolet (UV) light. The peptide density and surface stiffness of these coatings could be controlled and were each observed to affect the efficiency of hPSC adhesion. Peptide-modified PAAA coatings (cRGDfK-PAAA) that had been synthesised with higher numbers of UV exposures were observed to increase in thickness and to bind hPSCs less efficiently. On the other hand, PAPA coatings were observed to increase in stiffness rather than thickness; hPSCs were observed to consistently adhere to PAPA coatings that were synthesised with up to 60 UV exposures and modified with reaction solutions containing ~500-fold less peptide than has been reported in similar studies, which resulted in a surface peptide density of 3.6 pmol/cm². This was the first report of the efficacy of chemically-defined hPSC culture surfaces presenting only cRGDfK. The optimised polymer coatings were individually modified with a library of 35 other peptides reported to bind cells, but the hPSC-binding efficiency of cRGDfK-modified coatings could not be equalled. Although hPSCs adhered to surfaces modified with four other peptides, colony numbers were low and/or adhesion was unstable compared to cRGDfK-modified control wells, even when the other peptides were applied in excess. hPSC cultures maintained for ten passages in cRGDfK-PAPA-coated flasks passed a battery of pluripotency tests. Parallel hPSC cultures were maintained for benchmarking on flasks coated with Geltrex™ and with commercially available synthetic hPSC culture surfaces, StemAdhere™ and Synthemax™. Morphological assessment, flow cytometry for the pluripotency marker OCT4, PluriTest analysis of global gene expression, G-banding karyotype and teratoma formation assays produced comparable results regardless of surface type. A low level of karyotypic abnormalities were observed to arise in cultures maintained on each of the target surfaces, but not in cultures maintained on cRGDfK-PAPA. This may indicate that the cRGDfK-PAPA surface is protective of genetic damage. This thesis presents cRGDfK as an optimal ligand for mediating hPSC adhesion to polymer coatings and cRGDfK-PAPA is presented as a well-characterised, affordable and effective synthetic surface for hPSC culture. Furthermore a discussion is presented of the strengths and weaknesses of commercially available alternative hPSC culture surfaces. The surface used for maintaining hPSC cultures must be selected in any case based on individual requirements, considering parameters such as cost, surface stability and the need for cell scraping.