Peptides and polymers for stem cell modulation

Abstract

One of the requisites for a growth factor and a biomaterial in tissue engineering, cell therapy and regenerative medicine is the ability to control cell fate. Cells exist in a complex micro-environment consisting of extra-cellular matrix, growth factors, together with adjacent cells. Stem cell culture and modulation remains a challenge due to insufficient, undefined and costly culture systems. This thesis describes synthetic approaches that can modulate stem cell fate by the identification of new synthetic substrates for the growth of cancer, embryonic stem cells and potential short peptide sequences that can mimic the biological functions of the native cytokine used to culture stem cells. Glioma cancers exist as a heterogeneous population of cancer stem cells and cancer progenies. Scale up and spin coating of a polyurethane and polyacrylate polymers was done on agarose for the enrichment of the cancer stem cell population from glioma cells. A polyurethane, synthesised from poly(tetramethylene glycol) and 1,3-bis(isocyanatomethyl)cyclohexane spin (PU10) coated on an agarose surface, was identified to have a higher affinity for the cancer stem cell population over its progenies. By using this polymer to study the mechanism of the cancer stem cell adhesion, two niche components i.e. galectin, transferrin that are enriched by the polymer that contributed to the growth of the cancer stem cells were identified. A synthetic hydrogel (HG21) was identified as substrate for the culture of mouse embryonic stem cells (mESC) as a replacement for gelatin. mESCs were cultured on the hydrogel under undefined and defined conditions. Under both culture conditions, mESC pluripotency and naïve phenotype markers were verified. Marker profiles by immunostaining (Oct-4, Nanog), flow cytometry (SSEA-1) and qPCR (14 gene markers) of mESC grown on the hydrogel were comparable to gelatin, while enabling thermo-detachment for enzyme free passaging of mESC. To identify alternative substances to the cytokines used in stem cell culture, a microarray system was developed. The microarray system was developed initially with adhesion cellulose peptides printed onto polyacrylamide coated microscope slides. These slides were then screened for interaction with human embryonic stem cells (hESCs). After successful development of the cell based cellulose peptide microarray system, overlapping 25-mer peptides based on of basic fibroblast growth factor were synthesised, printed onto the same type of slide and screened with hESC. The screen identified “hit” peptides, which could potentially mimic the biological effects of the native cytokine on hESCs. These “hit” peptides were scaled up and tested in solution with hESC. In the linear form the peptides were not sufficient to sustain pluripotency and further optimisation is needed.