(321d) Retinoic Acid And Bmp Signaling Synergize To Efficiently Direct Human Embryonic Stem Cells To The Keratinocyte Lineage

Tissue engineered skin constructs are valuable tools for diagnostic testing, clinical therapy, and the study of studying epithelial differentiation, though their production requires large quantities of functional keratinocytes. While incorporation of primary cells has proven to be technically feasible, human embryonic stem cell (hESC)-derived keratinocytes are an attractive alternative given their enhanced proliferative capacity and pluripotency. However, current methods of differentiating hESCs to the keratinocyte lineage are inefficient and require feeder cells and/or undefined medium components. Here we employ quantitative analysis of differentiated hESC populations to identify key signaling factors involved in ectodermal lineage specification, demonstrating a novel, stage-specific effect of retinoic acid (RA) on hESC differentiation. When applied to undifferentiated hESC, RA effectively mediates expression of epithelial genes such as keratin 18 and the transcription factor p63. In conjunction with bone morphogenetic protein (BMP) signaling, RA efficiently directs hESCs to epithelial lineages, yielding essentially pure cultures expressing the basal keratinocyte marker keratin 14. Furthermore, hESC-derived keratinocytes are capable of expressing terminal differentiation markers and forming coherent epithelial sheets when presented with the appropriate cues. This method may therefore be used to produce relatively large quantities of functional, non-transformed keratinocytes for diagnostic and therapeutic applications under feeder-free, defined conditions.