The Journal of Dermatology Vol. 19: 793-796, 1992

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In vitro Keratin Expression ofMllir Cells Yukio Kitano, Katsuyuki Saito, Eriko Okamoto, Yuri ~o, Noriko Tanigaki-Obana*, KaOIU Ito**, Toshio Tazawa** and M~ Ito** Abstract

Human hair follicles were isolated from the scalp by dis}>~e andcoU~$¢Ilase treatment and dispersed into a cell suspension by trypsin. These cells proliferated well and could be subcultured 7 to 8 times. The medium used was MCDB 153 HAA m~furtherstJpPlementedWith some amino acids, hydrocortisone, insulin, EGF, and bovine brain extract. The concentration ofCa++ was adjusted to 0.1 mM. Immunohistochemically, these cells were proved to. possess keratins specific to hair forming cells.

Key words: hair follicle cell; tissue culture; serum-free medium; keratin Hair originates from a follicular structure which consists of three parts: an outer root sheath, an inner root sheath which includes Henle's layer and the inner root sheath cuticle, and the hair fiber itself which includes the hair cuticle, cortex, and medulla, The bulbar region of the hair follicle contains a pool of relatively undifferentiated epithelial cells, termed matrix cells, which give rise to the hair and its surrounding inner root sheath (1). The hair follicle from the human scalp produces approximately 0.35 mm of hair per day. A hair is a dead structure composed of keratinized cells that are compactly cemented together. The mitotic activity of the germinative tissue and the metabolic requirements of the cells which synthesize the specialized fibrous proteins to support the daily growth of hair are enormous. . A hair does not grow indefinitely; after 3 to 5 years, the so-called agagen phase, a period of degeneration starts, the catagen phase, and the follicle ceases to produce a hair. Recently, Cotsarelis et al. suggested that a certain part of the upper outer root sheath contained the only true follicular stem cells and proposed that hair matrix germinative cells are a 'transit' amplifying population with a limited proliferative potential and a set path of differentiation. On the other hand, a group of germinative cells at the base of the follicle end bulb Department of Dermatology, Hyogo College of Medicine,"'Fundamental ResearchLaboratory, R&D Division, Sunstar Inc., and "''''Department of Dermatology, Niigata University School of Medicine. Reprint requests to: Yukio Kitano, M.D., Department of .Dennatology, Hyogo College of Medicine, 1-1 Mukogawa, Nishinomiya, Hyogo 663,Japan.

region have long been considered to be an epidermal stem cell population with a pluripotential nature (2). Reynolds et al. isolated by microdissection a small but identifiable group of cells from complete follicles and cultured these so-called germinative cells. They appeared to be small and round and never formed large pavement arrangements typical ofepidermal type cells. When cultured in association with hair follicle dermal papilla cells, they were stimulated to proliferate and sometimes developed an organotypic-like structure (3). While detailed structural and chemical studies have advanced our knowledge of the hair and hair follicle characteristics, current understanding of mechanism underlying physiological and pathological changes in human hair growth is poor. This is in part due to the limited experimental methods for studying mechanism of hair growth (kinetics of formation, regulation of hair cycle, and differentiating factors). In recent years, several methods have been developed for establishing primary cultures of human hair keratinocytes. Weterings et al. were first able to grow human hair keratinoeytes by explanting plucked scalp hair follicles on bovine lens capsules (4). Similarly, Wells succeeded in explanting plucked human hair follicles directly on tissue culture plastic (5). Since plucking hair follicles usually left the lower part of the bulb anchored in the scalp, the cultured keratinocytes apparently originated from the outer root sheath of the hair follicle. There has been no report ofculture ofwellproven hair forming cells to date. Isolation and cultivation ofhairfollicle cells In our previous study, we showed that, after

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Fig. 1. Primary culture of hair follicle cells 24 h after plating. Phase contrast (x81) dispase treatment, the epidermal sheet can be separated from the dermis without disruption of intercellular connections (6). It is expected that collagenase will loosen the collagen fibers in the dermis. Therefore, in order to get the epidermal components of the hair organ intact, we treated the scalp with dispase and collagenase. The scalp with some subcutaneous tissues was soaked a,t 4°C overnight in 500 unit!ml dispase in Dulbecco's modified minimum essential medium (DMEM). The epidermis separated easily, leaving the hair follicles in the dermal side. The dermis, containing hair follicles, was treated with 0.25% collagenase in phosphate buffered saline (PBS) at 37°C for 1 h. Then the hair follicles were easily removed from the surrounding tissue and treated with 0.25% trypsin for 7 min to dispearse them into a cell suspension. The suspension was centrifuged at 200xg for 5 min. The cells were resuspended in DMEM supplemented with 20% fetal bovine serum (attachment medium), plated in a type I collagencoated culture dish, and incubated at 37°C in a humidified atmosphere of air containing 5% CO 2 , On the next day, the medium was changed to growth medium consisting of MCDB 153 HAA medium modified further by supplementing with Lhistidine (0.25 mM), L-isoleucine (0.75 mM), Lmethionine (0.09 mM), L-phenylalanine (0.09 mM), L-tryptophane (0.045 mM), and L-tyrosine (0.075 mM) as well as 0.5 ,ug/ml hydrocortisone, 5 ,ug/ml insulin, 5 ng/ml human epidermal growth factor (hEGF), and 35 ,ug protein/ml bovine brain extract. After dispase and collagenase treatment, the dermis became loose and somewhat gelatinous, and hairs could be easily plucked with the surrounding

Fig. 2. Primary culture of hair follicle cells 5 days after plating. The cells are small and polygonal. Phase contrast (x81)

Fig. 3. Hair follicle cells in the fourth subculture. There are many large cells which are often multinucleated. Phase contrast (x81) follicular structure; the out~r root sheath and most of the bulb remained entire. The end bulb was often lost and the sebaceous gland was partially destroyed. The isolated hair follicles were treated with 0.25% trypsin and dissociated into a cell suspension. Twenty-four hours after plating the suspended cells in the attachment medium, small polygonal cells were attached individually or in clumps (Fig. 1). The plating efficiency was a little poorer than our regular culture of human epidermal keratinocytes using the dispase-trypsin method. A plating cell density of 3 x lOs small smoothsurfaced round cella/em" was desirable for primary culture. With the use of a small culture dish, it was possible to establish a culture from 10 hair follicles.

Culture of Hair Cells

Fig. 4. Immunohistochemical staining of hair follicle cells with HKN-5. Secondary culture. (xllO)

At 24 h of cultivation, the medium was changed to growth medium. The concentration of Ca" was adjusted to 0.1 mM for regular culture. The cells were relatively dormant for a few days and then burst into rapid proliferation (Fig. 2). In seven to ten days, subculture was done by dispersing the cells with EDTA and trypsin-treatment. At the time of con fluency, the yield of cells was ca 2 x 104 cells/em", and 3 x lOscella/em" were plated to a new dish. Subculture could be repeated 7 to 8 times at intervals of 7 to 10 days. In the early passages, the cells were mostly small and polygonal; the number of elongated and/or large distended cells increased with repeated subculture (Fig. 3). Immunohistochemical study On the basis of the data from polyacrylamide gel electrophoresis (PAGE) and amino acid analysis, the components of hair fibrous proteins are biochemically distinct compared from those of epidermal fibrous proteins (7-9). On the other hand, immunological'studies on keratin proteins, especially by use of monoclonal antibodies, have revealed that some antigenic determinants of the proteins are common, whereas others are more specific (10-12). Among the monoclonal antibodies raised against human hair keratins, HKN-2 and HKN-4mark the electrophoretic bands ofboth hair and epidermal fibrous proteins, while HKN-5 and HKN-6 react only with the bands of hair fibrous proteins (10, 11). Immunohistochemically, HKN-5 and HKN-6 are hair-specific. HKN-5 stained the medulla, cortex, cuticle, inner root sheath in the k.eratogenous zone of anagen hairs, and the innermosr cells of the outer root sheath, but not hair

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Fig. 5. Immunohistochemical staining of hair follicle cells with HKN-6. Secondary culture. (xl 10)

matrix cells. HKN-6 reacted with the inner root sheath, hair cuticle, cortex, and medulla in the keratogenous zone of anagen hairs. These monoclonal antibodies did not react with any other skin components. For immunofluorescence histochemistry, cells grown on 18 mm round coverslips were washed with phosphate-buffered saline (PBS), fixed with cold methanol for 5 min, washed in PBS, and permeabilized with 0.2%Triton X-I 00 in PBS for 5 min. They were then reacted with anti-human hair keratin antibodies, HKN-2, HKN-5, and HKN-6, at 37°C for 30 min, washed with PBS, and stained with fluorescein-conjugated goat anti-mouse IgG. Microscopic examination was via Olympus fluorescence microscope BH-2 after mounting in Perma Fluor. All hair follicle cells in culture were positive for HKN-2. A monoclonal antibody, HKN-5, stained many cultured hair follicle cells (Fig. 4). Most small polygonal cells were not stained, but some of them were faintly positive, and a few were distinctly stained. Most of the large cells were stained by HKN-5, but the intensity of fluorescence varied. Another monoclonal antibody, HKN-6, recognizes the keratogenous zone of anagen hairs. The reaction sites of HKN-6 are limited to the inner root sheath and hair shaft; the outer root sheath is excluded. A few cultured hair follicle cells were stained with HKN-6 (Fig. 5). These included small polygonal cells, but large distended cells were stained in higher percentages. Immunohistochemical study disclosed that this culture contained cells which had keratins specific

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to hair and hair follicles. The cells stained with HKN-5 and HKN-6 were possibly derived from the hair bulb and inner root sheath and were hair forming cells. Since, in the previous report of culture of human hair keratinocytes, the cultured cells apparently originated from the outer root sheath, this is the first report of culture of well proven hair forming cells.

References 1) Montagna W: The Stnu:tun and Fundion of Skin, 2nd ed, Academic Press, London and New York, 1962, p 186. 2) Cotsarelis G, Sun T-T, Lavker RM: Label-retaining cells reside in the bulge area of pilosebaceous unit: Implications for follicular stem cells, hair cycle, and skin carcinogenesis, ou; 61: 1329-1337,1990. 3) Reynolds A], ]ahoda CAB: Hair follicle stem cells? A distinct germinative epidermal cell population is activated in vitro by the presence of hair dermal papilla cells,] ca Sci, 99: 373-385, 1991. 4) Weterings PlJM, Vermorken A]M, Bloemendal H: A method for culturing human hair follicle cells, Br] Dermatol, 104: 1-5, 1981. 5) Wells]: A simple technique for establishing cultures of epithelial cells, Br] Dermatol, 107: 481-482, 1982.

6) Kitano Y, Okada N: Separation of epidermal sheet by dispase, Br] Dermatol; 108: 555-560, 1983. 7) Baden HP, McGilvrayN, Lee LD, Baden L, KubilusJ: Comparison of stratum corneum and hair fibrous proteins,] Invest Dermatol, 75: 311-315, 1980. 8) Marshall RC: Characterization of the proteins of human hair and nail by electrophoresis,] Invest Dmnatol, 80: 519-524, 1983. 9) Heid HW, Werner E, Franke WW: The complement of native a-keratin polypeptides of hair-forming cells: A subset of eight polypeptides that differ from epithelial cytokeratins, Differentiation, 52: 101-119, 1986.

10) Ito M, Tazawa T, Ito K, Shimizu N, Katsuumi K, Sato Y: Immunological characteristics and histological distribution of human hair fibrous proteins studied with anti-hair keratin monoclonal antibodies HKN-2, HKN-4, and HKN-6,] Histochem Cytochem, M: 269275,1986. 11) Ito M,Tazawa T, Shimizu N, et al: Cell differentiation in human anagen hair and hair follicles with antihair keratin monoclonal antibodies,] InvestDermatol, 86: 563-569, 1986. 12) Heid HW, Moll I, Franke WW: Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues. I. Human and bovine hair follicles, Differentiation, 37: 137-157, 1988.

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In vitro keratin expression of hair cells.

Human hair follicles were isolated from the scalp by dispase and collagenase treatment and dispersed into a cell suspension by trypsin. These cells pr...
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