Direct conversion of mouse embryonic fibroblasts into functional keratinocytes through transient expression of pluripotency-related genes

Abstract

The insufficient ability of specialized cells such as neurons, cardiac myocytes, and epidermal cells to regenerate after tissue damage poses a great challenge to treat devastating injuries and ailments. Recent studies demonstrated that a diverse array of cell types can be directly derived from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or somatic cells by combinations of specific factors. The use of iPSCs and direct somatic cell fate conversion, or transdifferentiation, holds great promise for regenerative medicine as these techniques may circumvent obstacles related to immunological rejection and ethical considerations. However, producing iPSC-derived keratinocytes requires a lengthy two-step process of initially generating iPSCs and subsequently differentiating into skin cells, thereby elevating the risk of cellular damage accumulation and tumor formation. In this study, we describe the reprogramming of mouse embryonic fibroblasts into functional keratinocytes via the transient expression of pluripotency factors coupled with directed differentiation. The isolation of an iPSC intermediate is dispensable when using this method. Cells derived with this approach, termed induced keratinocytes (iKCs), morphologically resemble primary keratinocytes. Furthermore they express keratinocyte-specific markers, downregulate mesenchymal markers as well as the pluripotency factors Oct4, Sox2, and Klf4, and they show important functional characteristics of primary keratinocytes. iKCs can be further differentiated by high calcium administration in vitro and are capable of regenerating a fully stratified epidermis in vivo. Efficient conversion of somatic cells into keratinocytes could have important implications for studying genetic skin diseases and designing regenerative therapies to ameliorate devastating skin conditions.

Transcriptomic and Functional Evidence Show Similarities between Human Amniotic Epithelial Stem Cells and Keratinocytes

“…The goal of regenerative medicine is to replace diseased tissues with functional cells or cellular products. Many studies have sought to generate KCs from fibroblasts [ 29 , 52 ], adipose tissue stem cells [ 53 ], and mesenchymal cells via reprogramming [ 54 ]. Due to its critical role in epidermal development [ 55 ], TP63 was once combined with other TFs and used for this purpose [ 29 , 54 ].…”

Generation of Skin Organoids: Potential Opportunities and Challenges

“…Direct reprogramming techniques can convert adult cells from one type to another. The technology of fused assembloids is ripe to build different brain regions in cerebral organoids and this scheme is expected to bring new hope for reasonable source of functional skin ( Figure 1B ) ( Soufi et al, 2012 ; Iacovides et al, 2016 ; Qian et al, 2019 ).…”

Direct cell-fate conversion of somatic cells: Toward regenerative medicine and industries

“…In particular, initializing factors of iPSCs have frequently been used in combination with external signals. A wide variety of cells, e.g., O cells, 134) blood progenitor cells, 135) cardiomyo-cytes, 136) neural stem cells, 137),138) vascular endothelial cells, 139) neural crest cells, 140) hepatocytes, 140) oligodendrocyte progenitor cells, 141) and keratinocytes, 142) were previously generated and transplanted into model animals. Although these protocols are time-and cost-effective compared with iPSC-employing methods, the risk of tumorigenesis caused by cell initialization remains to be elucidated.…”