WITHDRAWN: Transdifferentiation of Human Neonatal Foreskin Fibroblasts into Keratinocyte-like Cells via Regulating the AKT/P53/WNT/LEF1 Axis

Abstract

Background: Wound healing is a dynamic, sequential, and complex physiological process, including a variety of cellular events, such as proliferation, adhesion, chemotaxis, and apoptosis. Skin fibroblasts (FBs) and keratinocytes (KCs) are the two most important cells involved in wound repair, and Relying on the proliferation and differentiation of keratinocytes to form epithelium to completely cover the wound is the ideal result for wound repair, so expanding the source of keratinocytes is a huge challenge. Objective: In this study, we examined the phenomenon that human neonatal foreskin fibroblasts (HFF) transdifferentiated into keratinocyte-like (KLCs) cells in conventional culture and evaluated the characteristics of KLCs and the potential mechanisms of the transdifferentiation process. Methods: The HFF and KCs were isolated with dynamic enzymolysis. HFF were routinely cultured in ordinary DMEM medium for more than 40 days, and observed the cell morphology. Western-blot, quantitative PCR (qPCR), immunofluorescence, and flow cytometry were adopted to assess the expression of the KCs markers cytokeratin 5, cytokeratin 14, cytokeratin 19, E-cadherin, Integrin β1 and the FBs marker Vimentin. Scratch wound assays, CCK-8 assays, and Transwell assays were conducted to test the function of KLCs. Mouse xenograft models were also used to evaluate the therapeutic effects and tumorigenicity of KLCs. High-throughput mRNA sequencing was also performed to explore the mechanism of cellular transformation. Results: HFF transdifferentiation started on day 25 and reached 98% by day 40. qPCR and Western-blot indicated significantly increased levels of the KCs markers (CK5, CK14, CK19, E-cadherin, and Integrin β1) in KLCs, and decreased FBs marker (Vimentin). Flow cytometry analysis demonstrated the number of cells expressing CK14 increased over time while the number of Vimentin-positive cells decreased. CCK8 results showed that the proliferation rates of KLCs and KCs were higher than HFF-1, but there was no obvious difference between KLCs and KCs. Scratch and Transwell assays revealed that the migration ability of KLCs and KCs was significantly lower than that of HFF. In vivo transplantation experiments indicated that there was no significant difference in the wound healing potential between KLCs and KCs. The transdifferentiation was regulated by the AKT/P53/WNT/LEF1 signaling pathway, and modulating the pathway could shorten the transdifferentiation time to 10 days. Conclusion: HFF can transdifferentiate into KLCs over time without intervention. This transdifferentiation process is regulated by the AKT/P53/WNT/LEF1 signaling pathway.