1) Identification of stem cells in the skin

The skin is the largest organ in the body. Hair follicles are mini-organs located in the skin that constantly renew themselves by alternate phases of growth, regression and rest. During this process, mature melanocytes (pigment cells) in hair follicles are replaced by a new cell population in each hair cycle. We previously identified the source of those melanocytes, “melanocyte stem cells” (McSCs), which are located in the hair follicle bulge and supply mature melanocytes required for hair and skin pigmentation (Nishimura EK et al. Nature, 2002). Subsequently, we identified similar McSC in sweat glands (Okamoto N et al. PCMR, 2014) .
We developed fate tracing technologies for McSCs and found that those cells are the origin of acral melanoma and succeeded in modeling acral melanoma in mice (Eshiba S et al.). Based on that, we developed a new method for differential diagnosis of acral melanoma from acral nevi (Eshiba S et al.). Further, we recently succeeded in identifying epidermal stem cells with sufficient self-renewing potential by using genetic tracing of stem cell clones (Liu N et al. Nature, 2019). As we have succeeded in identifying cultured human keratinocyte stem cells by deep learning-based automated cell tracking (Nanba D et al. in press), the system will provides a platform to a reliable and non-invasive technology for their quality control in regenerative medicine.

2) Mechanisms of stem cell maintenance

The underlying mechanisms of stem cell maintenance are a fundamental issue in stem cell biology and medicine. We have revealed that the niche microenvironment plays a dominant role in the fate determination of McSCs (Nishimura EK et al. Nature, 2002). That finding prompted us to further study the mechanisms involved and led us to demonstrate that hair follicle stem cells (HFSCs), which reside in the hair follicle bulge, serve as a functional niche for the maintenance of McSCs (Nishimura EK et al. Cell Stem Cell, 2010)(Tanimura S et al. Cell Stem Cell, 2011). We identified transforming growth factor β (TGF-β) secreted from HFSCs as niche-derived factors that is essential for McSC maintenance. Also we identified Mitf that encodes master regulator of melanocyte development and its downstream Bcl2 as critical for maintenance. Furthermore, the deficiency of those genes all leads to the progressive expression of hair graying phenotype. Therefore, we concluded that the incomplete maintenance of McSCs either by defective signaling from the niche or by intrinsic defects in stem cells, results in an insufficient supply of mature melanocytes expressing the progressive hair graying phenotype (Figure 1).

Figure 1: Aging and genomic stress abrogates the self-renewal of McSCs causing hair graying.

3) A self-renewal checkpoint underlies the quality maintenance of tissues

Physiological hair graying and hair thinning are typical outward signs of aging in mammals. We found that the incomplete maintenance of McSCs during the course of aging causes hair graying (Nishimura EK et al. Science, 2004). We then showed that genotoxic stress triggers/accelerates the aging process and abrogates the self-renewal of McSCs by triggering their differentiation (Inomata K et al. Cell, 2009). Further study of aged wild-type mice and progeroid mouse models, including ATM-deficient mice, revealed that a “self-renewal checkpoint”, which determines whether stem cells are qualified to self-renew or rather are forced to differentiate, maintains the quality of the stem cell pool and eliminates stressed/damaged stem cells from tissues (Inomata K et al. Cell, 2009). Similar checkpoint mechanisms have been found in HFSCs (Matsumura H et al. Science, 2016) and in epidermal stem cells (Liu N et al. Nature, 2019) by us and also in other somatic stem cells by other groups.

4) Dynamic elimination of aged stem cells causes hair follicle aging

To study the fate and dynamics of aged somatic stem cells, we performed in vivo fate tracing analysis of HFSCs and demonstrated that the dynamic elimination of HFSCs through their epidermal differentiation causes the stepwise miniaturization of hair follicles and eventual hair loss in mice. The DNA damage response in HFSCs causes proteolysis of Type XVII Collagen (COL17A1/BP180), a critical molecule for HFSC maintenance, to trigger HFSC aging that is characterized by the loss of stemness signatures and epidermal differentiation. Aged HFSCs are thus cyclically eliminated from the skin through their epidermal differentiation-mediated shedding from the skin surface, thereby causing hair follicle miniaturization (Figure 2).

Figure 2: Dynamic elimination of aged stem cells causes hair follicle aging.

The aging process can be recapitulated by Col17a1-deficiency and prevented by the forced maintenance of COL17A1 in HFSCs, demonstrating that COL17A1 in HFSCs orchestrates the stem cell-centric aging program of the epithelial mini-organ (Matsumura H et al. Science, 2016). Further, we analyzed the stem cell division axis in combination with fate tracing of HFSCs and identified the distinct stem cell division types that determine hair follicle regeneration and aging (Matsumura H et al. Science, 2016)(Figure 3).

Figure 3: Schematic model of the distinct types of stem cell divisions that determine hair follicle regeneration and aging.

5) Stem cell competition in the epidermis underlies skin homeostasis and aging

The skin protects living organisms from the outside world by acting as a barrier throughout the life-span, suggesting that the skin has more robust and flexible anti-aging mechanisms than mini-organs such as hair follicles. We have performed in vivo clonal analysis in mice by focusing on the expression of the hemidesmosomal protein COL17A1 by epidermal stem cells. Those studies revealed that the expression of COL17A1 fluctuates physiologically through genomic/oxidative stress-induced proteolysis, and that the resulting differential expression of COL17A1 in individual stem cells generates a driving force for cell competition (Figure 3). Clones that express high levels of COL17A1 divide symmetrically and outcompete/eliminate adjacent stressed clones that express low levels of COL17A1 and divide asymmetrically. Stem cells with higher potential or quality are thus selected for homeostasis, but their eventual loss of COL17A1 limits their competition, thereby causing aging. The resulting hemidesmosome fragility and stem cell delamination depletes adjacent melanocytes and fibroblasts to promote skin aging. Conversely, the forced maintenance of COL17A1 rescues skin organ aging, thereby indicating potential new approaches for anti-aging therapeutic intervention. We are searching for small molecules that provide somatic stem cells with high regenerative potential and quality that have clinical application.

Figure 4: Stem cell competition orchestrates skin homeostasis and aging. SCD: symmetric cell division; ACD : asymmetric cell division.

1)	Matsumura H, Liu N, Nanba D, Ichinose S, Takada A, Kurata S, Morinaga H, Mohri Y, De Arcangelis A, Ohno S, Nishimura EKDistinct types of stem cell divisions determine organ regeneration and aging in hair follicles. Nature Aging, 1: 190-204, 2021
2)	Al-Busani H, Al-Sobaihi S, Nojima K, Tanemura A, Yaguchi T, Kawakami Y, Matsumura H, Nishimura EK, Yokozeki H, Namiki TNUAK2 localization in normal skin and its expression in a variety of skin tumors with YAP. J Dermatol Sci., 97(2): 143-151, 2020
3)	Liu N, Matsumura H, Kato T, Ichinose S, Takada A, Namiki T, Asakawa K, Morinaga H, Mohri Y, De Arcangelis, Geroges-Labousse E, Nanba D, Nishimura EK. Stem cell competition orchestrates skin homeostasis and ageing. Nature, 568(7752):344-350, 2019
4)	Matsumura H, Mohri Y, Binh NT, Morinaga H, Fukuda M, Ito M, Kurata S, Hoeijmakers J, Nishimura EK. Hair follicle aging is driven by transepidermal elimination of stem cells via COL17A1 proteolysis. Science, 351(6273):535, 2016
5)	Okamoto N, Aoto T, Uhara H, Yamazaki S, Akutsu H, Umezawa A, Nakauchi H, Miyachi Y, Saida T, Nishimura EK. A melanocyte-melanoma precursor niche in sweat glands of volar skin. Pigment Cell & Melanoma research, 27(6):1039-1050, 2014
6)	Tanimura S, Tadokoro Y, Inomata K, Nishie W, Yamazaki S, Nakauchi H, Tanaka Y, McMillan JR, Sawamura D, Yancey K, Shimizu H, Nishimura EK. Hair follicle stem cells provide a functional niche for melanocyte stem cells. Cell Stem Cell, 8(2):177-187, 2011
7)	Nishimura EK, Suzuki M, Igras V, Du J, Lonning S, Miyachi Y, Roes J, Beermann F, Fisher DE. Key roles for transforming growth factor Beta in melanocyte stem cell maintenance. Cell Stem Cell, 6(2):130-40, 2010
8)	Inomata K, Aoto T, Binh NT, Okamoto N, Tanimura S, Wakayama T, Iseki S, Hara E, Masunaga T, Shimizu H, Nishimura EK. Genotoxic stress abrogates renewal of melanocyte stem cell by triggering their differentiation. Cell, 137(6):1088-99, 2009
9)	Nishimura EK*., Granter, S.R., Fisher DE. Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche Science, 307(5710):720-724, 2005
10)	Nishimura, E.K., Jordan, S.A, Oshima, H., Yoshida, H., Osawa, M., Jackson, I.J., Barrandon, Y., Yoshiki, M. Nishikawa, SI Dominant Role of the Niche in Melanocyte Stem Cell Fate Determination. Nature, 416(6883):854-60, 2002.