UC Irvine

In the study conducted, the histological and cellular characteristics of human skin equivalents (HSEs) were explored. These HSEs were created by seeding primary human keratinocytes on devitalized human dermis, treated with either Matrigel (GelHSE) or primary human dermal fibroblasts (FibHSE). A histological analysis was performed revealing similarities between HSEs and in vivo human epidermis, with notable differences such as a thicker living epidermal layer in FibHSEs and reduced keratinocyte proliferation.

Cellular states of keratinocytes from 28-day old HSEs were identified using droplet-enabled scRNA-seq. These transcriptomes were similar to in vivo states, but unique gene expression patterns were identified, including a novel cell state, HSE-1, primarily in the GelHSE cultures.

Despite the predominantly normal histology of HSEs, abnormalities such as an expansion of KRT14+ cell layers and disrupted epidermal differentiation were present. Evidence of a partial epithelial-to-mesenchymal transition (EMT) state was detected, and the expression of the PSCA gene in the HSE-1 cluster was observed, suggesting a residual embryonic program in the culture medium. An examination of the lineage trajectory of HSE epidermal differentiation was conducted, revealing two abnormalities: differentiated keratinocytes retained much of the basal keratinocytes’ gene expression signature and the GelHSE keratinocytes followed a basal to spinous to HSE-unique lineage. All of these changes were evident at the RNA and protein levels.

To address these abnormalities, HSEs were xenografted onto mice, leading to the emergence of three xenograft-specific clusters alongside the expected keratinocyte clusters. This process was found to partially rectify terminal differentiation, cell-cell adhesion, and basal programs, yet it also revealed two distinct transcriptional paths from basal to granular keratinocytes. Hypoxia was identified as a potential driver of the transcriptional changes observed in the xenograft-specific cells. The conducted research has enhanced the understanding of the cellular states and differentiation paths in HSEs and has suggested potential strategies for their enhancement, such as hypoxic culturing and xenografting.