In vivo regeneration of hair-bearing skin through reprogramming of mesenchymal cells

Purpose:
Mammalian skin appendages, such as hair follicles and sweat glands, are complex mini-organs formed during skin development1). However, scar tissue resulting from wound healing lacks these appendages, presenting a clinical challenge in skin regeneration2). We have developed a method to generate skin epithelial tissues through in vivo reprogramming of wound-resident mesenchymal cells using four transcription factors: DNp63A, GRHL2, TFAP2A, and cMYC. This process yields cells capable of forming stratified epithelia, which we term Induced Stratified Epithelium Progenitors (DGTM-iSEPs)3). Nevertheless, the regenerated skin lacked appendages. Considering that skin appendages form during development, we hypothesized that reprogramming adult mesenchymal cells to epithelial and mesenchymal cells akin to those in developing skin could enable in situ skin appendage generation.

Methods and Materials:
We employed a traditional skin reconstitution assay as the functional test to identify a set of reprogramming genes capable of regenerating skin appendages4). This assay involved transplanting a mixture of skin-derived epithelial cells (SECs) and subcutaneous mesenchymal cells (SMCs) into a silicone chamber attached to a skin ulcer on immunodeficient mice. We tested eligible genes by measuring dermal papilla markers (PROM1, CRABP1, VCAN) and alkaline phosphatase (ALP) expression following the transduction of each gene individually and in combination, using retroviruses.

Results:
Our initial goal was to identify genes that reprogram adult SECs into epithelial cells capable of reconstituting skin appendages alongside neonatal SMCs. Through the skin reconstitution assay, we found that DGTM+LEF1-induced stratified epithelium progenitors (DGTML-iSEPs) could regenerate skin appendages with neonatal SMCs. We also explored the gene combinations required to reprogram adult SMCs into mesenchymal cells with the potential for skin appendage regeneration. Co-transplanting FOXD1+PRDM1-transduced subcutaneous mesenchymal cells (FP-SMCs) and LEF1+SHH-transduced subcutaneous mesenchymal cells (LS-SMCs) with DGTML-iSEPs resulted in the formation of mature hair shafts, hair follicles, and sebaceous gland-like structures. GFP-labeled adult SMCs used in generating DGTML-iSEPs, FP-SMCs, and LS-SMCs, when co-transplanted with unlabeled cells, demonstrated that these cell types were integral to regenerated skin appendages.

Conclusions:
Our study demonstrates that transplanting a specific type of epithelial cell and two types of mesenchymal cells, all reprogrammed from adult mouse subcutaneous mesenchymal cells to resemble developing skin cells, can lead to the formation of skin-appendage-like structures. These findings could open new avenues for treating skin regeneration issues and common aging-associated skin appendage disorders, such as hair loss and dry skin. This approach also holds potential for the de novo generation of complex organs in vivo.

1) Sennett, R. et al. An integrated transcriptome atlas of embryonic hair follicle progenitors, their niche, and the developing skin. Dev. Cell 34, 577–591 (2015).
2) Lee, J. et al. Hair follicle development in mouse pluripotent stem cell-derived skin organoids. Cell Rep. 22, 242–254 (2018).
3) Kurita, M. et al. In vivo reprogramming of wound-resident cells generates skin epithelial tissue. Nature 561, 243–247 (2018).
4) Worst P. K., Mackenzie, I. C. & Fusenig, N. E. Reformation of organized epidermal structure by transplantation of suspensions and cultures of epidermal and dermal cells. Cell Tissue Res. 225, 65–77 (1982).

Research and Technology

PSTM 2024