Committee Members:Dr. Ling Wei (Advisor)Dr. Larry McIntire (Advisor)Dr. Marie CseteDr. Hang LuDr. Michel Maharbiz (UC Berkeley)Dr. Todd McDevittHuman embryonic stem cells provide an unmatched window into early differentiation and their indefinite proliferation and pluripotency make them an attractive prospect for cell replacement therapies. Embryonic stem cells are exquisitely sensitive to their microenvironment. This project explores three ways in which the microenvironment can affect hES cell behavior. Polydimethylsiloxane (PDMS) is commonly used in devices designed to control the microenvironment in cell culture, but it can have adverse effects on cells. We explored increased curing temperature and UV treatment as methods to improve hES cell growth on PDMS surfaces, increasing cell survival and the maintenance of pluripotency.Cell culture is most often carried out at 20% oxygen, much higher than the more physiologic range of 1-12%. We used a diffusion-based device to provide an oxygen gradient in this range to hES cells in culture. The results demonstrated that cells maintained pluripotency better at lowered oxygen.Directed neural differentiation of hES cells often involves suspension culture or feeder layers, introducing heterogeneity and complicating analysis. We explored a fully adherent, feeder-free neural differentiation protocol using small molecules and common medium supplements. We demonstrated efficient neural induction and differentiation of mature, functional neurons in vitro. In addition, we verified the utility of this protocol in translational studies by using neural precursor cell transplantation to improve outcomes in a murine model of ischemic stroke.The results of this work demonstrate the importance of controlling the microenvironment when seeking to control hES cell fate and provide insight into the best conditions for both maintenance of pluripotency and neural differentiation in developmental and therapeutic studies.