Directed differentiation of human embryonic stem cells to epidermal progenitors

Abstract

Human embryonic stem (hES) cells can differentiate into virtually all somatic cell types. In order to incorporate these derivatives into scientific or clinical applications, efficient methods of directing hES cell differentiation to pure subpopulations are required. Here we describe a robust strategy for generating cytokeratin 14+ (K14+)/p63+ keratinocyte progenitors from hES cells through stage-specific application of retinoic acid (RA) and bone morphogenetic protein-4 (BMP4). Induction of undifferentiated hES cells with RA stimulates expression of epithelial genes such as K18 and p63. Subculture of RA-treated cells in defined keratinocyte medium enables isolation of relatively pure K14+ epithelial populations; these cells also retain the capacity to terminally differentiate. The use of defined media throughout differentiation allows for detailed characterization of keratinocyte lineage specification from hES cells through the use of gene expression and immunofluorescence analyses.

Figure 1

Immunofluorescent staining of hES cell-derived keratinocyte progenitors. A, B) Subcultured keratinocytes (as in step 3.1.5. of protocol) immunostained against the cytoskeletal protein K14 (A) and the nuclear transcription factor p63 (B). C) Differentiated hES cells (after 3 weeks culture in step 3.1.4. of protocol) stained against β-catenin; note punctate staining at membranes. D) Keratinocyte progenitors cultured to confluence and treated with 0.8 mM Ca2+ (after 4 weeks of culture in step 3.1.4. of protocol), then stained against the terminal differentiation marker filaggrin; note localization within suprabasal layers. Scale bar denotes 50 μm.