CRISPR/dCas9-mediated activation of SOX18 reprograms human dermal fibroblasts into dermal papilla cells

Summary: Abstract Body: A major contributing factor to the failure of cell-based human hair follicle (HF) regeneration is the lack of highly specialized and inductive mesenchymal fibroblasts, located in the HF dermal papilla (DP). Thus, there is great demand to derive a sufficient amount of inductive DP cells (DPCs) from small amounts of tissue. Dermal fibroblasts (DFs) are abundant and can be isolated and expanded easily, making DFs an ideal cell source for HF engineering. Bioinformatic analysis identified five major transcription factors (TFs) central to DP phenotype and inductivity. Via CRISPR activation, each TF was enriched in DFs and their extent of reprogramming compared. Among them, SOX18-overexpressing DFs (SOX18-DFs) were the only transduced DFs that induced the expression of alkaline phosphatase (ALPL) at a level comparable to DPCs. In hydrogel and 3D spheroid culture, SOX18-DFs exhibit colony-forming behavior and maintain significantly higher ALPL expression for longer time than other TF-overexpressing DFs, comparable to passage 5 DPCs. RT-qPCR showed SOX18-DFs express DPC signature genes ALPL, FGF2, FGF7, FGF10, CTNNB1, and LEF1 significantly higher than control DFs (p < 0.05). We have previously shown that DPCs but not DFs interact with human epidermal keratinocytes (KCs) to form hair germ-like structures in organoid culture. Following this method, reprogrammed DFs were seeded in mixed suspension with keratinocytes for 3D co-culture. Among the TF-overexpressing DFs and controls, only organoids constructed with SOX18-DFs exhibit strong ALPL-positivity and advanced structure formation comparable to those constructed from DPCs. Application of the organoids to micropatterned engineered skin substitute showed that SOX18-DF organoids continue to develop ex vivo. Our findings demonstrate that genetic reprogramming of human DFs, including with SOX18, is a feasible approach to overcome the key bottleneck in HF engineering: generating human DPCs with sufficient activity and quantity. Linli Zhou¹, Lindsey Siegfried¹, Thomas Andl², Yuhang Zhang¹ 1. College of Pharmacy, University of Cincinnati, Cincinnati, OH, United States. 2. Burnett School of Biomedical Sciences, University of Central Florida, Orlando, FL, United States. Stem Cell Biology, Tissue Regeneration and Wound Healing