Hair growth promoting effects of human dermal papilla cells in pig.

Dermal papilla (DP) is important in maintaining the stem cell niche and stimulates the hair follicle (HF)'s re-entry into anagen during cycling. It is worth noting that both the isolated DP and cultured dermal papilla cells (DPCs) from mouse, rat and human induced follicle formation contact with the epithelial cells.1, 2 Phase II clinical trials were conducted in the early 2000s to evaluate the efficacy using DPCs with epidermal cells for hair regeneration, and some hair growth was observed.3 However, combinational cell therapy for hair growth failed to show expected cosmetic efficacy, leading companies to develop cell therapy focusing on rejuvenation of miniaturized HFs.4 Though numerous researches indicate the hair growth promoting effects of human DPCs (hDPCs) in rodents, preclinical or clinical application alone has not been reported in pig or human until now.2, 5 Pig is an increasingly popular biomedical model, and the atlas of prenatal hair follicle morphogenesis using the pig has been reported.6 For example, human and pig share high similarities in HF morphogenesis occurrence time (early/mid gestational) and marker gene expression patterns. Therefore, pig model facilitates the research on complex hair diseases and offers researchers a suitable model for human hair research. In a previous study, hDPCs cultured under hypoxia improved hair inductivity of hDPCs via nuclear NADPH oxidase 4-mediated reactive oxygen species generation.5 In that study, we tested the hair growth promoting effects of hDPCs and its underlying molecular mechanism in C3H and hairless mice. However, it is difficult to predict how often hDPCs needs to be injected and how long they will survive in human while using rodent models. Therefore, in the present study we studied the hair growth promoting effects of hDPCs in the pig, and primarily investigated whether or not intradermal injection of hDPCs could increase the hair number and thickness. We have used three pigs, and intradermally injected low and high dose of hDPCs in 6 sites of the back skin of pig (Figure 1A,B; Appendix S1). At first, HF number and thickness were measured after a single injection of hDPCs after 3 months. As expected, single injection of hDPCs increased the HF number for more than 3 months (Figure 1C,D), and the hair thickness for 1 month. However, there was no difference in HF number and thickness between low and high dose of hDPCs. In addition, we injected hDPCs twice (Figure S1) and three times (Figure S2) in a monthly interval, which also increased the HF number and thickness. We have also found that single injection of hDPCs was more effective in increasing HF number and thickness compared with multiple injection. These might be due to the fact that the survival of hDPCs was decreased by multiple injection in the pig which is demonstrated by tracing of hDPCs labelled by PKH26.5 The PKH26-labelled hDPCs of single (Figure 1H) and double (Figure S3) injection were detected, whereas not for the triple injection. PKH26-labelled hDPCs were mostly localized in the injection site, and were not detected in DP or the surrounding HF, which indicates that the hair growth promoting effects of hDPCs are primarily mediated by paracrine effects instead of building-block function. In addition, mixed lymphocyte reaction assays were performed to test for the immunogenicity of the injected hDPCs.7 Proliferation of lymphocyte and secreted interferon-γ concentration were very low when co-cultured with hDPCs (Figure S4). These indicate that immunological reactions of DPCs might be negligible in human. Three months after the intradermal injection, we sacrificed the pigs, and histologically analysed the skin of the injection sites. H&E staining of the back skin in the injection site showed that the HF number has increased in the hDPCs injection group (Figure 1E). DP area was also measured, and we have found that single injection of DPCs significantly increased the DP area in the pig HF (Figure 1F). In addition, we observed microvessels around HF using immunohistochemical staining of CD31, and the number of microvessles around HF was increased in the hDPC-treated group (Figure 1G). However, repeated injection of hDPCs was not as effective as single injection (data not shown). These results collectively indicate that hDPCs increased the HF number, thickness and the angiogenesis in pig. In addition, single injection of hDPCs might be effective and survive for more than 3 months in clinical study. JH: Methodology, Formal analysis, Investigation, Data curation. TNHL: Formal analysis. MZ: Validation, Project administration. HK: Validation. JHS: Conceptualization, Writing-Review & Editing, Supervision. All authors read and approved the final manuscript. The human hair follicles were kindly provided by MD. Jino Kim (New Hair Institute, Seoul, South Korea). This research was supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (HN22C0027, Republic of Korea) and Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Health & Welfare) (23C0125L1, Republic of Korea). The authors declare no conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1. The number of hair follicles and hair thickness were increased after double intradermal injection of hDPCs. Figure S2. The number of hair follicles and hair thickness were increased after triple intradermal injection of hDPCs. Figure S3. Survival of hDPCs was detected after double injection. Figure S4. The PBMC were co-cultured with hDPCs to test immunogenicity of injected hDPCs. Appendix S1. Materials and methods. 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