Defining the Mechanism by which Synthetic Polymer Surfaces Support Human Pluripotent Stem Cell Self-Renewal.

Abstract

Human pluripotent stem cells (hPSCs), which include embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have become a promising resource for regenerative medicine and research into early development because these cells are able to indefinitely self-renew and are capable of differentiation into specialized cell types of all three germ layers and trophoectoderm. However, a major limitation for successful therapeutic application of hPSCs and their derivatives is the potential xenogeneic contamination and instability of current culture conditions. Synthetic polymers, such as poly[2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide] (PMEDSAH), offer multiple advantages over mouse embryonic fibroblasts (MEFs) and Matrigel for hPSC culture. The main purpose of this dissertation is to define the mechanisms by which hPSCs are propagated on synthetic polymers. By physical modifications of PMEDSAH, we found that modifying substrate thickness changed the physical properties, and thus altered pluripotent stem cell behavior. Our data suggest that the 105 nm thick atom transfer radical polymerization (ATRP) PMEDSAH possesses the optimal gel architecture for hPSC expansion with its intermediate thickness, hydrophilicity, surface charge, and a moderate degree of inter-chain association. Our findings demonstrate the importance of polymer physical properties in hPSC expansion. The 105 nm thick ATRP PMEDSAH and similar modifications may be used to obtain scalable populations of clinical-grade hPSCs for regenerative medicine. Although a specific group of transcription factors, such as OCT4, SOX2 and NANOG, are known to play critical roles in hPSC pluripotency and reprogramming, other factors and the key signaling pathways regulating these important properties are not completely understood. In this dissertation, we also investigated the role of the PSC marker Developmental Pluripotency Associated 5 (DPPA5) in hPSCs. Our data demonstrate higher expression of DPPA5 in hPSCs under PMEDSAH and other feeder-free conditions, compared to MEFs. DPPA5 stabilizes protein levels and enhances the function of NANOG. Finally, DPPA5 increases the hiPSC-reprogramming efficiency. These results provide new molecular insight into the function of the DPPA5 in hPSCs. Our findings extend our understanding of the mechanism by which PMEDSAH and other feeder-free conditions support hPSC self-renewal, and offers improvements to current protocols in hPSC maintenance and reprogramming.