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Stem Cells of Pelage, Vibrissae, and Eyelash Follicles: The Hair Cycle and Tbmor Formation ROBERT M. LAVKER,4=GEORGE COTSARELIS,b ZHI-GANG WEI,b AND TUNG-TIEN SUN‘ bDepartment of Dermatology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104 ‘Epithelial Biology Unit Departments of Dermatology and Pharmacology Kaplan Cancer Center New York University School of Medicine New York, New York 10016
INTRODUCTION Stem cells are by definition present in all self-renewing These cells are believed to be long-lived, have great potential for cell division, and are ultimately responsible for homeostasis of continually renewing tissues. In addition, stem cells play a central role in wound healing, aging, and carcinogenesis. Thus, in order to better understand the growth of any self-renewing tissue, such as the hair follicle, it is important to study its stem cells. Based on previous studies of stem cells of the hemopoietic system and several stratified squamous and simple epithelia,5-I4 we know that stem cells possess many of the following properties: (i) they are relatively undifferentiated, both ultrastructurally and biochemically; (ii) they have a tremendous proliferative potential and are responsible for long-term maintenance and regeneration of the tissue; (iii) they rarely incorporate tritiated thymidine (3H-TdR) after a single pulse labeling, indicative that they are normally slow cycling; (iv) they can, however, be induced to enter the proliferative pool in response to wounding and to certain growth stimuli; and (v) when they undergo occasional cell division, they give rise to more rapidly proliferating “transient amplifying” (TA) cells, which incorporate 3H-TdR after a single exposure; these TA cells have a limited capacity for division before they become TA cell postmitotic or terminally differentiated in the scheme of “stem cell terminally differentiated cell;” and finally, (vi) stem cells are usually found in a well-protected, highly vascularized and innervated area. Using these as the criteria, we have over the past decade identified the putative stem cells of several stratified squamous epithelia, including epidermis and corneal epithelium as well as the hair
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a This work was supported by NIH Grants AR39674 and EY06769 (R.M.L.), and -34511, AR39749, and EY4722 (T-T.S.),and by the National Alopecia Areata Foundation (R.M.L. and T-T.S.). Address for correspondence: Robert M. Lavker, Ph.D., Duhring Laboratories, Department of Dermatology, University of Pennsylvania School of Medicine, Clinical Research Building, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104.
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follicle. In this paper we will compare the properties of the stem cells of these diverse but related tissues, and we will provide some new data on stem cells of several specialized hair follicles-the vibrissa and eyelash. The implication of our findings on keratinocyte biology will be discussed.
EPIDERMAL STEM CELLS During investigations on monkey palm and human trunk epidermis:JO we noted the existence of two morphologically distinct subpopulations of basal keratinocytes. One population was characterized by a “primitive” cytoplasm containing abundant melanosomes and a relatively flattened (“nonserrated”) dermal-epidermal junction. In contrast, the other population was characterized by a cytoplasm filled with keratin filaments and a highly convoluted (“serrated”) dermal-epidermal junction. 3H-TdR autoradiographic experiments indicated that the nonserrated basal keratinocytes did not incorporate the labeled nucleotide, suggesting that the nonserrated cells were slow cycling. However, a population of keratinocytes that actively incorporates 3H-TdR was identified immediately above these basal cells. We postulated that the nonserrated basal cells represented stem cells that gave rise to superabasally located TA cells. Consistent with this hypothesis, we found that these normally slow-cycling basal cells became heavily labeled in response to adjacent linear incision wounds, indicative that these cells could be recruited into the proliferative pool during tissue depleti~n.~,~
CORNEAL EPITHELIAL STEM CELLS
As previously mentioned, an important feature of stem cells is that they are normally slow cycling. To label these slow-cycling cells requires the administration of 3H-TdR for a prolonged period. Once labeled, cells that cycle slowly will retain the isotope for an extended period of time and thus can be identified as “labelretaining cells” ( L R C S ) . ~ J Although ~J~ this approach is not practical for studying monkey palm epithelia, it is feasible in murine animals. Using this approach we have successfully identified a subpopulation of slow-cycling corneal epithelial cells that are located at the edge of the cornea in a region known as the l i m b u ~That . ~ these limbal cells may represent corneal epithelial stem cells is supported by several pieces of evidence. First, Davenger and Evensen observed pigmented corneal epithelial streaks that were believed to result from a movement of pigmented limbal and/or conjunctival cells toward the center of the cornea. This centripetal migration was postulated to be the means by which corneal epithelium was maintait1ed.17-1~Second, Schermer et aLZoused a monoclonal antibody to a major 64-kD basic corneal epithelial keratin to demonstrate that this keratin was a marker for an advanced stage of corneal epithelial differentiation. This keratin was shown to be expressed suprabasally in limbal epithelium, but uniformly in central corneal epithelium. This finding was indicative that limbal basal cells were biochemically more primitive than corneal epithelial basal cells. These data formed the initial basis of a model in which corneal epithelial stem cells were postulated to be located in the basal layer of limbal epithelium.Z0 Third, we found that limbal epithelium could be preferentially stimulated to proliferate in response to either wounding or topical application of a tumor promotor, Taken together, these results provided strong support for the hypothesis that corneal epithelial stem cells were concentrated in the limbus.
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HAIR FOLLICLE STEM CELLS With respect to other self-renewing tissues (e.g., epidermis), the hair follicle is unique in that, instead of a relatively constant steady state of cell proliferation, hair follicle proliferation is tightly controlled and c y ~ l i c a l . After ~ ~ - ~a~period of active growth in anagen, the lowermost proliferative epithelial cells (matrix cells of the bulbar region) cease dividing, and they regress during catagen.= When regression is completed, the follicle enters a resting phase (telogen), and after a period of time proliferation begins, and the follicle reenters anagen. During anagen, matrix cells are known to proliferate extremely rapidly, with a doubling time of 18-24 hours.29 Thus, mitotic figures are readily observed in matrix epithelial cells, and a large proportion of these cells incorporate 3H-TdR after a single injection. Because associations are frequently made (erroneously) between sites of high proliferation and location of stem cells, the bulbar region has long been considered the site of follicular epithelial stem cells.27,29~30 We were thus surprised to find no LRCs in the hair bulb when we evaluated the distribution of slow-cycling cells (LRCs) in the hair follicle. Instead, we found a subpopulation of LRCs in the outer root sheath in the upper portion of the follicle, in a region known as the ‘‘bulge’’-the attachment site of the arector pili muscle.30~32-34 This area is below the opening of the sebaceous gland. It marks the lower end of the “permanent” portion of the hair follicle, since keratinocytes below the bulge degenerate during catagen and telogen. In addition to being slow cycling, cells comprising the bulge possess many stem cell properties. For example, they can be stimulated to proliferate by a tumor promotor-TPA. Ultrastructurally, they have a relatively primitive cytoplasm filled with ribosomes and relatively devoid of keratin filament bundles. Finally, they are located in a physically well-protected and wellnourished area.31 While the above data were obtained from the regular pelage hairs, we have obtained similar results from two specialized hairs-vibrissae and eyelash follicles. Vibrissae represent the major tactile organ of rodents and have been used extensively as a convenient model for the study of hair bi0logy.2~+~~ Eyelashes protect the eyes from dust and sunlight, and appear to be independent of sex h o r m o n e ~ . ~Both 2 , ~ ~of these structures are morphologically similar to pelage hair follicles, with an analogous outer and inner root sheath, dermal papilla, and fibrous capsule (FIGS.la, 2a). Although vibrissae, eyelash, and pelage hair follicles undergo similar cycles of growth (anagen), regression (catagen), and rest (telogen), the length of each cycle is different.z,24Vibrissae also differ from pelage hair in their larger size, presence of blood-filled sinuses, a surrounding fibrous band structure (the ringwulst), and extensive and specialized innervation” (FIG.la). Eyelash follicles differ from adjacent pelage hair follicles by their much larger size (FIG.2a). Because of the structural and cycling differences between pelage, eyelash, and vibrissa hairs we investigated the distribution of LRCs in these specialized hair structures in neonatal and adult mice. A population of slow-cycling cells was localized in the outer root sheath of the vibrissa follicle at the level of the ringwulst and ring sinus (FIG.lb). This area is analogous to the bulge in pelage hair. LRCs were not detected in the matrix keratinocytes or follicular papilla cells, which comprise the bulb region of the vibrissa follicle (FIG.lc). Similarly, in eyelash a population of LRCs was present exclusively in the upper portion of the outer root sheath in a region corresponding to the bulge (FIG.2b, c). These findings indicate that despite differences in size, length of growth cycle, and hormonal control, LRCs of the vibrissa, eyelash, and pelage hair reside in analogous regions, indicating that cells in the bulge area are kinetically unique in many hair types.
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These findings, in addition to a critical reevaluation of the literature, suggested that the bulge was the site of the hair follicle stem cell. Montagna suggested in 1962 that the outer root sheath, not the bulb, was the source of the germinative cells for each generation of hair follicles.3*This was based on his earlier work, which demonstrated that after destruction of the hair matrix by X-irradiation, outer root sheath cells could regenerate a complete hair Surgical removal of the lower half of human axillary hair follicles did not impede the formation of new follicles, indicative that follicular stem cells are located in the upper portion of the follicle.q0Similarly, after surgical removal of the lower half of rat vibrissa hair follicles, regeneration of new hair bulbs was observed in response to the implantation of a new dermal papilla.22.23~4* Together, these findings strongly support our notion that the upper portion of the follicle, not the lower bulb region, is the site of the germinative cells.
“BULGEACTIVATION” HYPOTHESIS The identification of the putative follicular stem cells in the bulge region of the hair follicle enabled us to develop a “bulge activation” h y p o t h e s i ~ ~that ~ . ~provides * a unifying concept that explains many puzzling aspects of hair biology (FIG. 3). Successful hair growth depends on several separate interactions between epithelial cells and specialized mesenchymal cells of the dermal papilla. We postulate that sometime during telogen or early anagen, the normally slow-cycling bulge cells are activated by dermal papilla cells that are in close proximity to the bulge at that time. The nature of this cell-cell interaction and the specific factors elaborated by the dermal papilla cells are not presently known. Activations results in the proliferation of the bulge cells, which form a down-growth of epithelial cells that eventually gives rise to the new matrix. As this down-growth evolves, the dermal papilla is pushed away from the bulge, and the bulge stem cells return to their slow-cycling state. During the remaining hair cycle the matrix proliferation necessary to elaborate the hair and inner root sheath is accomplished through the replication of matrix cells. These rapidly dividing cells are derived from stem cell divisions; they are therefore TA cells and have a limited capacity to proliferate before becoming postmitotic. This finite capacity of mature cells to proliferate may explain the events of catagen, when the matrix cells exhaust their proliferative potential and undergo terminal differentiation. We believe that another critical interaction between epithelial cells and the dermal papilla occurs during midanagen. During most phases of the hair cycle the dermal papilla cells appear to be relatively quiescent.43However, during midanagen (stage IV) they undergo a burst of cell proliferation, and new blood vessels form.” Since proliferation of matrix epithelial cells precedes that of dermal papilla cells, it is likely that at this stage of the hair cycle that matrix cells are capable of stimulating the growth of papillary mesenchymal cells.45The degree to which the dermal papilla is stimulated and subsequently enlarges, as measured by the volume of the dermal papilla, has been shown by Van Scott et al. in 1963 to be directly proportional to the diameter and length of the resulting hair.41 The third important dermal papilla-epithelial interaction occurs during early catagen, when the papilla condenses and is connected to the regressing matrix via a “connective tissue sheath.” As catagen progresses, the dermal papilla is pulled upward and eventually becomes positioned in the vicinity of the bulge. This sequence is crucial, as failure of the ascendance of the dermal papilla is known to be accompanied by the failure of the hair follicle to enter into the next cycle, presumably due to the inability of the bulge cells to become activated.&
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a.
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FIGURE 3. Hair cycle: the bulge activation hypothesis. Different phases of the hair cycle are shown, including (a) anagen V1,(b)catagen, (c) telogen, (a) anagen 11, and (e) anagen IV. h e c t o r pili muscle (AMP), bulge (B), cortex (C), dermal papilla (DP), epidermis (E), inner root sheath (IRS), matrix (M), medulla (Md), outer root sheath (ORS),and sebaceous gland (S) are key structures of the pilosebaceous unit. The quiescent (B) and activated (B*)states of the bulge cells are as indicated. The structures above the dashed line represent the permanent portion of the follicle; keratinocytes below the bulge degenerate during catagen and thus can be considered as “dispensable.” The four major elements of this hypothesis are: activation of the bulge by dermal papilla during telogen (c), activation of the dermal papilla by the matrix keratinocytes during anagen IV (e), finite proliferative capabilities of matrix cells as transient-amplifying cells during late anagen (a), and upward migration of the dermal papilla during catagen @). (From Cotsarelis et aL3’ Reprinted by permission of Cell.)
SKIN CARCINOGENESIS In addition to yielding new insights on the regulation of the hair cycle, the localization of putative follicular stem cells to the bulge region of the hair follicle has significant implications in understanding skin carcinogenesis. Previous studies on chemical carcinogenesis in mice have indicated that cancer development is greatly
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influenced by the stage of the hair c y ~ l e . Many ~ ~ - more ~ ~ skin cancers occur when a complete carcinogen is applied during telogen as opposed to anagen; thus it has become almost standard practice to use only telogen-phase animals in murine carcinogenesis experiments. The reason for an increased tumor rate with topical application of carcinogen during telogen has been ascribed to increased retention of carcinogen by the pilosebaceous unit. This has been explained in part as due to the fact that in telogen the inner root sheath cells, which normally seal the hair canal, are absent, thus giving greater accessibility of the carcinogen to the hair follicle.5° If, as discussed above, follicular stem cells are located in the bulge area, this implies that follicular stem cells are exposed to a much higher concentration of carcinogen for a longer period in telogen than in anagen. Thus, the correlation between tumor yield and the enhanced entry of carcinogen to the bulge raises the possibility that follicular stem cells, rather than the interfollicular epidermis, are involved in and largely responsible for chemically induced skin tumor formation in the mouse model. This hypothesis raises many intriguing questions regarding the relative contribution of the follicle and interfollicular epidermis to the formation of various human skin carcinomas.
PLURIPOTENT STEM CELLS The specific location of the follicular stem cells raises the intriguing question of whether these cells might be pluripotent, giving rise to not only hair follicles, but also to sebaceous glands and, in certain instances, the epidermis. Although cell proliferation occurs in the basal cell layer of the sebaceous gland,” these rapidly proliferating cells are most likely transient amplifying cells. Interestingly, we were unable to detect any LRCs in the sebaceous glands, suggesting that this gland’s stem cells reside elsewhere. The fact that the bulge area is immediately adjacent to the opening of the sebaceous gland raises the possibility that bulge cells also give rise to the sebaceous gland. With regards to the epidermis, it is well established that, following the loss of a large area of the epidermis by mechanical or thermal means, reepithelialization occurs by cells emerging from hair follicles.5*Since these folliclederived cells would later be responsible for the long-term maintenance of the epidermis, they would be stem cells and as such are most likely derived from the bulge.
CONCLUSIONS In this paper we have reviewed the evidence that has led us to suggest that the tips of the deep epidermal rete ridges, the limbal epithelium, and the bulge region of the hair follicle are sites of epidermal, corneal epithelial, and hair follicle stem cells, respectively. Comparisons of stem cells from these three epithelia with stem cells from other epithelia (e.g., dorsal tongue, intestinal epithelium) reveal a commonality of features with respect to location and biological properties.5 Briefly, they are usually located close to the vasculature, in well-protected and innervated areas. If they are in a sun-exposed area, they tend to be highly pigmented. They are usually slow cycling but can be recruited into the proliferative pool in response to wounding or certain growth stimuli. Finally, they are morphologically and biochemically “primitive.” The finding that hair follicle stem cells reside in the upper region of the permanent portion of the hair follicle, the bulge, has allowed us to develop a “bulge
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activation” hypothesis. This hypothesis redirects our thoughts concerning the various aspects of the hair cycle. Whereas most previous investigators have concentrated their efforts at understanding the events occurring during the active growing phases (anagen), we believe that major emphasis should be focused in the future on understanding the resting phase (telogen), when specific interactions between dermal papilla and the bulge cells result in the regrowth of hair. Finally, since many epidermal and follicular tumors are similar in their morphology and biological behavior, it seems reasonable that the bulge may play a central role in their development. The relatively close proximity of the bulge to the epidermis, as well as the recruitment of bulge cells to replenish the epidermis after major crises suggest a pluripotent role for bulge stem cells. REFERENCES 1. LAJTHA, L. G. 1979. Stem cell concepts. Differentiation 14: 23-34. 2. LEBLOND, C. P. 1981. The life history of cells in renewing systems. Am. 1. Anat. 160: 114-157. 3. POTTEN, C. S., R. SCHOFIELD & L. G. LAJTHA. 1979. A comparison of cell replacement in bone marrow, testis and three regions of surface epithelium. Biochem. Biophys. Acta 560: 281-299. EDS.1976. Stem Cells of Renewing Cell 4. CAIRNIE, A. B., P. K. LALA& D. G. OSMOND, Populations. Academic Press, New York, NY. 5. COTSARELIS, G., S-Z. CHENG, G. DONG,T-T. SUN& R. M. LAVKER. 1989. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells. Cell 57: 201-209. 6. QUESENBERRY, P. & L. LEVITT.1979. Hematopoietic stem cells. N. Engl. J. Med. 301: 755-760. 7. V A N BEKKUM, D. W., D. J. VAN DER ENGH,G. WAGENMAKER, S. S. L. BOL& J. W. M. VISSER.1979. Structural identity of the pluripotential hemopoietic stem cell. Blood Cells 5: 143-159. 8. ISCOVE, N. N., J. E. TILL,& E. A. MCCULLOCH. 1970. The proliferative states of mouse granulopoietic progenitor cells. Proc. SOC.Exp. Biol. Med. 134 33-36. 9. LAVKER, R. M. & T-T. SUN.1982. Heterogeneity in epidermal basal keratinocytes: Morphological and functional correlations. Science 215: 1239-1241. 10. LAVKER, R.M. & T-T. SUN.1983. Epidermal stem cells. J. Invest. Dermatol. 81 (Suppl): 121-127. 11. WRIGHT,N. & M. ALLISON, EDS. 1984. The Biology of Epithelial Cell Populations. Clarendon Press. Oxford. 12. HALL,P. A. & F. M. WATT. 1989. Stem cells: The generation and maintenance of cellular diversity. Development 106: 619-633. 13. HUME,W. J. 1983. Stem cells in oral epithelia. In Stem Cells: Their Identification and Characterization. C. S. Potten, Ed. 234-270. Churchill and Livingstone. Edinburgh. 14. BICKENBACH, J. R. & B. D. S. MACKENZIE. 1984. Identification and localization of label-retaining cells in hamster epithelia. J. Invest. Dermatol. 82: 618-622. J. R. 1981. Identification and behavior of label-retainingcells in oral mumsa 15. BICKENBACH, and skin. J. Dent. Res. 60:1611-1620. & T. J. SLAGA.1985. Evidence that the centrally and 16. MORRIS, R. J., S. M. FISCHER peripherally located cells in the murine epidermal proliferative unit are two distinct cell populations. J. Invest. Dermatol. 84: 277-281. 1971. Role of the pericorneal papillary structure in renewal 17. DAVENGER, M. & A. EVENSEN. of corneal epithelium. Nature 229 560-561. 18. BRON, A. J. 1973. Vortex patterns of the corneal epithelium. Trans. Ophthalmol. Soc.U.K. 93: 455-472. 19. GOLDBERG, M. F. & A. J. BRON.1982. Limbal palisades of Vogt. Trans. Am. Ophthalmol. SOC. 80: 155-171.
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20. SCHERMER, A., S . GALviN& T-T. SUN.1986. Differentiation-relatedexpression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J. Cell Biol. 103: 49-62. 1959. Influence of the dermal papilla on survival of 21. CROUNSE, R. G., & J. M. STENGLE. isolated human scalp hair roots in an heterologus host. J. Invest. Dermatol. 32: 477-479. 22. OLIVER, R. F. 1967. Ectopic regeneration of whisker in the hooded rat from implanted lengths of vibrissa follicle wall. J. Embryol. Exp. Morphol. 17: 27-34. 23. OLIVER, R. F. 1967. The experimental induction of whisker growth in the hooded rat by implantation of dermal papillae. J. Embryol. Exp. Morphol. 18: 43-51. 24. DRY,F. W. 1926. The coat of the mouse (Mus rnuscufus). J. Genet. 16: 287-340. 25. CHASE,H. B., H. RAUCH & V. W. SMITH. 1951. Critical stages of hair development and pigmentation in the mouse. Physiol. Zool. 24: 1-8. 26. CHASE,H. B. 1954. Growth of the hair. Physiol. Rev. 34: 113-126. 27. KLIGMAN, A. M. 1959. The human hair cycle. J. Invest. Dermatol. 33: 307-316. 28. STRAILE, W. C., H. B. CHASE& C. ARSENAULT. 1961. Growth and differentiation of hair follicles between periods of activity and quiescence. J. Exp. h l . 148: 205-216. 1963. Determinants of rate and kinetics 29. VANSCOTT,E. J., T. M. EKEL& R. AUERBACH. of cell division in scalp hair. J. Invest. Dermatol. 41: 269-273. 30. PINKUS, H. 1978. Embryology of hair. In The Biology of Hair Growth. W. Montagna & R. Ellis, Eds.: 1-32. Academic Press. New York, W . 31. CoTsARELis, G.,T-T. SUN & R.M. LAVKER. 1990. Label-retainingcells reside in the bulge area of pilosebaceous unit: Implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61: 1329-1337. 32. MONTAGNA, W. & K. S. CARLISLE. 1981. Considerationson hair research and hair growth. In Hair Research. C. E. Orfanos, W. Montagna & G. Stuttgen, Eds.: 7. Springer-Verlag. Berlin. 33. SCHWEIKERT, H. U. & J. D. WILSON. 1981. Androgen metabolism in isolated human hair roots. In Hair Research. C. E. Orfanos, W. Montagna & G. Stuttgen, Eds.: 210. Springer-Verlag. Berlin. 34. RICE,F. L., A. MANCE & B. L. MUNGER. 1986. A comparative light microscopic analysis of the sensory innervation of the mystacial pad. I. Innervation of vibrissal follicle-sinus complexes. J. Comp. Neurol. 252: 154-174. 35. UNNA,P. G. 1876. Beitrage zur histologie und entwicklungsgeschichteder mensclichen oberhaut und hrer anhangsgebilde. Arch. Microskop. Anat. Entwicklungsmech. 12: 665-741. 36. STOHR,P. 1903-1904. Entwicklungsgechichtedes menschlichen wolhaares. Anat. Hefte. Abt. 123: 1-66. 37. MADSEN, A. 1964. Studies on the “bulge” (Wulst) in superficial basal cell epitheliomas. Arch. Dermatol. 8 9 698-708. 38. MONTAGNA, W. 1962. The Structure and Function of Skin. Academic Press. New York,
NY. 39. MONTAGNA, W. & H. B. Chase. 1956. Histology and cytochemistry of human skin: X-irradiation of the scalp. Am. J. Anat. 99: 415-445. & C. MCKISTRY. 1979. Histologic study of the regeneration of 40. INABA,M., J. ANTHONY axillary hair after removal with subcutaneous tissue shaver. J. Invest. Dermatol. 72: 224-23 1. 41. IBRAHIM, L. & E. A. WRIGHT. 1982. A quantitative study of hair growth using mouse and rat vibrissal follicles. J. Embryol. Exp. Morphol. 72: 209-224. & R. M. LAVKER. 1991. Hair follicular stem cells: The bulge42. SUN,T-T., G. COTSARELIS activation hypothesis. J. Invest. Dermatol. %: 77-78s. 43. MOFFAT,G. H. 1968. The growth of hair follicles and its relation to the adjacent dermal structures. J. Anat. 102: 527-540. 1975. Modulation of dermal cell activity during 44. PIERARD, G . E. & M. DE LA BRASSINNE. hair growth in the rat. J. Cutaneous Pathol. 2: 35-41. A. F. & H. B. CHASE.1977. The incorporation of tritiatied uridine in hair germ 45. SILVER, and dermal papilla during dormancy (telogen) and activation (early anagen). J. Invest. Dermatol. 68: 201-205.
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46. MONTAGNA, W., H. B. CHASE& H. P. MELARAGNO. 1952. Skin of hairless mice. 1. Formation of cysts and the distribution of lipids. J. Invest. Dermatol. 1 9 83-94. 47. ANDREASEN, E. 1953. Significance of mouse hair cycle in experimental carcinogenesis. Acta. Pathol. Microbiol. Scand. 32: 165-169. 48. BORUM, K. 1954. The role of mouse hair cycle in epidermal carcinogenesis. Acta. Pathol. Microbiol. Scand. 34: 542-553. 49. ARGYRIS, T. S. 1980. Tumor promotion by abrasion-induced epidermal hyperplasia in the skin of mice. J. Invest. Dermatol. 75: 360-362. 50. BERENBLUM, I., N. HAREN-GHERE & N. TRAININ. 1959. An experimental analysis of the “hair cycle effect” in mouse skin carcinogenesis. Br. J. Cancer 12: 402-413. G. D. 1974. Cell kinetics of human sebaceous glands. J. Invest. Dermatol. 62: 51. WEINSTEIN, 144-146. 52. ARGYRIS, T. S. 1981. Regulation of epidermal hyperplastic growth. CRC Crit. Rev. Toxicol. 9: 151-200.
DISCUSSION OF THE PAPER E. FUCHS (University of Chicago, Chicago, Ill.): Have you tried to dissect out bulge cells? And, can you tell us a little bit about your experiments, successes, failures, etc? LAVKER: My lab has not actually been actively trying to take that approach. What we have been doing is looking at some of the kinetics that are going on during the early parts of the hair cycle. B. HOGAN (Vanderbilt Medical School, Nashville, Tenn.): Can you tell me approximately how many stem cells there are per unit? And, are they stem cells for the sebaceous gland, also? Are the stem cells separate ones, or are they sort of pluripotent in the sense of also giving rise to sebaceous glands? LAVKER: The answer is I don’t know. I would like to believe that they do give rise to what would be the basal sebocyte. The bulge is a very logical site for sebaceous stem cells only because we have been unable to detect labeled retaining cells or a slow-cycling cell amongst the basal sebocytes. As to the numbers of stem cells, I can tell you that approximately 20-30% of the [corneal] limbal epithelial cells are slow-cycling cells. We haven’t quantified the bulge cells in that respect. The work that has been done with labeled retaining cells in the mouse epidermis-work by McKenzie and Bickenbach-indicates that about 1%of the basal cells are label retaining. We certainly see a much greater number than that in the bulge. When we look for label-retaining cells in the epidermis, we occasionally will see one, but the bulge appears to be a repository of label-retaining cells. K. WARD(C.S.I.R.O., Blacktown, New South Wales, Australia): Do vellus follicles also have a bulge? Because they don’t have an arrector pili muscle, do they? LAVKER: We see a bulge in almost all of the hair follicles that we’ve looked at as well as the vibrissa. We find the label-retaining cells in that region of the vibrissa that is the bulge. So it seems to be consistent in all the hairs that we’ve looked at, whether they are vellus or not. U , LICHTI (National Institutes of Health, Bethesda, Md.): Can you spell out for me, again, why you think that stem cells in the resting follicle and in the growing follicle in the same location should be differently sensitive to carcinogen? Is it the accessibility? Or is it something intrinsically different about the cells under those two conditions?
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LAVKER: At the present time, I would think it is accessibility of the bulge to the carcinogen. C. JAHODA (University of Dundee, Dundee, Scotland, U.K.):The vibrissa follicle does not shorten, so the papilla doesn’t move out during the cycle. How does this accord with your theory? LAVKER: What I think is happening here is that the vibrissa follicle is in a permanent anagen state. This gets back to the concept of the transient amplifying cell, how many amplification divisions there are, and how much support do the transient amplifying cells need? In the vibrissa we do seem to find the label-retaining cells in the same anatomical position as the bulge. These cells no doubt undergo divisions when called upon, and they send down transient amplifying cells that probably have a hierarchy of divisions. Such cell growth supports the proliferative state. The vibrissa does go into telogen, but it is a very short one. JAHODA: But there is no movement of the papilla. LAVKER: There is no movement, no. We don’t see the slow-cycling cells, nor do we see the primitive-looking cells anywhere else in the vibrissa other than in the bulge. JAHODA: We see them in the base. And that’s why in some systems like the intestine I think there could be two groups of stem cells. Would you rule that out? LAVKER: There could be. At one site there could be noncycling or G-zero cells and at other sites hierarchies of transient amplifying cells, some of which have tremendous potential. UNIDENTIFIED SPEAKER: There has been development of the tumor suppressor genes, such as RB or P53 genes. In colon cancer, for example, the P53 genes become lost. Could it be that the stem cells might have a higher expression of these tumor suppressor genes? LAVKER: It is something that we have not looked into. J. BRIND (Orentech Foundation, Cold Spring, MY.): You had mentioned that the bulge is not as prominent in the scalp follicle as it is in some other areas. In what areas is this a more prominent feature? LAVKER: In areas across the bridge of the nose, around the eybrows, and on the forearm, we see a much more prominent bulge than in the scalp.
Stem Cells of Pelage, Vibrissae, and Eyelash Follicles: The Hair Cycle and Tbmor Formation ROBERT M. LAVKER,4=GEORGE COTSARELIS,b ZHI-GANG WEI,b AND TUNG-TIEN SUN‘ bDepartment of Dermatology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104 ‘Epithelial Biology Unit Departments of Dermatology and Pharmacology Kaplan Cancer Center New York University School of Medicine New York, New York 10016
INTRODUCTION Stem cells are by definition present in all self-renewing These cells are believed to be long-lived, have great potential for cell division, and are ultimately responsible for homeostasis of continually renewing tissues. In addition, stem cells play a central role in wound healing, aging, and carcinogenesis. Thus, in order to better understand the growth of any self-renewing tissue, such as the hair follicle, it is important to study its stem cells. Based on previous studies of stem cells of the hemopoietic system and several stratified squamous and simple epithelia,5-I4 we know that stem cells possess many of the following properties: (i) they are relatively undifferentiated, both ultrastructurally and biochemically; (ii) they have a tremendous proliferative potential and are responsible for long-term maintenance and regeneration of the tissue; (iii) they rarely incorporate tritiated thymidine (3H-TdR) after a single pulse labeling, indicative that they are normally slow cycling; (iv) they can, however, be induced to enter the proliferative pool in response to wounding and to certain growth stimuli; and (v) when they undergo occasional cell division, they give rise to more rapidly proliferating “transient amplifying” (TA) cells, which incorporate 3H-TdR after a single exposure; these TA cells have a limited capacity for division before they become TA cell postmitotic or terminally differentiated in the scheme of “stem cell terminally differentiated cell;” and finally, (vi) stem cells are usually found in a well-protected, highly vascularized and innervated area. Using these as the criteria, we have over the past decade identified the putative stem cells of several stratified squamous epithelia, including epidermis and corneal epithelium as well as the hair
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a This work was supported by NIH Grants AR39674 and EY06769 (R.M.L.), and -34511, AR39749, and EY4722 (T-T.S.),and by the National Alopecia Areata Foundation (R.M.L. and T-T.S.). Address for correspondence: Robert M. Lavker, Ph.D., Duhring Laboratories, Department of Dermatology, University of Pennsylvania School of Medicine, Clinical Research Building, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104.
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follicle. In this paper we will compare the properties of the stem cells of these diverse but related tissues, and we will provide some new data on stem cells of several specialized hair follicles-the vibrissa and eyelash. The implication of our findings on keratinocyte biology will be discussed.
EPIDERMAL STEM CELLS During investigations on monkey palm and human trunk epidermis:JO we noted the existence of two morphologically distinct subpopulations of basal keratinocytes. One population was characterized by a “primitive” cytoplasm containing abundant melanosomes and a relatively flattened (“nonserrated”) dermal-epidermal junction. In contrast, the other population was characterized by a cytoplasm filled with keratin filaments and a highly convoluted (“serrated”) dermal-epidermal junction. 3H-TdR autoradiographic experiments indicated that the nonserrated basal keratinocytes did not incorporate the labeled nucleotide, suggesting that the nonserrated cells were slow cycling. However, a population of keratinocytes that actively incorporates 3H-TdR was identified immediately above these basal cells. We postulated that the nonserrated basal cells represented stem cells that gave rise to superabasally located TA cells. Consistent with this hypothesis, we found that these normally slow-cycling basal cells became heavily labeled in response to adjacent linear incision wounds, indicative that these cells could be recruited into the proliferative pool during tissue depleti~n.~,~
CORNEAL EPITHELIAL STEM CELLS
As previously mentioned, an important feature of stem cells is that they are normally slow cycling. To label these slow-cycling cells requires the administration of 3H-TdR for a prolonged period. Once labeled, cells that cycle slowly will retain the isotope for an extended period of time and thus can be identified as “labelretaining cells” ( L R C S ) . ~ J Although ~J~ this approach is not practical for studying monkey palm epithelia, it is feasible in murine animals. Using this approach we have successfully identified a subpopulation of slow-cycling corneal epithelial cells that are located at the edge of the cornea in a region known as the l i m b u ~That . ~ these limbal cells may represent corneal epithelial stem cells is supported by several pieces of evidence. First, Davenger and Evensen observed pigmented corneal epithelial streaks that were believed to result from a movement of pigmented limbal and/or conjunctival cells toward the center of the cornea. This centripetal migration was postulated to be the means by which corneal epithelium was maintait1ed.17-1~Second, Schermer et aLZoused a monoclonal antibody to a major 64-kD basic corneal epithelial keratin to demonstrate that this keratin was a marker for an advanced stage of corneal epithelial differentiation. This keratin was shown to be expressed suprabasally in limbal epithelium, but uniformly in central corneal epithelium. This finding was indicative that limbal basal cells were biochemically more primitive than corneal epithelial basal cells. These data formed the initial basis of a model in which corneal epithelial stem cells were postulated to be located in the basal layer of limbal epithelium.Z0 Third, we found that limbal epithelium could be preferentially stimulated to proliferate in response to either wounding or topical application of a tumor promotor, Taken together, these results provided strong support for the hypothesis that corneal epithelial stem cells were concentrated in the limbus.
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HAIR FOLLICLE STEM CELLS With respect to other self-renewing tissues (e.g., epidermis), the hair follicle is unique in that, instead of a relatively constant steady state of cell proliferation, hair follicle proliferation is tightly controlled and c y ~ l i c a l . After ~ ~ - ~a~period of active growth in anagen, the lowermost proliferative epithelial cells (matrix cells of the bulbar region) cease dividing, and they regress during catagen.= When regression is completed, the follicle enters a resting phase (telogen), and after a period of time proliferation begins, and the follicle reenters anagen. During anagen, matrix cells are known to proliferate extremely rapidly, with a doubling time of 18-24 hours.29 Thus, mitotic figures are readily observed in matrix epithelial cells, and a large proportion of these cells incorporate 3H-TdR after a single injection. Because associations are frequently made (erroneously) between sites of high proliferation and location of stem cells, the bulbar region has long been considered the site of follicular epithelial stem cells.27,29~30 We were thus surprised to find no LRCs in the hair bulb when we evaluated the distribution of slow-cycling cells (LRCs) in the hair follicle. Instead, we found a subpopulation of LRCs in the outer root sheath in the upper portion of the follicle, in a region known as the ‘‘bulge’’-the attachment site of the arector pili muscle.30~32-34 This area is below the opening of the sebaceous gland. It marks the lower end of the “permanent” portion of the hair follicle, since keratinocytes below the bulge degenerate during catagen and telogen. In addition to being slow cycling, cells comprising the bulge possess many stem cell properties. For example, they can be stimulated to proliferate by a tumor promotor-TPA. Ultrastructurally, they have a relatively primitive cytoplasm filled with ribosomes and relatively devoid of keratin filament bundles. Finally, they are located in a physically well-protected and wellnourished area.31 While the above data were obtained from the regular pelage hairs, we have obtained similar results from two specialized hairs-vibrissae and eyelash follicles. Vibrissae represent the major tactile organ of rodents and have been used extensively as a convenient model for the study of hair bi0logy.2~+~~ Eyelashes protect the eyes from dust and sunlight, and appear to be independent of sex h o r m o n e ~ . ~Both 2 , ~ ~of these structures are morphologically similar to pelage hair follicles, with an analogous outer and inner root sheath, dermal papilla, and fibrous capsule (FIGS.la, 2a). Although vibrissae, eyelash, and pelage hair follicles undergo similar cycles of growth (anagen), regression (catagen), and rest (telogen), the length of each cycle is different.z,24Vibrissae also differ from pelage hair in their larger size, presence of blood-filled sinuses, a surrounding fibrous band structure (the ringwulst), and extensive and specialized innervation” (FIG.la). Eyelash follicles differ from adjacent pelage hair follicles by their much larger size (FIG.2a). Because of the structural and cycling differences between pelage, eyelash, and vibrissa hairs we investigated the distribution of LRCs in these specialized hair structures in neonatal and adult mice. A population of slow-cycling cells was localized in the outer root sheath of the vibrissa follicle at the level of the ringwulst and ring sinus (FIG.lb). This area is analogous to the bulge in pelage hair. LRCs were not detected in the matrix keratinocytes or follicular papilla cells, which comprise the bulb region of the vibrissa follicle (FIG.lc). Similarly, in eyelash a population of LRCs was present exclusively in the upper portion of the outer root sheath in a region corresponding to the bulge (FIG.2b, c). These findings indicate that despite differences in size, length of growth cycle, and hormonal control, LRCs of the vibrissa, eyelash, and pelage hair reside in analogous regions, indicating that cells in the bulge area are kinetically unique in many hair types.
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These findings, in addition to a critical reevaluation of the literature, suggested that the bulge was the site of the hair follicle stem cell. Montagna suggested in 1962 that the outer root sheath, not the bulb, was the source of the germinative cells for each generation of hair follicles.3*This was based on his earlier work, which demonstrated that after destruction of the hair matrix by X-irradiation, outer root sheath cells could regenerate a complete hair Surgical removal of the lower half of human axillary hair follicles did not impede the formation of new follicles, indicative that follicular stem cells are located in the upper portion of the follicle.q0Similarly, after surgical removal of the lower half of rat vibrissa hair follicles, regeneration of new hair bulbs was observed in response to the implantation of a new dermal papilla.22.23~4* Together, these findings strongly support our notion that the upper portion of the follicle, not the lower bulb region, is the site of the germinative cells.
“BULGEACTIVATION” HYPOTHESIS The identification of the putative follicular stem cells in the bulge region of the hair follicle enabled us to develop a “bulge activation” h y p o t h e s i ~ ~that ~ . ~provides * a unifying concept that explains many puzzling aspects of hair biology (FIG. 3). Successful hair growth depends on several separate interactions between epithelial cells and specialized mesenchymal cells of the dermal papilla. We postulate that sometime during telogen or early anagen, the normally slow-cycling bulge cells are activated by dermal papilla cells that are in close proximity to the bulge at that time. The nature of this cell-cell interaction and the specific factors elaborated by the dermal papilla cells are not presently known. Activations results in the proliferation of the bulge cells, which form a down-growth of epithelial cells that eventually gives rise to the new matrix. As this down-growth evolves, the dermal papilla is pushed away from the bulge, and the bulge stem cells return to their slow-cycling state. During the remaining hair cycle the matrix proliferation necessary to elaborate the hair and inner root sheath is accomplished through the replication of matrix cells. These rapidly dividing cells are derived from stem cell divisions; they are therefore TA cells and have a limited capacity to proliferate before becoming postmitotic. This finite capacity of mature cells to proliferate may explain the events of catagen, when the matrix cells exhaust their proliferative potential and undergo terminal differentiation. We believe that another critical interaction between epithelial cells and the dermal papilla occurs during midanagen. During most phases of the hair cycle the dermal papilla cells appear to be relatively quiescent.43However, during midanagen (stage IV) they undergo a burst of cell proliferation, and new blood vessels form.” Since proliferation of matrix epithelial cells precedes that of dermal papilla cells, it is likely that at this stage of the hair cycle that matrix cells are capable of stimulating the growth of papillary mesenchymal cells.45The degree to which the dermal papilla is stimulated and subsequently enlarges, as measured by the volume of the dermal papilla, has been shown by Van Scott et al. in 1963 to be directly proportional to the diameter and length of the resulting hair.41 The third important dermal papilla-epithelial interaction occurs during early catagen, when the papilla condenses and is connected to the regressing matrix via a “connective tissue sheath.” As catagen progresses, the dermal papilla is pulled upward and eventually becomes positioned in the vicinity of the bulge. This sequence is crucial, as failure of the ascendance of the dermal papilla is known to be accompanied by the failure of the hair follicle to enter into the next cycle, presumably due to the inability of the bulge cells to become activated.&
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a.
ANAGEN
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CATAGEN
c.
TELOGEN
/Y L (< 4ctivation
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ANAGEN II
FIGURE 3. Hair cycle: the bulge activation hypothesis. Different phases of the hair cycle are shown, including (a) anagen V1,(b)catagen, (c) telogen, (a) anagen 11, and (e) anagen IV. h e c t o r pili muscle (AMP), bulge (B), cortex (C), dermal papilla (DP), epidermis (E), inner root sheath (IRS), matrix (M), medulla (Md), outer root sheath (ORS),and sebaceous gland (S) are key structures of the pilosebaceous unit. The quiescent (B) and activated (B*)states of the bulge cells are as indicated. The structures above the dashed line represent the permanent portion of the follicle; keratinocytes below the bulge degenerate during catagen and thus can be considered as “dispensable.” The four major elements of this hypothesis are: activation of the bulge by dermal papilla during telogen (c), activation of the dermal papilla by the matrix keratinocytes during anagen IV (e), finite proliferative capabilities of matrix cells as transient-amplifying cells during late anagen (a), and upward migration of the dermal papilla during catagen @). (From Cotsarelis et aL3’ Reprinted by permission of Cell.)
SKIN CARCINOGENESIS In addition to yielding new insights on the regulation of the hair cycle, the localization of putative follicular stem cells to the bulge region of the hair follicle has significant implications in understanding skin carcinogenesis. Previous studies on chemical carcinogenesis in mice have indicated that cancer development is greatly
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influenced by the stage of the hair c y ~ l e . Many ~ ~ - more ~ ~ skin cancers occur when a complete carcinogen is applied during telogen as opposed to anagen; thus it has become almost standard practice to use only telogen-phase animals in murine carcinogenesis experiments. The reason for an increased tumor rate with topical application of carcinogen during telogen has been ascribed to increased retention of carcinogen by the pilosebaceous unit. This has been explained in part as due to the fact that in telogen the inner root sheath cells, which normally seal the hair canal, are absent, thus giving greater accessibility of the carcinogen to the hair follicle.5° If, as discussed above, follicular stem cells are located in the bulge area, this implies that follicular stem cells are exposed to a much higher concentration of carcinogen for a longer period in telogen than in anagen. Thus, the correlation between tumor yield and the enhanced entry of carcinogen to the bulge raises the possibility that follicular stem cells, rather than the interfollicular epidermis, are involved in and largely responsible for chemically induced skin tumor formation in the mouse model. This hypothesis raises many intriguing questions regarding the relative contribution of the follicle and interfollicular epidermis to the formation of various human skin carcinomas.
PLURIPOTENT STEM CELLS The specific location of the follicular stem cells raises the intriguing question of whether these cells might be pluripotent, giving rise to not only hair follicles, but also to sebaceous glands and, in certain instances, the epidermis. Although cell proliferation occurs in the basal cell layer of the sebaceous gland,” these rapidly proliferating cells are most likely transient amplifying cells. Interestingly, we were unable to detect any LRCs in the sebaceous glands, suggesting that this gland’s stem cells reside elsewhere. The fact that the bulge area is immediately adjacent to the opening of the sebaceous gland raises the possibility that bulge cells also give rise to the sebaceous gland. With regards to the epidermis, it is well established that, following the loss of a large area of the epidermis by mechanical or thermal means, reepithelialization occurs by cells emerging from hair follicles.5*Since these folliclederived cells would later be responsible for the long-term maintenance of the epidermis, they would be stem cells and as such are most likely derived from the bulge.
CONCLUSIONS In this paper we have reviewed the evidence that has led us to suggest that the tips of the deep epidermal rete ridges, the limbal epithelium, and the bulge region of the hair follicle are sites of epidermal, corneal epithelial, and hair follicle stem cells, respectively. Comparisons of stem cells from these three epithelia with stem cells from other epithelia (e.g., dorsal tongue, intestinal epithelium) reveal a commonality of features with respect to location and biological properties.5 Briefly, they are usually located close to the vasculature, in well-protected and innervated areas. If they are in a sun-exposed area, they tend to be highly pigmented. They are usually slow cycling but can be recruited into the proliferative pool in response to wounding or certain growth stimuli. Finally, they are morphologically and biochemically “primitive.” The finding that hair follicle stem cells reside in the upper region of the permanent portion of the hair follicle, the bulge, has allowed us to develop a “bulge
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activation” hypothesis. This hypothesis redirects our thoughts concerning the various aspects of the hair cycle. Whereas most previous investigators have concentrated their efforts at understanding the events occurring during the active growing phases (anagen), we believe that major emphasis should be focused in the future on understanding the resting phase (telogen), when specific interactions between dermal papilla and the bulge cells result in the regrowth of hair. Finally, since many epidermal and follicular tumors are similar in their morphology and biological behavior, it seems reasonable that the bulge may play a central role in their development. The relatively close proximity of the bulge to the epidermis, as well as the recruitment of bulge cells to replenish the epidermis after major crises suggest a pluripotent role for bulge stem cells. REFERENCES 1. LAJTHA, L. G. 1979. Stem cell concepts. Differentiation 14: 23-34. 2. LEBLOND, C. P. 1981. The life history of cells in renewing systems. Am. 1. Anat. 160: 114-157. 3. POTTEN, C. S., R. SCHOFIELD & L. G. LAJTHA. 1979. A comparison of cell replacement in bone marrow, testis and three regions of surface epithelium. Biochem. Biophys. Acta 560: 281-299. EDS.1976. Stem Cells of Renewing Cell 4. CAIRNIE, A. B., P. K. LALA& D. G. OSMOND, Populations. Academic Press, New York, NY. 5. COTSARELIS, G., S-Z. CHENG, G. DONG,T-T. SUN& R. M. LAVKER. 1989. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells. Cell 57: 201-209. 6. QUESENBERRY, P. & L. LEVITT.1979. Hematopoietic stem cells. N. Engl. J. Med. 301: 755-760. 7. V A N BEKKUM, D. W., D. J. VAN DER ENGH,G. WAGENMAKER, S. S. L. BOL& J. W. M. VISSER.1979. Structural identity of the pluripotential hemopoietic stem cell. Blood Cells 5: 143-159. 8. ISCOVE, N. N., J. E. TILL,& E. A. MCCULLOCH. 1970. The proliferative states of mouse granulopoietic progenitor cells. Proc. SOC.Exp. Biol. Med. 134 33-36. 9. LAVKER, R. M. & T-T. SUN.1982. Heterogeneity in epidermal basal keratinocytes: Morphological and functional correlations. Science 215: 1239-1241. 10. LAVKER, R.M. & T-T. SUN.1983. Epidermal stem cells. J. Invest. Dermatol. 81 (Suppl): 121-127. 11. WRIGHT,N. & M. ALLISON, EDS. 1984. The Biology of Epithelial Cell Populations. Clarendon Press. Oxford. 12. HALL,P. A. & F. M. WATT. 1989. Stem cells: The generation and maintenance of cellular diversity. Development 106: 619-633. 13. HUME,W. J. 1983. Stem cells in oral epithelia. In Stem Cells: Their Identification and Characterization. C. S. Potten, Ed. 234-270. Churchill and Livingstone. Edinburgh. 14. BICKENBACH, J. R. & B. D. S. MACKENZIE. 1984. Identification and localization of label-retaining cells in hamster epithelia. J. Invest. Dermatol. 82: 618-622. J. R. 1981. Identification and behavior of label-retainingcells in oral mumsa 15. BICKENBACH, and skin. J. Dent. Res. 60:1611-1620. & T. J. SLAGA.1985. Evidence that the centrally and 16. MORRIS, R. J., S. M. FISCHER peripherally located cells in the murine epidermal proliferative unit are two distinct cell populations. J. Invest. Dermatol. 84: 277-281. 1971. Role of the pericorneal papillary structure in renewal 17. DAVENGER, M. & A. EVENSEN. of corneal epithelium. Nature 229 560-561. 18. BRON, A. J. 1973. Vortex patterns of the corneal epithelium. Trans. Ophthalmol. Soc.U.K. 93: 455-472. 19. GOLDBERG, M. F. & A. J. BRON.1982. Limbal palisades of Vogt. Trans. Am. Ophthalmol. SOC. 80: 155-171.
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20. SCHERMER, A., S . GALviN& T-T. SUN.1986. Differentiation-relatedexpression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J. Cell Biol. 103: 49-62. 1959. Influence of the dermal papilla on survival of 21. CROUNSE, R. G., & J. M. STENGLE. isolated human scalp hair roots in an heterologus host. J. Invest. Dermatol. 32: 477-479. 22. OLIVER, R. F. 1967. Ectopic regeneration of whisker in the hooded rat from implanted lengths of vibrissa follicle wall. J. Embryol. Exp. Morphol. 17: 27-34. 23. OLIVER, R. F. 1967. The experimental induction of whisker growth in the hooded rat by implantation of dermal papillae. J. Embryol. Exp. Morphol. 18: 43-51. 24. DRY,F. W. 1926. The coat of the mouse (Mus rnuscufus). J. Genet. 16: 287-340. 25. CHASE,H. B., H. RAUCH & V. W. SMITH. 1951. Critical stages of hair development and pigmentation in the mouse. Physiol. Zool. 24: 1-8. 26. CHASE,H. B. 1954. Growth of the hair. Physiol. Rev. 34: 113-126. 27. KLIGMAN, A. M. 1959. The human hair cycle. J. Invest. Dermatol. 33: 307-316. 28. STRAILE, W. C., H. B. CHASE& C. ARSENAULT. 1961. Growth and differentiation of hair follicles between periods of activity and quiescence. J. Exp. h l . 148: 205-216. 1963. Determinants of rate and kinetics 29. VANSCOTT,E. J., T. M. EKEL& R. AUERBACH. of cell division in scalp hair. J. Invest. Dermatol. 41: 269-273. 30. PINKUS, H. 1978. Embryology of hair. In The Biology of Hair Growth. W. Montagna & R. Ellis, Eds.: 1-32. Academic Press. New York, W . 31. CoTsARELis, G.,T-T. SUN & R.M. LAVKER. 1990. Label-retainingcells reside in the bulge area of pilosebaceous unit: Implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61: 1329-1337. 32. MONTAGNA, W. & K. S. CARLISLE. 1981. Considerationson hair research and hair growth. In Hair Research. C. E. Orfanos, W. Montagna & G. Stuttgen, Eds.: 7. Springer-Verlag. Berlin. 33. SCHWEIKERT, H. U. & J. D. WILSON. 1981. Androgen metabolism in isolated human hair roots. In Hair Research. C. E. Orfanos, W. Montagna & G. Stuttgen, Eds.: 210. Springer-Verlag. Berlin. 34. RICE,F. L., A. MANCE & B. L. MUNGER. 1986. A comparative light microscopic analysis of the sensory innervation of the mystacial pad. I. Innervation of vibrissal follicle-sinus complexes. J. Comp. Neurol. 252: 154-174. 35. UNNA,P. G. 1876. Beitrage zur histologie und entwicklungsgeschichteder mensclichen oberhaut und hrer anhangsgebilde. Arch. Microskop. Anat. Entwicklungsmech. 12: 665-741. 36. STOHR,P. 1903-1904. Entwicklungsgechichtedes menschlichen wolhaares. Anat. Hefte. Abt. 123: 1-66. 37. MADSEN, A. 1964. Studies on the “bulge” (Wulst) in superficial basal cell epitheliomas. Arch. Dermatol. 8 9 698-708. 38. MONTAGNA, W. 1962. The Structure and Function of Skin. Academic Press. New York,
NY. 39. MONTAGNA, W. & H. B. Chase. 1956. Histology and cytochemistry of human skin: X-irradiation of the scalp. Am. J. Anat. 99: 415-445. & C. MCKISTRY. 1979. Histologic study of the regeneration of 40. INABA,M., J. ANTHONY axillary hair after removal with subcutaneous tissue shaver. J. Invest. Dermatol. 72: 224-23 1. 41. IBRAHIM, L. & E. A. WRIGHT. 1982. A quantitative study of hair growth using mouse and rat vibrissal follicles. J. Embryol. Exp. Morphol. 72: 209-224. & R. M. LAVKER. 1991. Hair follicular stem cells: The bulge42. SUN,T-T., G. COTSARELIS activation hypothesis. J. Invest. Dermatol. %: 77-78s. 43. MOFFAT,G. H. 1968. The growth of hair follicles and its relation to the adjacent dermal structures. J. Anat. 102: 527-540. 1975. Modulation of dermal cell activity during 44. PIERARD, G . E. & M. DE LA BRASSINNE. hair growth in the rat. J. Cutaneous Pathol. 2: 35-41. A. F. & H. B. CHASE.1977. The incorporation of tritiatied uridine in hair germ 45. SILVER, and dermal papilla during dormancy (telogen) and activation (early anagen). J. Invest. Dermatol. 68: 201-205.
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46. MONTAGNA, W., H. B. CHASE& H. P. MELARAGNO. 1952. Skin of hairless mice. 1. Formation of cysts and the distribution of lipids. J. Invest. Dermatol. 1 9 83-94. 47. ANDREASEN, E. 1953. Significance of mouse hair cycle in experimental carcinogenesis. Acta. Pathol. Microbiol. Scand. 32: 165-169. 48. BORUM, K. 1954. The role of mouse hair cycle in epidermal carcinogenesis. Acta. Pathol. Microbiol. Scand. 34: 542-553. 49. ARGYRIS, T. S. 1980. Tumor promotion by abrasion-induced epidermal hyperplasia in the skin of mice. J. Invest. Dermatol. 75: 360-362. 50. BERENBLUM, I., N. HAREN-GHERE & N. TRAININ. 1959. An experimental analysis of the “hair cycle effect” in mouse skin carcinogenesis. Br. J. Cancer 12: 402-413. G. D. 1974. Cell kinetics of human sebaceous glands. J. Invest. Dermatol. 62: 51. WEINSTEIN, 144-146. 52. ARGYRIS, T. S. 1981. Regulation of epidermal hyperplastic growth. CRC Crit. Rev. Toxicol. 9: 151-200.
DISCUSSION OF THE PAPER E. FUCHS (University of Chicago, Chicago, Ill.): Have you tried to dissect out bulge cells? And, can you tell us a little bit about your experiments, successes, failures, etc? LAVKER: My lab has not actually been actively trying to take that approach. What we have been doing is looking at some of the kinetics that are going on during the early parts of the hair cycle. B. HOGAN (Vanderbilt Medical School, Nashville, Tenn.): Can you tell me approximately how many stem cells there are per unit? And, are they stem cells for the sebaceous gland, also? Are the stem cells separate ones, or are they sort of pluripotent in the sense of also giving rise to sebaceous glands? LAVKER: The answer is I don’t know. I would like to believe that they do give rise to what would be the basal sebocyte. The bulge is a very logical site for sebaceous stem cells only because we have been unable to detect labeled retaining cells or a slow-cycling cell amongst the basal sebocytes. As to the numbers of stem cells, I can tell you that approximately 20-30% of the [corneal] limbal epithelial cells are slow-cycling cells. We haven’t quantified the bulge cells in that respect. The work that has been done with labeled retaining cells in the mouse epidermis-work by McKenzie and Bickenbach-indicates that about 1%of the basal cells are label retaining. We certainly see a much greater number than that in the bulge. When we look for label-retaining cells in the epidermis, we occasionally will see one, but the bulge appears to be a repository of label-retaining cells. K. WARD(C.S.I.R.O., Blacktown, New South Wales, Australia): Do vellus follicles also have a bulge? Because they don’t have an arrector pili muscle, do they? LAVKER: We see a bulge in almost all of the hair follicles that we’ve looked at as well as the vibrissa. We find the label-retaining cells in that region of the vibrissa that is the bulge. So it seems to be consistent in all the hairs that we’ve looked at, whether they are vellus or not. U , LICHTI (National Institutes of Health, Bethesda, Md.): Can you spell out for me, again, why you think that stem cells in the resting follicle and in the growing follicle in the same location should be differently sensitive to carcinogen? Is it the accessibility? Or is it something intrinsically different about the cells under those two conditions?
LAVKER et al.: HAIR FOLLICLE STEM CELLS
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LAVKER: At the present time, I would think it is accessibility of the bulge to the carcinogen. C. JAHODA (University of Dundee, Dundee, Scotland, U.K.):The vibrissa follicle does not shorten, so the papilla doesn’t move out during the cycle. How does this accord with your theory? LAVKER: What I think is happening here is that the vibrissa follicle is in a permanent anagen state. This gets back to the concept of the transient amplifying cell, how many amplification divisions there are, and how much support do the transient amplifying cells need? In the vibrissa we do seem to find the label-retaining cells in the same anatomical position as the bulge. These cells no doubt undergo divisions when called upon, and they send down transient amplifying cells that probably have a hierarchy of divisions. Such cell growth supports the proliferative state. The vibrissa does go into telogen, but it is a very short one. JAHODA: But there is no movement of the papilla. LAVKER: There is no movement, no. We don’t see the slow-cycling cells, nor do we see the primitive-looking cells anywhere else in the vibrissa other than in the bulge. JAHODA: We see them in the base. And that’s why in some systems like the intestine I think there could be two groups of stem cells. Would you rule that out? LAVKER: There could be. At one site there could be noncycling or G-zero cells and at other sites hierarchies of transient amplifying cells, some of which have tremendous potential. UNIDENTIFIED SPEAKER: There has been development of the tumor suppressor genes, such as RB or P53 genes. In colon cancer, for example, the P53 genes become lost. Could it be that the stem cells might have a higher expression of these tumor suppressor genes? LAVKER: It is something that we have not looked into. J. BRIND (Orentech Foundation, Cold Spring, MY.): You had mentioned that the bulge is not as prominent in the scalp follicle as it is in some other areas. In what areas is this a more prominent feature? LAVKER: In areas across the bridge of the nose, around the eybrows, and on the forearm, we see a much more prominent bulge than in the scalp.
