| Given their capacity for unlimited propagation as well as differentiation into any adult cell type, human pluripotent stem cells (hPSCs) hold tremendous promise for a wide range of applications, including development of cell-based therapies, drug and toxicity screening, and studying human developmental pathways. However, in order to realize the full potential of these cells, current processes for generation of desired cell types must be expanded to a larger scale, which will require more precise understanding of and control over mechanisms of self-renewal and differentiation.;Our approach was to utilize control of the cell microenvironment in order to engineer processes to better regulate growth and differentiation of hPSCs. We first used a microwell array system to study the effects of 3-D culture on growth and metabolism of hPSCs and found differences in cell size, cell cycle progression, and proliferative capacity in 3-D microwells as compared to 2-D substrates, providing various opportunities for further exploration of potential pathways that promote self-renewal or prime cells for differentiation. Next we used the microwell system to show that modulation of intercellular interactions impacted canonical Wnt/beta-catenin signaling in both undifferentiated hPSCs and differentiating embryoid bodies (EBs). In addition, we were able to link this modulation of Wnt/beta-catenin signaling to the enhanced cardiogenesis observed in EBs generated from microwells. These results demonstrate that the microwell platform can be used to study pathways affected by intercellular interactions in hPSCs and to elucidate how these pathways affect cell fate.;Finally, we designed a system that mimicked the embryonic brain microenvironment in order to produce endothelial cells possessing molecular markers and phenotypic characteristics of the blood-brain barrier (BBB) from hPSCs. This study was the first demonstration of organ-specific endothelial differentiation of hPSCs, and these brain-specific endothelial cells have potential applications in drug discovery and studies of human brain development. Taken together, these findings provide evidence that understanding the effects of specific microenvironmental cues in regulating hPSC fate decisions is critical in enabling the design of culture and differentiation processes to produce any desired cell type. |
