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Skin graft with dermis and appendages generated in vivo by cell competition

ABSTRACT: Autologous skin grafting is a standard treatment for skin defects such as burns. No artificial skin substitutes are functionally equivalent to autologous skin grafts. The cultured epidermis lacks the dermis and does not engraft deep wounds. Although reconstituted skin, which consists of cultured epidermal cells on a synthetic dermal substitute, can engraft deep wounds, it requires the wound bed to be well-vascularized and lacks skin appendages. In this study, we successfully generate complete skin grafts with PSC-derived epidermis with appendages on p63 knockout embryos' dermis. Donor PSC-derived keratinocytes encroach the embryos' dermis by eliminating p63 knockout keratinocytes based on cell-extracellular matrix adhesion mediated cell competition. Although the chimeric skin contains allogenic dermis, it is engraftable as long as autologous grafts. Furthermore, we could generate semi-humanized skin segments by human keratinocytes injection into the amnionic cavity of p63 knockout mice embryos. Niche encroachment opens the possibility of human skin graft production in livestock animals.

ORGANISM(S): Mus musculus

PROVIDER: GSE252023 | GEO | 2024/02/10

REPOSITORIES: GEO

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Project description:Cultured skin substitutes, prepared using keratinocytes, fibroblasts and biopolymers, can facilitate closure of massive burn wounds by increasing the availability of autologous tissue for grafting. However, because they contain only two cell types, skin substitutes cannot replace all of the functions of native human skin. To better understand the physiological and molecular differences between cultured skin substitutes and native skin, we undertook a comprehensive analysis of gene expression in native skin, cultured keratinocytes, cultured fibroblasts, and skin substitutes using Affymetrix gene chip microarrays. Goals: Our analysis focused on identifying gene signatures that were highly characteristic of each cell and tissue type, and those that are regulated by the formation of cultured skin substitute from the individual components. Normalization: We used a normalization and referencing strategy that consisted of BioConductor/RMA Express RMA processing of the entire series of cel files followed by a per gene normalization in which the median value of expression for each gene was derived from the cultured samples only, and this was used as a reference for all samples including the cultured skin substitute. This approach allowed for the identification of genes that were higher and lower-expressed in the cultured skin relative to the individual cell types that were also expressed strongly or weakly in normal skin relative to the median value established by the three cell types. Results Summary:We identified six major clusters of coordinately regulated genes that were the most differentially expressed between groups. These clusters correspond to biomarker pools representing expression signatures for native skin, fibroblasts, keratinocytes, and cultured skin. The expression analysis revealed that entire clusters of genes were either up-regulated or down-regulated upon combination of fibroblasts and keratinocytes in cultured skin grafts. Further, several categories of genes were overexpressed in cultured skin substitutes compared with native skin, including genes associated with hyperproliferative skin or activated keratinocytes. The observed pattern of expression indicates that cultured skin substitutes in vitro, which display a well-differentiated epidermal layer, exhibit skin-like differentiation relative to gene expression patterns in the individual cells. This consists of both the activation of normal skin signature genes and the suppression of keratinocyte and fibroblast signatures. There is also a signature consistent with a hyperproliferative phenotype similar to wounded native skin. Keywords: Cell interaction and co-culture response expression profile

Project description:Hair follicle formation depends on reciprocal epidermal-dermal interactions and occurs during skin development, but not in adult life. This suggests that the properties of dermal fibroblasts change during postnatal development. To examine this, we used a PdgfraEGFP mouse line to isolate GFP-positive fibroblasts from neonatal skin, adult telogen and anagen skin and adult skin in which ectopic hair follicles had been induced (EF skin) by transgenic epidermal activation of beta-catenin. We also isolated epidermal cells from each mouse. The gene expression profile of EF epidermis was most similar to that of anagen epidermis, consistent with activation of beta-catenin signalling. In contrast, adult dermis with ectopic hair follicles more closely resembled neonatal dermis than adult telogen or anagen dermis. In particular, genes associated with mitosis were upregulated and extracellular matrix-associated genes were downregulated in neonatal and EF fibroblasts. We confirmed that sustained epidermal beta-catenin activation stimulated fibroblasts to proliferate to reach the high cell density of neonatal skin. In addition, the extracellular matrix was comprehensively remodelled, with mature collagen being replaced by collagen subtypes normally present only in developing skin. The changes in proliferation and extracellular matrix composition originated from a specific subpopulation of fibroblasts located beneath the sebaceous gland. Our results show that adult dermis is an unexpectedly plastic tissue that can be reprogrammed to acquire the molecular, cellular and structural characteristics of neonatal dermis in response to cues from the overlying epidermis. We have isolated the following populations of cells from mouse back skin by flow cytometry: 1A) GFP+ WT neonatal dermal fibroblasts, 1B) ItgA6+ WT neonatal epidermal keratinocytes, 2A) GFP+ WT telogen dermal fibroblasts, 2B) ItgA6+ WT telogen epidermal keratinocytes, 3A) GFP+ D2 transient activation (anagen) dermal fibroblasts, 3B) ItgA6+ D2 transient activation (anagen) epidermal keratinocytes, 4A) GFP+ D2 sustained activation (ectopic follicles) dermal fibroblasts, 4B) ItgA6+ D2 sustained activation (ectopic follicles) epidermal keratinocytes

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Skin graft with dermis and appendages generated in vivo by cell competition

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