Human embryonic stem cell-derived keratinocytes exhibit an epidermal transcription program and undergo epithelial morphogenesis in engineered tissue constructs

Abstract

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 14+ (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 Ca(2+), 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.

FIG. 1.

Retinoic acid-induced differentiation of hES cells yields K14+ populations of similar purity to primary keratinocytes. Flow cytometry analysis of K14 expression in hEK (A) and PFK (B) cultures demonstrated that both achieved >95% K14+ purity. hES, human embryonic stem; hEKs, hES cell-derived keratinocytes; K14, keratin 14+; PFKs, primary foreskin keratinocytes.

FIG. 2.

Terminal differentiation of hEK cells in submerged cultures. (A) Phase contrast and (B) immunofluorescent images of confluent hES cell-derived epithelial cells stained against Mucin 1. (C, D) Confluent culture of hES cell-derived epithelia stained for K14 (C) and K19 (D). Images were taken at different focal planes. Note suprabasal cells stained positive for K19. Scale bars = 50 μm. (E) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis/western blot analyses of hES cell-derived epithelia treated with the specified media for 24 h.

FIG. 3.

HE staining and immunohistochemistry analyses of organotypic skin cultures cultured at the air–liquid interface for 2–3 weeks. (A, C, D, G, J, M) Organotypic cultures engineered with hES derivatives. (B, E, H, K, N) Organotypic tissues engineered with foreskin keratinocytes. (F, I, L, O) Human skin. (A–C) HE staining depicts stratified and cornified epithelial morphology in hES cell-derived epithelium (A, C) and foreskin keratinocyte engineered tissue (B). Intracellular bridges are evident in (C) (see arrows). (D–O) Immunohistochemistry analysis against the listed antigen reveals localization of markers within the epithelium of engineered hES cell-derived epithelium (D, G, J, M), foreskin keratinocyte-derived epithelium (E, H, K, N), and primary human skin (F, I, L, O). Images were taken from at least three independent experiments. Negative controls containing no primary antibody were included for all analysis. Arrows indicate specific areas of filaggrin staining (M, N). Scale bars = 50 μm for all images except (C). Scale bar = 16 μm for (C). HE, hematoxylin and eosin.