| Derived from the inner cell mass of human blastocysts, human embryonic stem cells (hESCs) are a naturally immortal cell line capable of differentiation to the three embryonic germ layers. hESC interactions with their microenvironmental niche are complex and manipulation of this niche, including introduction of chemicals and growth factors, and altering growth dynamics by culturing cells under physical constraints, can have profound effects on hESC development.; We initially focused on introducing foreign molecules, protein, and DNA to hESCs, by optimizing electroporation parameters and conditions for various molecules. We discovered that moderate voltage of 300 V was required for efficient loading of all molecules, but there were significant differences in pulsing times and numbers. Small molecules were able to maintain high viability and molecular loading using multiple short pulses. Proteins and DNA required much longer pulsing times for efficient loading. At the expense of cell viability, multiple pulses increased the protein and DNA loading levels per cell, but not overall electroloading efficiency.; To monitor hESC interactions with their extracellular matrix, we coated a biocompatible nematic liquid crystal, TL205, with a Matrigel layer. hESCs exerted forces on the Matrigel as they attempted to grow and spread contracting the ECM proteins and stunting growth. Results were visualized using polar and fluorescence microscopy to monitor not only Matrigel rearrangement, but also the corresponding response of the liquid crystals.; We investigated cell-cell and cell-substrate signaling by culturing hESCs within 3-D microwells. While constrained, hESCs remained viable and undifferentiated for up to 19 days. Additionally, resulting aggregates had a homogeneous size distribution, were easily passaged, and retained pluripotency.; Using the microwell system, we investigated the effect of EB size and structure, known to influence differentiation potential, on cardiomyocyte differentiation. The percentage of beating EBs increased from 2.8% in EBs from unconstrained Matrigel cultures to 31% in EBs derived from 300 mum square microwells. Though 300 mum microwells were optimal, every size yielded higher beating EB percentages than unconstrained controls. In addition, the percentage of cells in the total population expressing cardiac markers was higher in microwell cultures than controls. |
