(6cp) Directed Differentiation and Tissue Engineering of Keratinocytes Derived from Human Embryonic Stem Cells

Engineered tissue constructs with regenerative capacity require the incorporation of relatively large quantities of functional, lineage committed progenitors. Given their enhanced proliferative capacity and pluripotency, human embryonic stem cells (hESCs) are an attractive source of cells for tissue engineering. However, highly efficient processes must be developed to direct hESCs through developmental pathways to the appropriate lineages in order to exploit the technological potential of these cells. Formulation of such processes requires integration of engineering strategies with developmental biology concepts to effectively produce cells and tissues for scientific, diagnostic, and therapeutic use. Here we employ quantitative analysis of differentiated hESC populations to identify key signaling factors involved in ectodermal lineage specification; our primary focus is the generation of epithelial precursors that can be used to engineer regenerative constructs for the skin or related tissues. Application of retinoic acid (RA) to undifferentiated hESC populations, in conjunction with bone morphogenetic protein (BMP) signaling, efficiently mediates epithelial rather than neural differentiation. This differentiation process can be effectively used to generate essentially pure populations of hESC-derived keratinocytes under defined, feeder-free conditions. When coupled with the self-renewal capacity of hESCs, this method can produce relatively large quantities of functional, non-transformed keratinocytes. Furthermore, hESC-derived keratinocytes exhibit the capacity to terminally differentiate and form coherent epithelial sheets when presented with the appropriate cues. This differentiation process may therefore be used to produce epithelial tissues for scientific or clinical use.