Engineering of Epithelial Tissues From Pluripotent Human Cells

Pluripotent cells offer great potential as a scaleable source of tissue for regenerative medicine and as model systems of studying human cell biology. To harness this potential, pluripotent cell derivatives must be incorporated into tissue engineering approaches to better gauge their functionality in a more in vivo-like microenvironment. Previously we identified a highly efficient method of directing human embryonic stem (hES) cells to epithelial lineages by modulating retinoic acid (RA) and bone morphogenetic protein (BMP) signaling. Here we have conducted a functional characterization of hES cell-derived epithelial progenitors. Microarray analysis of these human ES cell-derivatives and primary foreskin keratinocytes (PFKs) demonstrated that both cells exhibited a similar global transcriptional program. However, significant differences were observed in the expression of genes associated with signal transduction, extracellular matrix, and chemotaxis. When cultivated at the air-liquid interface in organotypic skin cultures, hES cell-derived epithelia survived and proliferated for several weeks and were able to stratify and terminally differentiate. Organ cultures containing hES cell derivatives achieved a similar morphology and marker expression pattern (p63, involucrin, CK10) to constructs formed using PFKs, though some differences were observed compared to human skin. In addition, we have applied our RA differentiation method to induced pluripotent stem (iPS) cells, generating p63+/CK14+ epithelial cells at purities >90%. When cultured in a three-dimensional microenvironment, hES and iPS cell-derived epithelia form uniform spheres, undergo morphogenesis, and exhibit cellular polarity indicative of epithelial tissues in vivo. Distinct populations of CK14+ basal cells surrounded by the basement membrane protein laminin 5 were present at the periphery of each sphere. Our results demonstrate the functionality and potential of pluripotent cell derivatives as tools for understanding epithelial cell biology.