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. 2018 Dec 6;50(12):1-15.
doi: 10.1038/s12276-018-0185-z.

A novel and safe small molecule enhances hair follicle regeneration by facilitating metabolic reprogramming

Myung Jin Son  1   2 , Jae Kap Jeong  3   4 , Youjeong Kwon  5   6   7 , Jae-Sung Ryu  5 , Seon Ju Mun  5   6 , Hye Jin Kim  5   8 , Sung-Wuk Kim  3   9 , Sanghee Yoo  9 , Jiae Kook  9 , Hongbum Lee  9 , Janghwan Kim  5   6 , Kyung-Sook Chung  10   11
Affiliations

A novel and safe small molecule enhances hair follicle regeneration by facilitating metabolic reprogramming

Myung Jin Son et al. Exp Mol Med. .

Abstract

Targeting hair follicle regeneration has been investigated for the treatment of hair loss, and fundamental studies investigating stem cells and their niche have been described. However, knowledge of stem cell metabolism and the specific regulation of bioenergetics during the hair regeneration process is currently insufficient. Here, we report the hair regrowth-promoting effect of a newly synthesized novel small molecule, IM176OUT05 (IM), which activates stem cell metabolism. IM facilitated stemness induction and maintenance during an induced pluripotent stem cell generation process. IM treatment mildly inhibited mitochondrial oxidative phosphorylation and concurrently increased glycolysis, which accelerated stemness induction during the early phase of reprogramming. More importantly, the topical application of IM accelerated hair follicle regeneration by stimulating the progression of the hair follicle cycle to the anagen phase and increased the hair follicle number in mice. Furthermore, the stem cell population with a glycolytic metabotype appeared slightly earlier in the IM-treated mice. Stem cell and niche signaling involved in the hair regeneration process was also activated by the IM treatment during the early phase of hair follicle regeneration. Overall, these results show that the novel small molecule IM promotes tissue regeneration, specifically in hair regrowth, by restructuring the metabolic configuration of stem cells.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. IM176OUT05 (IM) improves the acquisition and maintenance of stem cell pluripotency.
a Chemical structure of IM. b Dose-response curve of the inhibition of the ETC by IM in the A549 lung carcinoma cell line. Cells were treated with serially diluted IM for 24 h, and the basal OCR was measured using a Sea Horse XF Analyzer. c MEFs and d HFFs were reprogrammed into iPSCs with OSKM reprogramming factors in the presence of 10 nM IM or rotenone. Representative images of AP+ colonies are shown (top). The total number of AP+ colonies was counted on day 14 (D14, MEFs) or day 28 (D28, HFFs) of reprogramming (bottom). e mESCs cultured under the non-self-renewing conditions (−LIF) or self-renewing conditions (+LIF) were treated with 10 nM IM for 4 days. Representative images of AP+ colonies (top) and the total number of AP+ colonies are shown (bottom). f hESCs were maintained under the self-renewing condition (CM). hESCs cultured under non-self-renewing conditions (UM) were treated with 10 nM IM for 6 days. Representative images of AP+ colonies (top) are shown, and the relative AP expression was quantified by scanning densitometry (bottom). *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test)
Fig. 2
Fig. 2. IM inhibits mitochondrial OXPHOS and increases lactate production by inducing the expression of glycolysis- and pluripotency-related genes.
a Schematic diagram of the reprogramming process. b OSKM-transduced MEFs were reseeded in poly-l-lysine-coated 96-well XF plates on D4. On the following day (D5), the medium was replaced with mESC medium and each indicated chemical. After 2 days (D7), the OCR was measured using an XFe96 Flux analyzer. An ATP synthase inhibitor (1.5 μM oligomycin, ETC complex V inhibitor), uncoupler (5 μM FCCP), and complex I inhibitor (0.5 μM rotenone) + complex III inhibitor (0.5 μM antimycin A) were sequentially added at each indicated time point. c Lactate production was measured in each treatment group on day 7 of reprogramming. d The ATP concentration was quantified in each treatment group on days 7 (D7) and 10 (D10) of reprogramming. e The expression of the indicated gene in each treatment group was measured using real-time PCR analysis on days 7 and 10 of reprogramming. β-Actin expression was used as an internal control. f ChIP assays were performed on day 7 of reprogramming with or without IM treatment. MEFs and miPSCs were used as negative/positive controls for each histone mark. Histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 trimethylation (H3K27me3) were precipitated, and the Nanog and Oct4 promoter loci were determined by real-time PCR. Input samples were used as a relative control. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test)
Fig. 3
Fig. 3. IM promotes hair regrowth in mice.
C57BL/6 mice in the telogen phase (7 weeks old) were depilated. Placebo control (−), 1% IM, or 1% minoxidil was topically applied daily to the dorsal skin. Representative photos of mice showing skin color darkness and hair regrowth on days 8, 10, 12, and 16 (left). The level of pigmentation was quantified by the intensity of the darkness of the back skin in the same area (right). *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test)
Fig. 4
Fig. 4. IM facilitates the cycle of hair follicle regeneration in mice.
a Representative images of H&E-stained longitudinal sections of each treated mouse skin on days 0 and 20 after depilation. b Progression of the hair follicle cycle on day 20 was quantitatively evaluated. Individual hair follicles were classified based on the classification by Chase. c Representative images of H&E-stained transverse sections of each treated mouse skin on days 0, 7, 14, and 20 after depilation. d Hair follicles were counted on days 7 and 20. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test). Scale bar = 200 μm
Fig. 5
Fig. 5. IM promotes the expression of hair follicle cycling-related markers.
a Immunohistochemistry of K15, β-catenin, and Ki67 in each treated mouse skin on day 7. Enlarged images of K15 in the top panels (dotted line) are shown in the second panels. b FACS analysis of K15/β-catenin and Ki67/Shh in single cells of each treated mouse skin on day 7. c Immunohistochemistry of K15, β-catenin, and Shh in each treated mouse skin on day 20. d FACS analysis of K15/β-catenin and Ki67/Shh in single cells of each treated mouse skin on day 20. DAPI staining was used to identify the nuclei (blue). Scale bar = 50 μm
Fig. 6
Fig. 6. IM stimulates the expression of stem cell metabolism-related and hair follicle regeneration-related genes during the early phases of hair regrowth.
Immunohistochemistry of K15, PDK, Ki67, and PDH in each treated mouse skin on a day 7 and b day 20. DAPI staining was used to identify the nuclei (blue). Scale bar = 50 μm. c FACS analysis of K15/PDK and Ki67/PDH in single cells of each treated mouse skin on day 7. d Expression of the indicated gene in each treated mouse was quantified by real-time PCR analysis on days 7 and 20 after depilation. β-Actin expression was used as an internal control. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test)
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