Hair follicle stem cell marker nestin expression in regenerating hair follicles of patients with alopecia areata

ARTICLE

ejd.2011.1306

Auteur(s) : Yasuyuki AMOH amo@med.kitasato-u.ac.jp, Hideki MAEJIMA, Shiro NIIYAMA, Sumiyuki MII, Yuko HAMADA, Norimitsu SAITO, Kensei KATSUOKA

Department of Dermatology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami Ward, Sagamihara 252-0373, Japan

Reprints: Y. AMOH

The neuron-specific-stem cell marker nestin is expressed in the hair follicle stem cell (hfPS) area (hfPSA) of transgenic mice with nestin-regulatory-element-driven GFP [4-8]. During early anagen or the growth phase of the hair follicle, nestin-expressing cells, as marked by GFP fluorescence in nestin-GFP transgenic mice, appear specifically in the hfPSA, located immediately below the sebaceous glands and above the hair follicle bulge area [4-8]. Using immunohistochemistry, the nestin-expressing hfPS located in the hfPSA have also been shown to be keratin 15 (K15) negative. Furthermore, hfPS isolated from mouse and human skin is able to differentiate into Schwann cells and form myelin sheaths in C57BL/6 immunocompetent mice with a severed sciatic nerve [9, 12]. In this experimental model, the hfPS promoted axonal growth and rejoining of the pre-existing neurons. These results suggest that hair follicle stem cells provide an easily accessible and autologous source of stem cells for transplantation.

We recently demonstrated that the anagen hair follicles located in the human scalp contain a population of cells that are both nestin positive and K15 negative [11]. Within the hair follicle, the nestin-positive and K15-negative cells are located immediately below the sebaceous glands and above the hair follicle bulge area. These cells formed cell colonies and differentiated into neurons, glial cells, keratinocytes and smooth muscle cells [12]. In addition, anagen hair follicles removed from the human scalp by depilation contained nestin-negative and K15-positive cells, but not nestin-positive and K15-negative cells. The plucked hair follicles also formed cell colonies and differentiated into K15-positive keratinocytes. Taken together, the nestin-positive and K15-negative cells present in the hair follicle appear to represent progenitor cells for neurons, glial cells, keratinocytes and smooth muscle cells, while the nestin-negative and K15-positive cells present in the depilated hair follicles represent keratinocyte progenitor cells [11]. In the present study, we observed nestin immunoreactivity in the early and middle anagen phases of hair follicle development in patients with alopecia areata.

Materials and methods

Human scalp skin of hair follicles from individuals with normal skin and alopecia areata

For immunohistochemical staining, human scalp skin samples were obtained from surgical specimens of 5 samples with normal human scalp skin (3 males and 2 females; mean age, 27.0 years; range, 10-48 years) and 8 patient samples with alopecia areata (4 males and 4 females; mean age, 37.3 years; range, 21-53 years). All experiments were performed according to the Helsinki guidelines and were in compliance with national regulations for the experimental use of human material. The surgical specimens of human scalp skin were taken from patients, who gave their informed consent. Hematoxylin and eosin (H&E) staining was used to verify the presence of alopecia areata.

Immunohistochemical analysis of normal and alopecia areata skin

Immunohistochemical stainings for nestin, K15 and CD34 in longitudinal sections from skin biopsies of human scalp were undertaken using the DAB peroxidase substrate kit (brown) (VECTOR, Burlingame, CA) and/or the Perma Green alkaline phosphatase kit (blue) following the manufacturer's instructions (DBS, Pleasanton, CA). The primary antibodies used were: anti-nestin polyclonal antibody (1:20; IBL, Gunma, Japan), anti-K15 monoclonal antibody (1:100; Lab Vision, Fremont, CA) and anti-CD34 monoclonal antibody (1:10; Nichirei, Tokyo, Japan).

Results

Middle anagen hair follicles with growing hair follicle cells demonstrate nestin-positive and K15-negative cells in the hfPSA.

Upon examination of longitudinal sections from skin biopsies of human scalp, we found that hfPS cells located in middle anagen phase hair follicles were nestin-positive and K15-negative (figure 1). These cells were located within close proximity of the root of the sebaceous glands and were above the K15-positive hair follicle bulge area (figure 1). The nestin-positive and K15-negative hair follicle cells were thought to represent pluripotent stem cells located in the hair follicle stem cell area.

Damaged hair bulbs in patients with alopecia areata are nestin and K15 negative.

H&E staining of skin biopsies from patients with alopecia areata demonstrated dense lymphocytic infiltration into the hair bulb. The damaged hair bulbs in these sections were nestin and K15 negative (figure 2).

Catagen and regenerating early anagen phase hair follicles contain nestin-positive and K15-negative cells in the dermal papilla of patients with alopecia areata.

We next observed the nestin-positive, K15-negative hair follicle cells surrounding the outer root sheath cells of catagen hair follicles in patients with alopecia areata (figure 3). The regenerating hair follicles contained nestin-positive and K15-negative cells in the dermal papilla and the area directly surrounding the hair bulb (figure 4).

Regenerating hair follicles contain nestin-positive and K15-negative cells in the dermal papilla of patients with alopecia areata.In the present study, we observed lymphocytic infiltration in the area of the nestin- and K15-negative cells of the dermal papilla in patients with alopecia areata. We also found that the K15-positive keratinocyte progenitor cells located in the hair follicle bulge area remained undamaged in the patients with alopecia areata (figure 5A). The regenerating hair follicles identified in the patients with alopecia areata were found to contain nestin-positive and K15-negative cells in the dermal papilla and the area surrounding the hair bulb (figure 5B).

Discussion

We have previously investigated the expression of nestin in the skin using nestin-driven GFP transgenic mice and demonstrated that during early anagen, or the growth phase of the hair follicle, nestin-expressing cells, marked by GFP fluorescence in nestin-GFP transgenic mice, were located in the permanent upper hair follicle immediately below the sebaceous glands and above the hair follicle bulge region [1-5]. Nestin was highly expressed in the hair follicle stem cells and the dermal vasculature network [6]. We subsequently described how the nestin-expressing stem cells were located in the hfPSA in the same nestin-driven GFP transgenic mice. Using immunohistochemical analysis, it has also been shown that the nestin-expressing stem cells located in the hfPSA are CD34 positive and K15 negative [4-7]. We isolated the nestin-positive, CD34-positive, and K15-negative stem cell colony from the hfPSA in nestin-driven GFP-transgenic mice, and found that this area contained pluripotent stem cells that differentiated into neurons, glia, keratinocytes, smooth muscle cells and melanocytes. Furthermore, it has been demonstrated that two months following transplantation, the hfPS cells are able to aid in the rejoining of severed sciatic nerves in both nude mice and C57BL/6 immunocompetent mice. In these animal models, GFAP-positive glial cells were found to differentiate from the hfPS in the presence of pre-existing neuron fibers in sciatic nerve tissue. A myelin sheath formed by Schwann cells was also found to surround the growing axons. The myelin sheath formed by the Schwann cells was thought to support the growth and rejoining of the damaged axons [9]. These findings suggested that the hfPS provide an accessible, autologous source of stem cells for transplantation. Yu et al. [10] have also reported the presence of pluripotent stem cells in human hair follicles. These stem cells in the hair follicles of human scalp were found to be positive for nestin and the embryonic stem cell transcription factors Nanog and Oct 4, and were able to differentiate into neurons, smooth muscle cells and melanocytes [10].We have also demonstrated that depilated anagen phase hair follicles removed from human scalp contain nestin-negative and K15-positive cells, but not nestin-positive and K15-negative cells. Only the upper portion of the plucked hair follicle was able to form cell colonies and differentiate into K15-positive keratinocytes. These results suggested that the nestin-positive and K15-negative cells located throughout the entire hair follicle represented progenitor cells for neurons, glial cells, keratinocytes and smooth muscle cells, while the nestin-negative and K15-positive cells located in the depilated hair follicles represent keratinocyte progenitor cells only [11, 12].

In the present study, we observed the expression of the stem cell marker nestin during hair follicle cycling in patients with alopecia areata. In the normal human scalp skin, the vast majority of hair follicles were found to be present in the late anagen phase. Unfortunately, it is often difficult to observe nestin expression in the cells of hair follicles in the late anagen phase. Generally, nestin-expressing cells are observed in proliferating cells located in the hfPSA of newly growing hair follicles. As a result of these difficulties, we observed the hair follicles that were in the early and middle anagen phases in samples isolated from patients with alopecia areata. We found that the hair follicles in the middle anagen phase that were undergoing growth had nestin-positive and K15-negative cells in the hfPSA. In contrast, the hair follicles undergoing degradation in the alopecia areata samples showed significant lymphocytic infiltration within the nestin- and K15-negative dermal papilla cells. Neither the nestin-positive hfPSA nor the K15-positive hair follicle bulge area was damaged in alopecia areata. In addition, the regenerating early anagen phase hair follicles without inflammation had nestin-positive and K15-negative cells in the dermal papilla and the area surrounding the hair bulb. Moreover, nestin-positive and K15-negative cells with lymphocytic infiltration surround the outer root sheath cells of catagen phase hair follicles in patients with alopecia areata.

Previously, we observed the hair follicle development and the behavior of hair follicle stem cells using ND-GFP transgenic mice. Nestin-expressing cells were located in mesenchymal-drived dermal papilla, and resided in the epithelial-derived matrix and pre-cortex regions of the follicular bulb. The nestin-expressing hair follicle stem cells in the dermal papilla were growing upwards, and formed the outer-root sheath cells in the first hair cycle. In the first cycle, nestin-expressing hair follicle stem cells moved from the dermal papilla to nestin-expressing hfPSA. The nestin-expressing hair follicle stem cells in the dermal papilla have important roles in the development of nestin-expressing hfPSA [13]. These results suggest that nestin-expressing cells in the human hair follicle may play an important role not only in the nestin-expressing hfPSA, but also in the nestin-expressing dermal papilla in the regenerating hair follicle.

Disclosure

Grant support: The authors express their heartfelt thanks to Ms. Yuko Hamada and Ms. Maho Kanoh (Department of Dermatology, Kitasato University School of Medicine, Kanagawa, Japan) for their helpful advice and encouragement. This work was partially supported by a Grant-in-Aid for encouragement of young scientists 20790814 from the Ministry of Education, Science, Sports, and Culture of Japan, the All Kitasato Project Study 2007-2009, and a grant from the Kanagawa Nanbyou Foundation to Y. Amoh. Conflict of interest: none.

References

1 R.M. Hoffman The hair follicle as a gene therapy target Nat Biothechnol 2000; 18: 20-21.

2 H Oshima, A Rochat, C Kedzia, K Kobayashi, Y. Barrandon Morphogenesis and Renewal of Hair Follicles from Adult Pluripotent Stem Cells Cell 2001; 104: 233-245.

3 G Cotsarelis, TT Sun, R.M. Lavker Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis Cell 1990; 61: 1329-1337.

4 Y Amoh, L Li, K Katsuoka, R.M. Hoffman Pluripotent nestin-expressing hair follicle stem cells J Dermatol 2009; 36: 1-9.

5 L Li, J Mignone, M Yang et al. Nestin expression in hair follicle sheath progenitor cells Proc Natl Acad Sci 2003; 100: 9958-9961.

6 Y Amoh, L Li, M Yang et al. Nascent blood vessels in the skin arise from nestin-expressing hair-follicle cells Proc Natl Acad Sci 2004; 101: 13291-13295.

7 Y Amoh, L Li, M Yang et al. Pluripotent nestin-positive, keratin-negative hair-follicle bulge stem cells can form neurons Proc Natl Acad Sci 2005; 102: 5530-5534.

8 Y Amoh, L Li, K Katsuoka, R.M. Hoffman Chemotherapy targets the hair-follicle vascular network but not the stem cells J Invest Dermatol 2007; 127: 11-15.

9 Y Amoh, L Li, R Campillo et al. Implanted hair follicle stem cells form Schwann cells which support repair of severed peripheral nerves Proc Natl Acad Sci 2005; 102: 17734-17738.

10 H Yu, D Fang, SM Kumar et al. Isolation of a novel population of pluripotent adult stem cells from human hair follicles Am J Pathol 2006; 168: 1879-1888.

11 Y Amoh, L Li, K Katsuoka, R.M. Hoffman Hair follicle pluripotent stem cells promote repair of spinal cord injury and recovery of walking function Cell Cycle 2008; 7: 1865-1869.

12 Y Amoh, M Kanoh, S Niiyama et al. Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral-nerve injury: An alternative to ES and iPS cells Journal of Cellular Biochemistry 2009; 107: 1016-1020.

13 Y Amoh, L Li, K Katsuoka, R.M. Hoffman Embryonic development of hair follicle pluripotent stem (hfPS) cells Medical Molecular Morphology 2010; 43: 123-127.