| Stem cells hold great promise for regenerative medicine. In a developing embryo or an adult body, the fate of stem cells is tightly regulated by their microenvironments which provide a unique combination of various extracellular stimuli such as soluble factors, extracellular matrix proteins, cell-cell interactions, mechanical stress, pH, ionic strength, temperature, etc. Successful clinical applications of stem cells will require reproducing key signaling cues that govern self-renewal, proliferation, and differentiation of stem cells in vitro. While conventional cell culture methods provide limited means to investigate extracellular cues, micro/nano technologies can offer a wide spectrum of tools to apply such cues to stem cells in a controlled manner. Therefore, stem cell research is likely to significantly benefit from a micro/nano technology-based platform that allows for screening combinations of various extracellular cues. In addition, the screening process for enabling combinations will be challenged by a large multi-dimensional parameter space created by the number and intensity of possible cues. In this dissertation, we first describe development of key microfluidic tools, a micro-deionizer and a cell microarray device, which would be important toward realization of the above-mentioned micro/nano technology-based platform. More importantly, we have developed a fully defined culture of human embryonic stem (hES) cells which could enable manufacturing of clinical-grade hES cells. Current state-of-the-art methods of hES cell culture are either poorly defined, may contain pathogenic or xenogenic contaminants, or cannot support single cell culture, limiting the successful transition to therapeutic applications. Therefore, we developed defined culture systems by identifying unique combinations of multiple small molecules which synergistically maintain hES cells in an undifferentiated state by blocking pathways leading to differentiation. We employed Feedback System Control (FSC) to quickly identify such combinations by efficiently exploring a large parameter space created by combinations of the small molecules. Our investigations led to discovery of a set of small molecule cocktails which allowed for maintenance of hES cell in an undifferentiated state for 10 passages or more. These unique small molecule cocktails could provide a fully defined system for clinically enabling applications hES cells. |
