Epidermal Cells Methods and Protocols
In recent years, our ability to understand and manipulate epidermal cells has increased tremendously, opening significant new possibilities in both basic science research and in regenerative medicine, including would healing and transplantation. Epidermal
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NIH-PA Author Manuscript
Published in final edited form as: Methods Mol Biol. 2010 ; 585: 83. doi:10.1007/978-1-60761-380-0_7.
Directed Differentiation of Human Embryonic Stem Cells to Epidermal Progenitors Christian M. Metallo, Lin Ji, Juan J. de Pablo, and Sean P. Palecek University of Wisconsin-Madison, Department of Chemical and Biological Engineering, 1415 Engineering Drive, Madison, WI 53706
Abstract
NIH-PA Author Manuscript
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 stagespecific 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.
Keywords Human embryonic stem cells; directed differentiation; ectoderm; retinoic acid; bone morphogenetic protein; p63; epithelial progenitors; keratinocytes
1. Introduction NIH-PA Author Manuscript
Human embryonic stem (hES) cells are capable of proliferating extensively and differentiating to form cells of the three embryonic germ layers (1). As such, these pluripotent cells can serve as a model system for studying early events during human development and are a powerful resource of non-transformed human cells for use in diagnostic and potentially therapeutic applications. Successful exploitation of hES cell derivatives requires the ability to direct hES cell differentiation to specific lineages in defined, efficient, and scalable systems (2). While researchers have identified strategies of obtaining endodermal, mesodermal, and neuroectodermal progenitors from hES cells (3-5), efficient methods of deriving non-neural epithelia have only recently been identified (6,7). The method described here involves treatment of hES cells with retinoic acid (RA) and bone morphogenetic protein-4 (BMP-4) in defined medium at specific stages of differentiation to direct hES cells to form relatively pure populations of epithelial cells and more definitive keratinocytes. Early induction of undifferentiated hES cell cultures is necessary to ensure efficient differentiation to epithelial lineages by RA and BMP4. This process is marked by significant increases in cytokeratin 18 (K18) and p63 transcription and relative decreases in pluripotency (e.g. Oct-4, SSEA-4, Nanog) and neural gene transcription (7), as measured by q
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