In Vitro Cell. Dev. Biol. 28A:695-698, November-December 1992 © 1992 Tissue Culture Association 0883-8364/92 $01.50+0.00
Letter to the E d i t o r SKIN HISTOCULTURE ASSAY FOR STUDYING THE HAIR CYCLE
Dear Editor: The poor understanding of the basic molecular mechanisms governing the growth, loss and pigmentation of hair is partly due to the paucity of relevant in vitro models for studying these phenomena. Elucidation of the biological clock that governs the cyclic activity of the hair follicle (telogen-anagen-catagen-telogen) would be a major breakthrough in hair research. Specifically, effective pharmacological manipulation of hair growth would greatly be facilitated by knowledge of the signals that initiate, drive and terminate anagen. Yet, there is currently no assay available that allows the in vitrostudy of the total growth phase, let alone cycling of adult hair follicles, or anagen-associated pigment production over an extended period of time. Though the growth of non-embryonic mouse, rat and human anagen hair follicles in vitro has been reported in various culture systems (cf. 11,18-20), none of these assays has utilized homogeneous populations of mature follicles of a well-defined stage of the growth cycle. Also, all assays were associated with considerable tissue traumatization by enzymatic digestion or mechanical manipulation and/or loss of the physiological follicular tissue environment, thus eliminating the intercellular communication between follicle cells on the one hand, and e.g. epidermal keratinocytes, fibroblasts, macrophages and mast cells on the other. This could be a serious limitation, considering that the epidermis (13,14), mast cells (15), macrophages (12,25) or other localimmunologicalfactors (16) may contribute to the regulation of hair growth. Previously, we have used the C 57 B1-6 mouse for hair growth studies in vivo and in skin organ culture on metal grids (13,14,16,17). In this assay', anagen is induced in telogen mice pharmacologically (16) or by depilation (13). In mice, all truncal melanocytes are confined to the hair follicles, melanogenesis is strictly coupled to the anagen phase of the hair cycle, and follicular growth patterns are synchronized (1,22). Thus, the development of anagen can easily be recognized by observing the gradual change in skin color from white/pink (telogen) to grey (mid anagen) and finally to black (late anagen) (1,13,16,22). This assay provides large populations of homogeneous, mature mouse follicles of defined hair cycle stages that can readily be studied in skin organ culture by incubating biopsies of whole skin under in vivo-like conditions at the air-liquid interphase on metal grids (13,17). However, keratinocyte viability is rather limited under these conditions so that it is not possible to follow follicle development and the formation and pigmentation of anagen hair shafts over an extended period of time. Thus, we have turned to a simple, yet effective histoculture technique which uses collagen-containing sponge-gel supports to mainrain tissue viability (5,8), Most recently, we observed hair shaft growth in mouse skin histocultured with this technique at approximately the in vivo rate, though we could not tell what stage of the hair cycle the follicles had been in at the beginning of tissue culture, since white-mouse skin was used (9,27). We have also observed
hair growth from human scalp (10) where the follicles are not synchronous which makes it difficult to study the hair cycle. Combining the C 57 BI-6 mouse model and the sponge-gel histoculture technique, we demonstrate here that follicles of the earliest anagen stage develop into full anagen follicles and produce pigmented hair shafts in vitro. C 57 BI-6 mice (female, syngeneic, 6 - 8 weeks old) were purchased from Charles River, Kingston, NY, housed in community cages in the Albany Medical College Facility, AntiCancer Inc. and University Hospital R. Virchow, Freie Universitaet Berlin with 12hour light periods and fed ad libitum with water and rat/mouse chow "3000" (Agway, Syracuse). Anagen was induced in telogen mice (recognizable by their pink, skin color) by depilation with a wax/rosin mixture under anaesthesia as previously described (13,14,17,22). Two days and 14 days after anagen-induction, back skin was harvested under sterile conditions from mice that had been sacrificed by cervical dislocation under ether narcosis. Before dissecting the skin at the level of the subcntis, it was flushed twice with 70% ethanol. Harvested skin was then incubated in Ham's F-10 medium at + 4 ° C for overnight shipment from Albany, NY to San Diego, CA or back skin was prepared for histoculture immediately in San Diego as described below. For some experiments, animals were also anagen-indueed in San Diego or Berlin, and their skin used directly within 2 hours after harvesting for histoculture. Skin was harvested at 2-day and 14-day post anagen-induetion from five C 57 B1-6 mice for each experiment. The skin was put in histocuhure under sterile conditions according to the method developed earlier by Hoffman et al. (5,8), based on the work by Leighton (7). The 2-day-post anagen-induced skin was non-pigmented and showed neither macroscopic nor microscopic evidence for hair shaft formation (Fig. 1 A and 2 A). The 14-day-post-anagen-induced skin was light grey and showed no-hair out growth from shafts which were shaved before histoculture. Briefly, after dissecting the subcutis proximal to the parniculus carnosus, small pieces of skin were cut out with a 4 mm biopsy punch or re-cut into 2 equal fragments in Eagle's MEM medium. They were then placed dermis-down onto 1 X 1 X 1 cm pieces of collagen-containing gel (Gelfoam gelatin sponge, Upjohn Co., Kalamazoo, MI) that had been prehydrated for at least 4 hours with culture medium (MEM + 10% FBS + 50 #g/ml gentamycin). From each mouse, 24 skin fragments were studied, i.e. 4 per sponge-gel, distributed between 6 gels placed individually in 6 well-plates with 2 ml medium added per well such that the gels were not covered and the skin was above the liquid medium. Cultures were maintained for up to 16 days at 37 ° C, 100% humidity and gassed with a mixture of 95% air and 5% CO2. Tissue fixed in 10% buffered formalin was processed for routine histology and stained with hematoxylin/eosin or Giemsa according to standard proeedures. The length of hair shafts was measured under a dissection microscope with a ruler. Viable cells were selectively labeled with 15 #M of the dye 2',7'695
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FIG. 1. Formation of pigmented hair shafts in histocuhure. Macroscopic appearance of histocultured C 57 B1-6 mouse skin, taken from mice in which anagen had been induced by depilation 2 days prior to tissue harvesting. The number of days of histocuhure on collagen-containing gels is indicated. Note the production of pigmented hair shafts and the shift in skin color from white/pink to grey/black that is associated with the development of mature anagen follicles. A, Day 0; B, Day 5; C, Day 8; D, Day 12. Bar = 0.5 mm. Fro. 2. Histology of anagen development in histocuhured skin. Hematoxylin/eosin stains of formalin-fixed, paraffin-embedded skin after various times of histocuhure. Note the absence of melanin production in the early-anagen follicles at the beginning of histoculture, and the development of fully mature follicles (antigen VI) which have generated regularly pigmented hair shafts. A, at 0 h of histocuhure; B, at 8 days of histocuiture; C, at 16 days of histocuhure. Bar = 20 #m. Fie. 3. Fluorescent double dye-labeling of viable and dead cells of mouse skin histocuhured for 5 days. BCEC-AM: green = living cells; PI: red = dead cells. Bar = 20 #m. Fit;. 4. Autoradiography of proliferating follicle ceils in histocuhure. The [3H]thymidine uptake after 8 days of skin histocuhure is shown, visualized as bright green by epi-polarization (8). Note the extensive labeling of hair matrix cells. Bar = 10 ttm. Fit;. 5. Hair growth with progressing pigment production from 14-day anagen-induced C 57 B1-6 mouse skin after histoculture at 0 (.4), 7 (B) and 14 (C) days of histocuhure. Note the large number of hair shafts growing out and their increased length with color change from grey to dark over time. Bar = 0.5 mm.
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HAIR CYCLE IN VITRO bis-(2carboxyethyl)-5-(n-6)carboxyfluorescein acetomethyl ester (BCECF-AM), which is activated to fluorescence by nonspecific esterases present only in living cells. Non-viable cells, whose plasma membranes are leaky, were labeled with 15 #M of propidium iodide (PI), a dye that enters only cells with non-intact membranes. Since the emission spectra of these dyes are different, they could be used simultaneously on the same specimen. The double-dye-treated cultures were analyzed by fluorescence and confocal microscopy within 30 minutes of staining (for details, see 15). Histocultured skin was labeled for 3 days with 4 gCi/ml [3H]thymidine, washed with PBS, fixed with 10% buffered formalin and processed for autoradiography as previously described (8). After exposure to the photoemulsion and fixation, slides were stained with hematoxylin/eosin, and analyzed under epi-illumination polarization so that replicating cells could be identified by the presence of silver grains over their nuclei, visualized as bright green in the epipolarization system (for details, see 8). When depigmented mouse skin without visible hair shafts, in which anagen had shortly before been induced by depilation 2 days previously, was cultured on sponge-gel supports, hair shaft formation was observed macroscopicalty in about 33% of all skin fragments after 5 days of histocuhure. The percentage of hair-growing skin fragments did not increase over time. Figure 1 shows that hair shafts were fully pigmented, and increased in length over time. After 5, 8 and 12 days of histocuhure, the average hair length had reached 0.44 + 0.07 mm, 0.60 + 0.07 mm and 0.67 + 0.08 mm respectively in four sets of explants in each of two experiments (Fig. 6). The maximum hair elongation reached 2.5 mm after 12 days histoculture. The density of hair growth was approximately 4 hairs/ mm 2 after 5 days histocuhure and did not increase during the time of histoculture. In parallel with hair shaft formation, visible skin color changed from white to dark grey (Fig. 1). Freshly harvested and histocultured skin fragments showed hair shaft elongation and pigmentation comparable to the in vivo situation with hair shafts becoming visible approximately 8 - 1 0 days after anagen induction by depilation (1,13,22). The beginning of visible hair shaft formation in histoculture thus reflects the in vivo-situation. When shaved skin was harvested and cultured 14 days post-anagen induction on sponge-gel supports, hair shaft elongation was 2 Day-Anagen-induced In Vitro C57B1.6 M o u s e
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FIG. 6. Hair growth of 2-day anagen-indueed C 57 B1-6 mouse skin histoculturedon collagen-gelsponge in vitro. Note the increased length over time.
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FIG. 7. Hair growth of 14-day anagen-indueed C 57 B1-6 mouse skin histoeuhured on collagen-gelsponge in vitro. Note the increased hair length over time. observed grossly in 50% of all skin fragments after 7 days of histoculture. Pigmented hair shafts increased in both length and density over time. The average length of hair growth reached 0.51 + 0.07 mm and 0.77 + 0.08 mm, respectively after 7 and 14 days histoculture (Fig. 7) with color change from grey to black (Fig. 5). The density of hair growth increased from 7 hairs/mm 2 after 7 days histocuhure to 11 hairs/mm 2 after 14 days histoculture in 5 sets of explants. Comparatively, more hair growth and faster growth occurred around the edge area of the skin histoculture than in the central area on both 2-day and 14-day-post-anagen-induced skin. The rate of hair growth of the 2-day and 14-day-post-anagen-induced skin was similar at about 0.1 mm/per day in the first 7 days, while the density of hair growth in 14-day-post-anagen-induced skin was much higher than 2-day-post-anagen-induced skin. The density of in-vitro growing hairs increased only in the 14-day post-anagen-induced cultures. It is important to note that, though 33% of the 2-day post-anagen-induced and 50% of the 14-day post-anagen-induced histocultured skin fragments exhibited hair shaft elongation and pigmentation, the number of actually growing shafts per biopsy and the maximal length achieved varied from experiment to experiment, ranged from 6 to 145 shafts per biopsy (mean 21 shafts per responsive biopsy). Histologically, at the start of histoculture, the 2-day post-anageninduced skin (harvested 2 days-post-depilation)exhibited the characteristic follicles of early anagen [anagen stage I-II according to Chase (12)] having neither hair shafts nor pigment production (Fig. 2 A cf. 14,22). Fig. 2 B,C, representing the same specimen as Fig. 2 A, show that after 8 and 16 days of histoculture, respectively, normal-appearing, mature follicles of anagen stage VI have developed, with the formation of regularly pigmented hair shafts and full melanin production in the epithelial bulbs. Note in Fig. 2 B and C the typical, band-like melanin-distribution pattern of the terminallydifferentiated keratinocytes forming the hair shaft. The 14-day post-anagen-induced skin also retained normal follicle architecture even after 14 days histoculture in vitro (data not shown). In order to demonstrate the viability of hair follicle keratinocytes in histoculture, fluorescent dye double-staining was performed and assessed by confocal laser scanning microscopy (8). After 5 days of
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histoculture, most follicle cells are viable (Fig. 3). To identify proliferating cells, [SH]thymidine incorporation was measured by autoradiography. Figure 4 shows that the majority of [SH]thymidinelabeled cells were the hair matrix cells after 8 days histocuhure. After 16 days of histoculture, DNA-synthesis still occurred in the hair bulb, though the majority of the proliferating cells were found in the outer root sheath (data not shown) thus possibly explaining the reduced rate of in vitro hair growth by Day 16. The histocuhure system described here can now be utilized to analyze the inherent apparatus of follicle growth control, and to define the growth requirements of mature anagen hair follicles growing in their natural tissue environment. It should be a useful tool for dissecting the relative importance of cytokines as well as of thyroid, pituitary and sex hormones for modulating the length of the anagen phase. In contrast to recently developed mixed-cell culture systems which study melanocytes seeded into in vitro-reconstituted skin-"equivalents" (e.g. 21), histocuhure of mouse skin does not require any prior enzymatic treatment of the tissue, and occurs in the presence of other resident skin cells like mast cells, which are absent in the mixed-cell culture models. Thus, the growth conditions of melanocytes in histocuhure seem closer to the physiological situation than of any other currently available system. That even after 16 days of histoculture there is histological evidence for melanin production (Fig. 2) suggests that melanocytes are still alive and melanogenically active at this time. The long-term histocuhure of C 57 BI-6 mouse skin in defined stages of the hair cycle may serve as a powerful tool for dissecting the relative contributions made by the cell populations and signal molecules implicated in the regulation of hair growth, and for analyzing the intrinsic apparatus that controls hair follicle cycling. ACKNOWLEDGEMKNTS This study was supported in part by a NIH/SB1R grant #R43 CA53995 to AntiCancer Inc., by DFG to R. P. and by Lawrence M. Gelb Research Award to A. S.
11. Link, R. E.; Pans, R.; Stenn, K. S., et al. Epithelial growth by rat vibrissae follicles in vitro requires mesenchymal contact via native extracellular matrix. J. Invest. Derm. 95:202-207; 1990. 12. Parakkal, P. F. Role of macrophages in collagen resorption during hair growth cycle. J. Ultrastruct. Res. 29:210-217; 1969. 13. Pans, R.; Stenn, K. S.; Link, R. E. Telogen skin contains an inhibitor of hair growth. Br. J. Dermatol. 122:777-784; 1990. 14. Pans, R.; Stenn, K. S.; Elgjo, K. The epidermal pentapoptide pyroGluGlu-Asp-Ser-GlyOH inhibits murine hair growth in vivo and in vitro. Dermatologica 183:173-178; 1991. 15. Pans, R.; Maurer, M.; Slominski, A., et al. Mast cells and hair growth: the murine hair cycle as a model for studying growth-regulatory functions of mast cells. Arch. Dermatol. Res. 284:31A; 1992. 16. Pans, R.; Stenn, K. S.; Ling, R. E. The induction of anagen hair follicle growth in telogen mouse skin by cyclosporine A administration. Lab. Invest. 60:365-369; 1989. 17. Pans, R. Hair growth inhibition by heparin in mice: a model system for studying the modulation of epithelial cell growth by glycosaminoglycans? Br. J. Derm. 124:415-422; 1991. 18. Philpott, M. P.; Green, M. R.; Kealey, T. Studies on the biochemistry and morphology of freshly isolated and maintained hair follicles. J. Cell Sci. 93:409-418; 1989. 19. Philpott, M. P.; Green, M. R.; Kealey, T. Human hair growth in vitro. J. Cell Sci. 97:463-471; 1990. 20. Rogers, G.; Martinet, N.; Steinert, P., et al. Cultivation of mufine hair follicles as orgaooids in a collagen matrix. J. Invest. Dermatol. 89:369-379; 1987. 21. Scott, G. A.; Haake, A. R. Kerafinocytes regulate melanocyte number in human fetal and neonatal skin equivalents. J. Invest. Dermatol. 97:776-782; 1991. 22. Slominski, A.; Pans, R.; Costantino, R. Differential expression and activity of melanogenesis-related proteins during induced hair growth in mice. J. Invest. Derm. 96:172-179; 1991. 23. Slominski, A.; Pans, R.; Mazurkiewicz, J. Proopiomelanocortin expression in the skin during induced hair growth in mice. Experientia. 48:50-54; 1992. 24. Slominski, A.; Paus, R.; Wortsman, J. Can some melanotropins modulate keratioocyte proliferation? J. Invest. Dermatol. 97:747; 1991. 25. Westgate, G. E.; Craggs, R. I.; Gibson, W. T. Immune privilege and hair growth. J. Invest. Dermatol. 97:417-421; 1991. 26. Yaar, M.; Gilchrest, B. A. Human melanocyte growth and differentiation: a decade of new data. J. Invest. Dermatol. 97:611-618; 1991. 27. Li, L.; Pans, R.; Margolis, L. B., et al. Hair growth in vitro from histocuitured skin. In Vitro Cell. Dev. Biol. 28A:479-481; 1992.
REFERENCES 1. Chase, H. B. Growth of the hair. Physiol. Rev. 34:113-126; 1954. 2. Dawber, R.; Rook, A., eds. Diseases of the hair and scalp. Oxford: Blackwell; 1992. 3. du Cros, D. L.; Isaacs, K.; Moore, G. P. M. Localization of epidermal growth factor immunoreactivity in sheep skin during wool follicle development. J. Invest. Dermatol. 98:109-115; 1992. 4. Ebling, F. J. G. The biology of hair. Dermatol. Clinics 5:476-481; 1987. 5. Hoffman, R. M. Three-dimensional histoculture: origin and applications in cancer research. Cancer Cells 3:86-92; 1991. 6. Johnson, E. Inherent rhythms of activity in the hair follicle and their control. In: Lyne, A. G.; Short, B. F., eds. Biology of the skin and hair growth. New York: Elsevier; 1965:491-505. 7. Leighton, J. A sponge matrix method for tissue culture. J. Nail. Cancer Inst. 12:545-561; 1951. 8. Li, L.; Margolis, L. B.; Hoffman, R. M. Skin toxicity determined in vitro by three-dimensional, native-state histoculture. Proc. Nail. Acad. Sci. USA 88:1908-1912; 1991. 9. Li, L.; Hoffman, R. M. Hair growth and hair follicle-cell proliferation in histocuhured mouse skin. Ann. NY Acad. Sci. 642:506-509; 1991. 10. Li, L.; Margolis, L. B.; Pans, R., et al. Hair shaft elongation, follicle growth and spontaneous regression in long-term, sponge-gel supported histocuhure of human scalp skin. Proc. Nail. Acad. Sci. USA 89:8764-8768; 1992.
Lingna Li Ralf Paus
Andrzej Slominski Robert M. I-Ioffman 1 AntiCancer Inc. (L. L., R. M. H.) 5325 Metro Street San Diego, CA 92110; Dept. of Dermatology (R. P.) University Hospital R. Virchow Freie Universitaet Berlin, D-1000 Berlin 65; Dept. of Microbiology, Immunology & Molecular Genetics (A. S.) Albany Medical College Albany, NY 12208; and Laboratory of Cancer Biology (R. M. H.) University of California, San Diego La Jolla, CA 92093 (Received 11 August 1992)
l To whom correspondence should be addressed at Laboratory of Cancer Biology, University of California, San Diego, 0609F, La Jolla, CA 92093.
Letter to the E d i t o r SKIN HISTOCULTURE ASSAY FOR STUDYING THE HAIR CYCLE
Dear Editor: The poor understanding of the basic molecular mechanisms governing the growth, loss and pigmentation of hair is partly due to the paucity of relevant in vitro models for studying these phenomena. Elucidation of the biological clock that governs the cyclic activity of the hair follicle (telogen-anagen-catagen-telogen) would be a major breakthrough in hair research. Specifically, effective pharmacological manipulation of hair growth would greatly be facilitated by knowledge of the signals that initiate, drive and terminate anagen. Yet, there is currently no assay available that allows the in vitrostudy of the total growth phase, let alone cycling of adult hair follicles, or anagen-associated pigment production over an extended period of time. Though the growth of non-embryonic mouse, rat and human anagen hair follicles in vitro has been reported in various culture systems (cf. 11,18-20), none of these assays has utilized homogeneous populations of mature follicles of a well-defined stage of the growth cycle. Also, all assays were associated with considerable tissue traumatization by enzymatic digestion or mechanical manipulation and/or loss of the physiological follicular tissue environment, thus eliminating the intercellular communication between follicle cells on the one hand, and e.g. epidermal keratinocytes, fibroblasts, macrophages and mast cells on the other. This could be a serious limitation, considering that the epidermis (13,14), mast cells (15), macrophages (12,25) or other localimmunologicalfactors (16) may contribute to the regulation of hair growth. Previously, we have used the C 57 B1-6 mouse for hair growth studies in vivo and in skin organ culture on metal grids (13,14,16,17). In this assay', anagen is induced in telogen mice pharmacologically (16) or by depilation (13). In mice, all truncal melanocytes are confined to the hair follicles, melanogenesis is strictly coupled to the anagen phase of the hair cycle, and follicular growth patterns are synchronized (1,22). Thus, the development of anagen can easily be recognized by observing the gradual change in skin color from white/pink (telogen) to grey (mid anagen) and finally to black (late anagen) (1,13,16,22). This assay provides large populations of homogeneous, mature mouse follicles of defined hair cycle stages that can readily be studied in skin organ culture by incubating biopsies of whole skin under in vivo-like conditions at the air-liquid interphase on metal grids (13,17). However, keratinocyte viability is rather limited under these conditions so that it is not possible to follow follicle development and the formation and pigmentation of anagen hair shafts over an extended period of time. Thus, we have turned to a simple, yet effective histoculture technique which uses collagen-containing sponge-gel supports to mainrain tissue viability (5,8), Most recently, we observed hair shaft growth in mouse skin histocultured with this technique at approximately the in vivo rate, though we could not tell what stage of the hair cycle the follicles had been in at the beginning of tissue culture, since white-mouse skin was used (9,27). We have also observed
hair growth from human scalp (10) where the follicles are not synchronous which makes it difficult to study the hair cycle. Combining the C 57 BI-6 mouse model and the sponge-gel histoculture technique, we demonstrate here that follicles of the earliest anagen stage develop into full anagen follicles and produce pigmented hair shafts in vitro. C 57 BI-6 mice (female, syngeneic, 6 - 8 weeks old) were purchased from Charles River, Kingston, NY, housed in community cages in the Albany Medical College Facility, AntiCancer Inc. and University Hospital R. Virchow, Freie Universitaet Berlin with 12hour light periods and fed ad libitum with water and rat/mouse chow "3000" (Agway, Syracuse). Anagen was induced in telogen mice (recognizable by their pink, skin color) by depilation with a wax/rosin mixture under anaesthesia as previously described (13,14,17,22). Two days and 14 days after anagen-induction, back skin was harvested under sterile conditions from mice that had been sacrificed by cervical dislocation under ether narcosis. Before dissecting the skin at the level of the subcntis, it was flushed twice with 70% ethanol. Harvested skin was then incubated in Ham's F-10 medium at + 4 ° C for overnight shipment from Albany, NY to San Diego, CA or back skin was prepared for histoculture immediately in San Diego as described below. For some experiments, animals were also anagen-indueed in San Diego or Berlin, and their skin used directly within 2 hours after harvesting for histoculture. Skin was harvested at 2-day and 14-day post anagen-induetion from five C 57 B1-6 mice for each experiment. The skin was put in histocuhure under sterile conditions according to the method developed earlier by Hoffman et al. (5,8), based on the work by Leighton (7). The 2-day-post anagen-induced skin was non-pigmented and showed neither macroscopic nor microscopic evidence for hair shaft formation (Fig. 1 A and 2 A). The 14-day-post-anagen-induced skin was light grey and showed no-hair out growth from shafts which were shaved before histoculture. Briefly, after dissecting the subcutis proximal to the parniculus carnosus, small pieces of skin were cut out with a 4 mm biopsy punch or re-cut into 2 equal fragments in Eagle's MEM medium. They were then placed dermis-down onto 1 X 1 X 1 cm pieces of collagen-containing gel (Gelfoam gelatin sponge, Upjohn Co., Kalamazoo, MI) that had been prehydrated for at least 4 hours with culture medium (MEM + 10% FBS + 50 #g/ml gentamycin). From each mouse, 24 skin fragments were studied, i.e. 4 per sponge-gel, distributed between 6 gels placed individually in 6 well-plates with 2 ml medium added per well such that the gels were not covered and the skin was above the liquid medium. Cultures were maintained for up to 16 days at 37 ° C, 100% humidity and gassed with a mixture of 95% air and 5% CO2. Tissue fixed in 10% buffered formalin was processed for routine histology and stained with hematoxylin/eosin or Giemsa according to standard proeedures. The length of hair shafts was measured under a dissection microscope with a ruler. Viable cells were selectively labeled with 15 #M of the dye 2',7'695
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FIG. 1. Formation of pigmented hair shafts in histocuhure. Macroscopic appearance of histocultured C 57 B1-6 mouse skin, taken from mice in which anagen had been induced by depilation 2 days prior to tissue harvesting. The number of days of histocuhure on collagen-containing gels is indicated. Note the production of pigmented hair shafts and the shift in skin color from white/pink to grey/black that is associated with the development of mature anagen follicles. A, Day 0; B, Day 5; C, Day 8; D, Day 12. Bar = 0.5 mm. Fro. 2. Histology of anagen development in histocuhured skin. Hematoxylin/eosin stains of formalin-fixed, paraffin-embedded skin after various times of histocuhure. Note the absence of melanin production in the early-anagen follicles at the beginning of histoculture, and the development of fully mature follicles (antigen VI) which have generated regularly pigmented hair shafts. A, at 0 h of histocuhure; B, at 8 days of histocuiture; C, at 16 days of histocuhure. Bar = 20 #m. Fie. 3. Fluorescent double dye-labeling of viable and dead cells of mouse skin histocuhured for 5 days. BCEC-AM: green = living cells; PI: red = dead cells. Bar = 20 #m. Fit;. 4. Autoradiography of proliferating follicle ceils in histocuhure. The [3H]thymidine uptake after 8 days of skin histocuhure is shown, visualized as bright green by epi-polarization (8). Note the extensive labeling of hair matrix cells. Bar = 10 ttm. Fit;. 5. Hair growth with progressing pigment production from 14-day anagen-induced C 57 B1-6 mouse skin after histoculture at 0 (.4), 7 (B) and 14 (C) days of histocuhure. Note the large number of hair shafts growing out and their increased length with color change from grey to dark over time. Bar = 0.5 mm.
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HAIR CYCLE IN VITRO bis-(2carboxyethyl)-5-(n-6)carboxyfluorescein acetomethyl ester (BCECF-AM), which is activated to fluorescence by nonspecific esterases present only in living cells. Non-viable cells, whose plasma membranes are leaky, were labeled with 15 #M of propidium iodide (PI), a dye that enters only cells with non-intact membranes. Since the emission spectra of these dyes are different, they could be used simultaneously on the same specimen. The double-dye-treated cultures were analyzed by fluorescence and confocal microscopy within 30 minutes of staining (for details, see 15). Histocultured skin was labeled for 3 days with 4 gCi/ml [3H]thymidine, washed with PBS, fixed with 10% buffered formalin and processed for autoradiography as previously described (8). After exposure to the photoemulsion and fixation, slides were stained with hematoxylin/eosin, and analyzed under epi-illumination polarization so that replicating cells could be identified by the presence of silver grains over their nuclei, visualized as bright green in the epipolarization system (for details, see 8). When depigmented mouse skin without visible hair shafts, in which anagen had shortly before been induced by depilation 2 days previously, was cultured on sponge-gel supports, hair shaft formation was observed macroscopicalty in about 33% of all skin fragments after 5 days of histocuhure. The percentage of hair-growing skin fragments did not increase over time. Figure 1 shows that hair shafts were fully pigmented, and increased in length over time. After 5, 8 and 12 days of histocuhure, the average hair length had reached 0.44 + 0.07 mm, 0.60 + 0.07 mm and 0.67 + 0.08 mm respectively in four sets of explants in each of two experiments (Fig. 6). The maximum hair elongation reached 2.5 mm after 12 days histoculture. The density of hair growth was approximately 4 hairs/ mm 2 after 5 days histocuhure and did not increase during the time of histoculture. In parallel with hair shaft formation, visible skin color changed from white to dark grey (Fig. 1). Freshly harvested and histocultured skin fragments showed hair shaft elongation and pigmentation comparable to the in vivo situation with hair shafts becoming visible approximately 8 - 1 0 days after anagen induction by depilation (1,13,22). The beginning of visible hair shaft formation in histoculture thus reflects the in vivo-situation. When shaved skin was harvested and cultured 14 days post-anagen induction on sponge-gel supports, hair shaft elongation was 2 Day-Anagen-induced In Vitro C57B1.6 M o u s e
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,~
11.30.2 11.1 11Day
FIG. 7. Hair growth of 14-day anagen-indueed C 57 B1-6 mouse skin histoeuhured on collagen-gelsponge in vitro. Note the increased hair length over time. observed grossly in 50% of all skin fragments after 7 days of histoculture. Pigmented hair shafts increased in both length and density over time. The average length of hair growth reached 0.51 + 0.07 mm and 0.77 + 0.08 mm, respectively after 7 and 14 days histoculture (Fig. 7) with color change from grey to black (Fig. 5). The density of hair growth increased from 7 hairs/mm 2 after 7 days histocuhure to 11 hairs/mm 2 after 14 days histoculture in 5 sets of explants. Comparatively, more hair growth and faster growth occurred around the edge area of the skin histoculture than in the central area on both 2-day and 14-day-post-anagen-induced skin. The rate of hair growth of the 2-day and 14-day-post-anagen-induced skin was similar at about 0.1 mm/per day in the first 7 days, while the density of hair growth in 14-day-post-anagen-induced skin was much higher than 2-day-post-anagen-induced skin. The density of in-vitro growing hairs increased only in the 14-day post-anagen-induced cultures. It is important to note that, though 33% of the 2-day post-anagen-induced and 50% of the 14-day post-anagen-induced histocultured skin fragments exhibited hair shaft elongation and pigmentation, the number of actually growing shafts per biopsy and the maximal length achieved varied from experiment to experiment, ranged from 6 to 145 shafts per biopsy (mean 21 shafts per responsive biopsy). Histologically, at the start of histoculture, the 2-day post-anageninduced skin (harvested 2 days-post-depilation)exhibited the characteristic follicles of early anagen [anagen stage I-II according to Chase (12)] having neither hair shafts nor pigment production (Fig. 2 A cf. 14,22). Fig. 2 B,C, representing the same specimen as Fig. 2 A, show that after 8 and 16 days of histoculture, respectively, normal-appearing, mature follicles of anagen stage VI have developed, with the formation of regularly pigmented hair shafts and full melanin production in the epithelial bulbs. Note in Fig. 2 B and C the typical, band-like melanin-distribution pattern of the terminallydifferentiated keratinocytes forming the hair shaft. The 14-day post-anagen-induced skin also retained normal follicle architecture even after 14 days histoculture in vitro (data not shown). In order to demonstrate the viability of hair follicle keratinocytes in histoculture, fluorescent dye double-staining was performed and assessed by confocal laser scanning microscopy (8). After 5 days of
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histoculture, most follicle cells are viable (Fig. 3). To identify proliferating cells, [SH]thymidine incorporation was measured by autoradiography. Figure 4 shows that the majority of [SH]thymidinelabeled cells were the hair matrix cells after 8 days histocuhure. After 16 days of histoculture, DNA-synthesis still occurred in the hair bulb, though the majority of the proliferating cells were found in the outer root sheath (data not shown) thus possibly explaining the reduced rate of in vitro hair growth by Day 16. The histocuhure system described here can now be utilized to analyze the inherent apparatus of follicle growth control, and to define the growth requirements of mature anagen hair follicles growing in their natural tissue environment. It should be a useful tool for dissecting the relative importance of cytokines as well as of thyroid, pituitary and sex hormones for modulating the length of the anagen phase. In contrast to recently developed mixed-cell culture systems which study melanocytes seeded into in vitro-reconstituted skin-"equivalents" (e.g. 21), histocuhure of mouse skin does not require any prior enzymatic treatment of the tissue, and occurs in the presence of other resident skin cells like mast cells, which are absent in the mixed-cell culture models. Thus, the growth conditions of melanocytes in histocuhure seem closer to the physiological situation than of any other currently available system. That even after 16 days of histoculture there is histological evidence for melanin production (Fig. 2) suggests that melanocytes are still alive and melanogenically active at this time. The long-term histocuhure of C 57 BI-6 mouse skin in defined stages of the hair cycle may serve as a powerful tool for dissecting the relative contributions made by the cell populations and signal molecules implicated in the regulation of hair growth, and for analyzing the intrinsic apparatus that controls hair follicle cycling. ACKNOWLEDGEMKNTS This study was supported in part by a NIH/SB1R grant #R43 CA53995 to AntiCancer Inc., by DFG to R. P. and by Lawrence M. Gelb Research Award to A. S.
11. Link, R. E.; Pans, R.; Stenn, K. S., et al. Epithelial growth by rat vibrissae follicles in vitro requires mesenchymal contact via native extracellular matrix. J. Invest. Derm. 95:202-207; 1990. 12. Parakkal, P. F. Role of macrophages in collagen resorption during hair growth cycle. J. Ultrastruct. Res. 29:210-217; 1969. 13. Pans, R.; Stenn, K. S.; Link, R. E. Telogen skin contains an inhibitor of hair growth. Br. J. Dermatol. 122:777-784; 1990. 14. Pans, R.; Stenn, K. S.; Elgjo, K. The epidermal pentapoptide pyroGluGlu-Asp-Ser-GlyOH inhibits murine hair growth in vivo and in vitro. Dermatologica 183:173-178; 1991. 15. Pans, R.; Maurer, M.; Slominski, A., et al. Mast cells and hair growth: the murine hair cycle as a model for studying growth-regulatory functions of mast cells. Arch. Dermatol. Res. 284:31A; 1992. 16. Pans, R.; Stenn, K. S.; Ling, R. E. The induction of anagen hair follicle growth in telogen mouse skin by cyclosporine A administration. Lab. Invest. 60:365-369; 1989. 17. Pans, R. Hair growth inhibition by heparin in mice: a model system for studying the modulation of epithelial cell growth by glycosaminoglycans? Br. J. Derm. 124:415-422; 1991. 18. Philpott, M. P.; Green, M. R.; Kealey, T. Studies on the biochemistry and morphology of freshly isolated and maintained hair follicles. J. Cell Sci. 93:409-418; 1989. 19. Philpott, M. P.; Green, M. R.; Kealey, T. Human hair growth in vitro. J. Cell Sci. 97:463-471; 1990. 20. Rogers, G.; Martinet, N.; Steinert, P., et al. Cultivation of mufine hair follicles as orgaooids in a collagen matrix. J. Invest. Dermatol. 89:369-379; 1987. 21. Scott, G. A.; Haake, A. R. Kerafinocytes regulate melanocyte number in human fetal and neonatal skin equivalents. J. Invest. Dermatol. 97:776-782; 1991. 22. Slominski, A.; Pans, R.; Costantino, R. Differential expression and activity of melanogenesis-related proteins during induced hair growth in mice. J. Invest. Derm. 96:172-179; 1991. 23. Slominski, A.; Pans, R.; Mazurkiewicz, J. Proopiomelanocortin expression in the skin during induced hair growth in mice. Experientia. 48:50-54; 1992. 24. Slominski, A.; Paus, R.; Wortsman, J. Can some melanotropins modulate keratioocyte proliferation? J. Invest. Dermatol. 97:747; 1991. 25. Westgate, G. E.; Craggs, R. I.; Gibson, W. T. Immune privilege and hair growth. J. Invest. Dermatol. 97:417-421; 1991. 26. Yaar, M.; Gilchrest, B. A. Human melanocyte growth and differentiation: a decade of new data. J. Invest. Dermatol. 97:611-618; 1991. 27. Li, L.; Pans, R.; Margolis, L. B., et al. Hair growth in vitro from histocuitured skin. In Vitro Cell. Dev. Biol. 28A:479-481; 1992.
REFERENCES 1. Chase, H. B. Growth of the hair. Physiol. Rev. 34:113-126; 1954. 2. Dawber, R.; Rook, A., eds. Diseases of the hair and scalp. Oxford: Blackwell; 1992. 3. du Cros, D. L.; Isaacs, K.; Moore, G. P. M. Localization of epidermal growth factor immunoreactivity in sheep skin during wool follicle development. J. Invest. Dermatol. 98:109-115; 1992. 4. Ebling, F. J. G. The biology of hair. Dermatol. Clinics 5:476-481; 1987. 5. Hoffman, R. M. Three-dimensional histoculture: origin and applications in cancer research. Cancer Cells 3:86-92; 1991. 6. Johnson, E. Inherent rhythms of activity in the hair follicle and their control. In: Lyne, A. G.; Short, B. F., eds. Biology of the skin and hair growth. New York: Elsevier; 1965:491-505. 7. Leighton, J. A sponge matrix method for tissue culture. J. Nail. Cancer Inst. 12:545-561; 1951. 8. Li, L.; Margolis, L. B.; Hoffman, R. M. Skin toxicity determined in vitro by three-dimensional, native-state histoculture. Proc. Nail. Acad. Sci. USA 88:1908-1912; 1991. 9. Li, L.; Hoffman, R. M. Hair growth and hair follicle-cell proliferation in histocuhured mouse skin. Ann. NY Acad. Sci. 642:506-509; 1991. 10. Li, L.; Margolis, L. B.; Pans, R., et al. Hair shaft elongation, follicle growth and spontaneous regression in long-term, sponge-gel supported histocuhure of human scalp skin. Proc. Nail. Acad. Sci. USA 89:8764-8768; 1992.
Lingna Li Ralf Paus
Andrzej Slominski Robert M. I-Ioffman 1 AntiCancer Inc. (L. L., R. M. H.) 5325 Metro Street San Diego, CA 92110; Dept. of Dermatology (R. P.) University Hospital R. Virchow Freie Universitaet Berlin, D-1000 Berlin 65; Dept. of Microbiology, Immunology & Molecular Genetics (A. S.) Albany Medical College Albany, NY 12208; and Laboratory of Cancer Biology (R. M. H.) University of California, San Diego La Jolla, CA 92093 (Received 11 August 1992)
l To whom correspondence should be addressed at Laboratory of Cancer Biology, University of California, San Diego, 0609F, La Jolla, CA 92093.
