| Summary: | Human embryonic stem (hES) cells are an attractive source of cellular material for scientific, diagnostic, and
potential therapeutic applications. Protocols are now available to direct hES cell differentiation to specific lineages
at high purity under relatively defined conditions; however, researchers must establish the functional
similarity of hES cell derivatives and associated primary cell types to validate their utility. Using retinoic acid to
initiate differentiation, we generated high-purity populations of keratin 14þ (K14) hES cell-derived keratinocyte
(hEK) progenitors and performed microarray analysis to compare the global transcriptional program of hEKs
and primary foreskin keratinocytes. Transcriptional patterns were largely similar, though gene ontology analysis
identified that genes associated with signal transduction and extracellular matrix were upregulated in hEKs. In
addition, we evaluated the ability of hEKs to detect and respond to environmental stimuli such as Ca2þ, serum,
and culture at the air–liquid interface. When cultivated on dermal constructs formed with collagen gels and
human dermal fibroblasts, hEKs survived and proliferated for 3 weeks in engineered tissue constructs. Maintenance
at the air–liquid interface induced stratification of surface epithelium, and immunohistochemistry results
indicated that markers of differentiation (e.g., keratin 10, involucrin, and filaggrin) were localized to
suprabasal layers. Although the overall tissue morphology was significantly different compared with human
skin samples, organotypic cultures generated with hEKs and primary foreskin keratinocytes were quite similar,
suggesting these cell types respond to this microenvironment in a similar manner. These results represent an
important step in characterizing the functional similarity of hEKs to primary epithelia.
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