Generation and Differentiation of Human Embryonic Stem Cell-Derived Keratinocyte Precursors

Abstract

Human embryonic stem cells (hESC) hold tremendous potential in the future of tissue engineering, offering promise as a source of virtually unlimited quantities of desired cell and tissue types. We have identified soluble chemical and extracellular matrix factors that permit isolation of keratinocyte precursors from hESCs. Culturing embryoid bodies (EB) formed from hESCs in a defined serum-free keratinocyte growth medium on a gelatin matrix generated keratin 14 (K14) expressing cells with an epithelial morphology. These K14 expressing cells could be subcultured in medium supplemented with hydrocortisone and induced to stratify and terminally differentiate by addition of calcium. Optimum times for obtaining K14 expressing cells were found for EB formation and for differentiation and growth of cultures after EB plating. EB formation was not necessary to generate keratinocyte precursors; direct transfer of hESC colonies to keratinocyte growth medium permitted differentiation into the keratinocyte lineage. With further studies to optimize generation and purification of hESC-derived keratinocyte precursors, these cells could provide a source of epidermal cells for skin tissue engineering applications in vitro or in vivo.

A Pure Population of Ectodermal Cells Derived from Human Embryonic Stem Cells

“…We and others have reported epidermal differentiation of mouse [2][3][4] and human [5][6][7][8] ES. The procedures used included the formation of embryoid bodies [2,6,8], direct culture as monolayers [3,9], and the use of fixed stromal-derived feeder along with bone morphogenetic protein 4 (BMP-4) treatment [3,4,8].…”

“…The procedures used included the formation of embryoid bodies [2,6,8], direct culture as monolayers [3,9], and the use of fixed stromal-derived feeder along with bone morphogenetic protein 4 (BMP-4) treatment [3,4,8]. The epidermal differentiation was spontaneous [2,7], induced by BMP-4 [3,4] or collagen IV [5], or induced through the cultivation of nodules derived from teratoma [9]. Whatever the protocol used, however, the commitment was inefficient, resulting in a highly heterogeneous cell population along with remaining undifferentiated ES, which could still produce teratoma.…”

Retinoic Acid and Bone Morphogenetic Protein Signaling Synergize to Efficiently Direct Epithelial Differentiation of Human Embryonic Stem Cells

“…Previous studies have demonstrated that spontaneous keratinocyte differentiation in EBs occurs inefficiently [2,4]. These hESC derivatives exhibited a lower proliferative capacity compared with primary keratinocytes, and immortalization was required to extend the lifespan of these cultures [3].…”

“…Human ESCs can differentiate to the keratinocyte lineage [2], although current methods are inefficient and isolated keratinocytes have a reduced expansion potential [3,4]. The hESC differentiation process toward keratinocytes is marked by expression of p63, a transcription factor necessary for maintenance of regenerative epithelia (i.e., the epidermis) [5][6][7], and keratin 14 (K14), an intermediate filament present in the basal layer of stratified squamous epithelia [8].…”

Temporal changes in Hox gene expression accompany endothelial cell differentiation of embryonic stem cells

“…[38][39][40] Bone morphogenetic protein receptor (BMPR) marks the mesenchymal lineage, whereas K14 is associated with epithelial differentiation. 38,39 Real-time PCR revealed that mesenchymal and epithelial marker expression gradually increased over time in culture ( Fig.…”