| Human pluripotent stem cells (hPSCs) offer a potential cell source for cell-based model systems to study development and disease, as well as for regenerative medicine due to their unique ability to self-renew or differentiate to any somatic cell type. Before the full potential of hPSCs can be realized, robust protocols must be developed to direct their fate. Researchers have identified various factors which can direct hPSC fate, such as those necessary for directing epithelial commitment of hPSCs. We have focused our efforts in refining methods for generating epithelial and epidermal cells from hPSCs and utilizing these somatic cells for various tissue engineering applications. We first developed a defined approach for generating epithelial and epidermal cells from hPSCs by identifying an optimal starting cell density to maximize yield and maintain high purity of simple epithelial progenitors. In addition, we demonstrated that the use of defined substrates, in lieu of undefined, xenogeneic substrates, can successfully facilitate efficient epithelial differentiation to produce functional keratinocyte progenitor cells that can terminally-differentiate to recapitulate epidermal tissue architecture in vitro. In a general application to be applied to any hPSC differentiation platform, we developed an approach to improve efficiency in and translate lab-scale hPSC differentiation systems to large-scale processes. Our integrated experimental and computational approach, using two epithelial differentiation systems as models, required the development of an ODE-based model and a fit of this model to data representing dynamics of various cell types present in culture. This fit was performed by estimating rate constants of cell fate decisions (self-renewal, differentiation, death). Sensitivity analyses on predicted rate constants indicated which cell fate decisions were limiting to the final cell yield. Finally, in a more specific application, we used our epithelial differentiation strategy for a more specific application in disease modeling. We derived patient-specific hiPSCs from patients with a genetic skin disorder and our preliminary findings indicate that the hiPSC-derived keratinocyte progenitors can recapitulate a diseased epidermal phenotype in vitro, providing a system to study this disease from a patient-specific cell source. |
