3D In Vitro Model of a Functional Epidermal Permeability Barrier from Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

Abstract

SummaryCornification and epidermal barrier defects are associated with a number of clinically diverse skin disorders. However, a suitable in vitro model for studying normal barrier function and barrier defects is still lacking. Here, we demonstrate the generation of human epidermal equivalents (HEEs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). HEEs are structurally similar to native epidermis, with a functional permeability barrier. We exposed a pure population of hESC/iPSC-derived keratinocytes, whose transcriptome corresponds to the gene signature of normal primary human keratinocytes (NHKs), to a sequential high-to-low humidity environment in an air/liquid interface culture. The resulting HEEs had all of the cellular strata of the human epidermis, with skin barrier properties similar to those of normal skin. Such HEEs generated from disease-specific iPSCs will be an invaluable tool not only for dissecting molecular mechanisms that lead to epidermal barrier defects but also for drug development and screening.

Induced Pluripotent Stem Cell Differentiation and Three-Dimensional Tissue Formation Attenuate Clonal Epigenetic Differences in Trichohyalin

“…Human neonatal foreskin fibroblasts BJ (ATCC, CRL-2522) were reprogrammed using modified synthetic mRNA as described [10]. At day 2-17 of reprogramming, some cells also were treated with 1 mM pifithrin-a.…”

“…At day 2-17 of reprogramming, some cells also were treated with 1 mM pifithrin-a. Two clones with a similar growth rate, one derived in the absence (iKCL004) and one in the presence of 1 mM pifithrin-a (iKCL011), were further characterized; pluripotency marker expression and differentiation into three germ layers in vitro and in vivo (teratomas) revealed no obvious difference between the lines [10].…”

“…Differentiation into keratinocytes and generation of HEEs were described previously [10]. Transepithelial electrical resistance was measured with epithelial voltohmmeter EVOM (World Precision Instruments) as described [10,21]. Normal human keratinocytes (NHK) were cultured in EpiLife (Life Technologies).…”

“…A total amount of 750 ng of biotin-labeled aRNA in a 5 mL volume was then used for HumanHT-12 Expression BeadChip whole-genome gene expression direct hybridization assay system (Illumina) according to manufacturer's instructions, run on iScan system (Illumina) and analyzed as described [10]. All samples were analyzed as biological replicates from three independent experiments.…”

“…Two iPSC clones, iKCL004 and iKCL011, that we used to build human skin equivalents [human epidermal equivalent (HEE)] with a functional permeability barrier [10] showed subtle differences, which prompted us to investigate in depth the genetic and epigenetic footprint of both lines. Since the focus of our work was keratinocyte differentiation culminating in the stratum corneum derived epidermal permeability barrier, we concentrated on the epidermal differentiation complex (EDC) on chromosome 1, which contains multiple genes involved in epidermal cornification [11][12][13][14][15][16][17][18][19].…”