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Gene expression profile of hESC/hIPC-derived keratinocytes

ABSTRACT: To induce the differentiation, undifferentiated human pluripotent stem cells (both hESC andh iPSC; T0 time point) were transferred into 20% O2 atmosphere environment and treated with mTESR1 basal media supplemented with 1 μM ATRA (Sigma-Aldrich) and 25 ng/ml BMP4 (R&D) for 7 days (Induction). To select for the cells that acquired early ectodermal fate, cells were harvested and re-plated onto freshly prepared 3D human dermal fibroblast ECM at a density of 5-10x10^3 cells per cm2 and grown in DMEM:Ham F12 (3:1) (Life Technologies) supplemented with 1 μM ATRA and 25 ng/ml BMP4 for a further 7 days (Selection). To enrich for putative epidermal progenitors, rapid-adhesion to type IV collagen-coated dishes was used, and the rapidly-adhering cells were cultured in DK SFM supplemented with 1 μM ATRA for 7 days (Enrichment). After that, the cells were cultured in EpiLife medium (Life Technologies) for a further 7 days (Expansion) before final harvest (T3 time point) and analysis. We analyzed here gene expression profiles of undifferentiated hESC/hiPSC (T0), hESC/hIPC-derived keratinocytes (T3) and primary normal human keratinocytes from skin biopsy (NHK). We found that hESC/hIPC-derived keratinocytes are similar to NHK.

ORGANISM(S): Homo sapiens

PROVIDER: GSE55898 | GEO | 2015/04/01

SECONDARY ACCESSION(S): PRJNA241270

REPOSITORIES: GEO

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Project description:Targets of Retinoic Acid (RA) and 3,4-didehydroretinoic acid (ddRA) were identified in primary human epidermal keratinocytes grown in the presence of atRA or ddRA for 4 and 24 hours. Retinoids (natural forms and synthetic derivatives of vitamin A) are used as therapeutic agents for numerous skin diseases such as keratinization disorders (e.g. ichthyoses) and psoriasis. Two endogenous ligands for retinoic acid receptors exist, retinoic acid (atRA) and 3,4-didehydroretinoic acid (ddRA). In primary human epidermal keratinocytes many transcriptional targets for atRA are known, whereas the targets for ddRA are unknown. In an attempt to determine the targets, we compared the effect of atRA and ddRA on transcriptional profiles in undifferentiated and differentiating human primary keratinocytes. First, as expected, many genes were induced or suppressed in response to keratinocyte differentiation. Furthermore, the two retinoids affected substantially more genes in differentiated keratinocytes (more than 350) than in proliferating keratinocytes (20). In differentiating keratinocytes markers of cornification were suppressed suggesting a de-differentiating effect by the two retinoids. When comparing the expression profile of atRA to that of ddRA, no differently regulated genes were found. The array analysis also found that a minor number of miRNAs and a large number of non-coding transcripts were changed during differentiation and in response to the two retinoids. Furthermore, the expression of all, except one, genes known to cause autosomal recessive congenital ichthyosis (ARCI) were found to be induced by differentiation. These results comprehensively document that atRA and ddRA exert similar transcriptional changes in keratinocytes and also add new insights into the molecular mechanism influenced by retinoids in the epidermis. Furthermore, it suggests which ARCI patients could benefit from therapy with retinoids. Proliferating and differentiated keratinocytes were harvested after 4 and 24 hours after treatment with atRA, ddRA and vehicle for RNA extraction and hybridization on Affymetrix microarrays. Triplicate samples were generated for each time point and each treatment.

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Gene expression profile of hESC/hIPC-derived keratinocytes

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