Person:

Hashmi, Basma

Development of three dimensional (3D) microenvironments that direct stem cell differentiation into functional cell types remains a major challenge in the field of regenerative medicine. Here, we describe a new platform to address this challenge by utilizing a robotic microarray spotter for testing stem cell fates inside various miniaturized cell-laden gels in a systematic manner. To demonstrate the feasibility of our platform, we evaluated the osteogenic differentiation of human mesenchymal stem cells (hMSCs) within combinatorial 3D niches. We were able to identify specific combinations, that enhanced the expression of osteogenic markers. Notably, these ‘hit' combinations directed hMSCs to form mineralized tissue when conditions were translated to 3D macroscale hydrogels, indicating that the miniaturization of the experimental system did not alter stem cell fate. Overall, our findings confirmed that the 3D cell-laden gel microarray can be used for screening of different conditions in a rapid, cost-effective, and multiplexed manner for a broad range of tissue engineering applications.

Increasing demands for organ transplants and the depleting supply of available organs has heightened the need for alternatives to this growing problem. Tissue engineers strive to regenerate organs in the future; however doing so requires a fundamental understanding of organ development and its key processes. The first chapter of this thesis provides a brief overview of developmentally inspired engineering, specifically in the context of how I approach this challenge in this thesis. The second chapter provides an in depth review of current and past work focused on organ regeneration from a developmentally-inspired perspective, and using tooth formation as a model system. The third chapter describes the design and fabrication of a thermoresponsive polymer inspired by an embryonic induction mechanism, and demonstrates its ability to induce tooth differentiation in vitro and in vivo. This is effectively a 3D `shrink wrap'-like polymer sponge that constricts when it is warmed to body temperature and induces compaction of cells contained within it, hence recapitulating the mesenchymal condensation process that has been shown to be a key induction mechanism that triggers formation of various epithelial organs, including tooth in the embryo. The fourth chapter describes the fabrication of a novel microarray screening platform consisting of a unique set of ECM proteins (collagen VI, tenascin, and combination of the two at different coating densities) on an array of soft substrates (~130-1500 Pa) that are physiologically relevant to the embryonic microenvironment. This technology demonstrated the capacity to analyze combinatorial effects of these ECM proteins and soft substrates on cell density, cell area and odontogenic differentiation in murine mandible embryonic mesenchymal cells. The fifth chapter of this thesis summarizes and discusses the advantages, limitations and future potential of the findings described in the previous two chapters in the context of organ engineering and regeneration. Taken together, the work and results presented in this thesis have led to the development of new insights, approaches and tools for studying organ formation and potentially inducing organ regeneration, which are inspired by key developmental mechanisms used during embryonic organ formation.

A biologically inspired thermoresponsive polymer has been developed that mechanically induces tooth differentiation in vitro and in vivo by promoting mesenchymal cell compaction as seen in each pore of the scaffold. This normally occurs during the physiological mesenchymal condensation response that triggers tooth formation in the embryo.