Human embryonic stem (hES) cells and human induced pluripotent stem (hips) cells can differentiate into all cell types of the body and therefore hold great therapeutic value. Applications ranging from in situ regenerative medicine, ex vivo tissue generation, and drug screening have consequently been proposed for these pluripotent cells. However, the current culturing techniques for both hES and hiPS cells hinder the clinical feasibility of their use. Pluripotent cell culture protocols have historically involved the use of animal products, which can introduce pathogens as well as limit the scalability and reproducibility of these platforms. While media formulations have been optimized to include only human or recombinant proteins, the current gold standard for human stem cell culture substrate is still Matrigel, an excretion from a mouse tumor that resembles the extra cellular matrix. It is therefore crucial to engineer materials that replace Matrigel in human pluripotent stem cell culture.

Despite the complexity of the native cell niche, key characteristics of the cell niche must be considered and chosen for emulation in constructing a synthetic system to replace Matrigel. Mechanical environment and paracrine signaling were deemed to be parameters that were both crucial for such an application and feasible to incorporate. Toward that end, a process to create tunable cell culture surfaces to replace Matrigel, and to conduct fundamental studies on the effects of mechanical environment and paracrine signaling was developed in this dissertation. A number of strategies for patterning hydrogels were considered for the creation of this synthetic human pluripotent substrate, as the hydrogel could provide mechanical cues to the cells and the patterning would control paracrine signaling by regulating cell colony size and spacing. Ultimately, a strategy involving concurrent UV illumination and microcontact printing of a UV activated crosslinker onto a low swelling hydrogel was selected, as it showed the highest pattern fidelity of the strategies explored, due to its minimization of ink diffusion. Hydrogels as compliant as 140 Pa could be patterned with this technique, and such hydrogels were shown to support hES cells in the short term.

To emphasize the limitations of Matrigel based cell culture platforms, the physical properties of Matrigel on variety of typical cell culture surfaces were studied, and it was found that the substrate beneath Matrigel was capable of influencing cell behavior such as proliferation and pluripotency, by altering the physical properties of Matrigel.

The need to create surfaces with defined cell colony sizes and spacing, thereby introducing standardization into the cell culture platform, was further emphasized by the results of our short term study conducted on the effects of mechanical environment on hES cells. While proliferation was influenced by substrate modulus, pluripotent gene expression was too variable to show any significant differences as a function of substrate modulus.

The patterning methodology developed in this dissertation for creating a modular synthetic culture surface for human pluripotent stem cells can be used not only for the maintenance of the pluripotency of the cells, but also for the directed differentiation of the cells. As a step toward using these platforms for directed differentiation protocols, the patterned surfaces were shown to support cardiomyocytes directly differentiated from iPS cells from a patient with long QT3 syndrome, with cells beating synchronously within a cell colony.