Encapsulation as a 3D model for human embryonic stem cell propagation and differentiation
Type 1 diabetes is a chronic disease caused by the destruction of the beta cells of the pancreas. Current treatments for are whole pancreas or islet transplantation, which is limited by scarcity of cadaveric pancreata. Therefore, developing a cell replacement therapy with renewable sources of surrogate beta cells is essential. Human embryonic stem cells (hESCs) have an indefinite replication capacity and can be directed to differentiate into many types of specialized cells. Currently, efficient protocols to generate beta cells rely on a step-wise ontogenic approach which mimics the in vivo pancreatic development. This approach requires a three dimensional (3D) culture system that can induce cell-cell and cell-matrix interactions which are essential for functional maturation. This project investigated the use of cell encapsulation technology to maintain hESC propagation and enhance their differentiation into beta cells in a 3D system. This system also offers potential for immune-isolation and prevention of teratoma formation by hESCs during transplantation. For the first time conditions were optimized for encapsulation of single cell population of the hESCs in alginate microcapsules with an extended proliferation. A 5-stage ontogeny-based protocol for differentiation to beta cells was then applied to these cells. Encapsulated hESCs were successfully differentiated into definitive endoderm (DE); however, this 3D model was not efficient to derive functional beta cells due to low cell viability maintained during the latter differentiation stages. To enhance this differentiation process, further investigation was performed by using glucagon-like peptide-1 (GLP-1) which has been shown to stimulate differentiation of stem and progenitor cells to an endocrine phenotype, induce insulin expression and prevent beta cell apoptosis. The present study demonstrated that undifferentiated and differentiated hESCs expressed GLP-1 receptor (GLP-1R). Attempts were made by supplementing the GLP-1R agonist, exendin-4 (Ex-4) to the culture media during each stage of differentiation. However, Ex-4 supplementation had no additive effect on cell viability. In contrast, further investigation demonstrated a significant reduction of the DE gene expression levels. Examination of miRNA profiles of hESC-derived DE revealed that Ex-4 supplementation resulted in the increase in pluripotency-associated miRNAs. Furthermore, in apoptosis-inducing medium, Ex-4 in combination with bFGF resulted in a significant downregulation of pro-apoptotic markers in hESCs. While these data revealed the possible role of Ex-4 in the maintenance of pluripotency and prevention of apoptosis in hESCs, future work is necessary to clarify the mechanism of GLP-1R-ligand interactions and their relevance to hESC development. Findings from this thesis demonstrate the establishment of a novel 3D model for hESC propagation and differentiation. Encapsulated hESCs successfully proliferated and differentiated into DE. Further optimization of the differentiation protocol and modification of biomaterial surface are required to generate functional beta cells. However, this study provides the possibility of using the alginate microcapsules for hESC 3D propagation and differentiation into other lineages and may be useful for cell replacement therapy upon transplantation.
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