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Stem Cells in Hair Follicles Cytoskeletal Studies E. B. LANE,"C. A. WILSON,bB. R. HUGHES,' AND I. M. LEIGHc T R C Cell Structure Research Group Cancer Research Campaign Laboratories Department of Biochemistry Medical Sciences Institute University of Dundee Dundee DDl 4HN, United Kingdom bDepartment of Dermatology Slade Hospital Oxford, United Kingdom CImperial Cancer Research Fund Skin Tumour Laboratory London Hospital Medical College London E l 2BL, United Kingdom

INTRODUCTION The intermediate filaments are structural components of the cytoskeleton that show highly differentiation-specific expression patterns of the 30 to 40 members of this multigene family. Even among the 20 "soft" keratins expressed in human epithelia, specific antibody markers can be used to identify subpopulations of cells, in an otherwise homogeneous epithelium, that are probably fulfilling quite distinct physiological roles. Immunohistochemical analyses of keratin expression patterns in human hair follicles have revealed a population of cells with a distinct phenotype that may harbor a multipotential progenitor population of keratinocytes. There are several reasons for anticipating such a population of cells. Hair follicles are known to have an exceptional capacity for proliferation, which suggests that a substantial number of stem cells may reside within the hair follicle. However, hair growth follows a cyclical pattern, with a period of about loo0 days' growth in human scalp hair, following which the hair follicle involutes and then regenerates both the epithelial cells of the outer root sheath and the trichocytes that give rise to the hair shaft and inner root sheath. Hair follicles are recognized as being important contributors to the regenerative capacity of the epidermis. The rate of regeneration of an epidermal site is proportional to the numbers of retained hair follicles; experimental outgrowths from hair follicles can regenerate full-thickness epidermis showing normal differentiation.' The growth of the hair itself is particularly sensitive to X-irradiation and cytotoxic drugs, although hair regrowth follows rapidly: studies of irradiated skin have shown that there is a significant proliferative cell population within the hair follicle that can contribute to epidermal regeneration.2~~ Furthermore, there is a strong phenotypic correlation between basal cell carcinomas and cells of the hair follicle outer root sheath, as well as some peculiar kinetics observed in the 197

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experimental development of skin tumors, which implicate hair follicle involvement. The hair follicle is a highly specialized and structurally complicated skin appendage and provides a striking example of differentiation-restricted expression of keratin intermediate filament proteins. This is, therefore, a good place to look for clues to the differences in properties that must exist between the keratins selected for expression in different body sites. As many as six morphologically distinct concentric columns of cells can be distinguished in the deep follicle (FIG.l), each one immunohistochemically distinct, with appropriate antibodies. The innermost of these zones represents the hair shaft itself, rising from the hair matrix of trichocyte cells in the bulb. The outermost layer is the living jacket of outer root sheath cells, which surrounds the bulb as a thin layer, then widens upwards but variably to a bulge, and constricts again at the isthmus. This follicular expansion has been termed .~ the isthmus, the outer root sheath becomes thicker as the the Wulst r e g i ~ nAbove infundibulum, where the sebaceous duct feeds into the hair canal and the follicle then opens onto the surface. Outer root sheath cells appear to be differentiating horizontally, towards the center of the follicle, to give rise to most or all of the outer layers, the Henle layer, Huxley layer (possibly analogous to the granular layer of interfollicular epidermis), and the outer and inner cuticle (possibly analogous to the stratum corneum). The result of this growth pattern is the formation of a hard cornified tube, through which the column of cells produced by the hair matrix is pushed as the hair grows. It is at the point of entry into this narrowing tube that the cytoplasmic bundles of keratin filaments can be seen by electron microscopy to become perpendicularly aligned, parallel to the direction of the hair shaft (FIG. 1). Interestingly, a similar process of “thinning,” to align fibers for maximum strength of the final cable, is an essential step in the manufacture of man-made cables. Using a range of well-defined monospecific antibodies to keratins for immunohistochemical staining of frozen tissue sections, highly reproducible patterns were observed that could distinguish these states of differentiation from each other. These staining patterns are described briefly below.

MATERLALS AND METHODS Fresh samples of hair-bearing skin from eyelid, scrotum, trunk skin, and scalp were biopsied following informed consent, in the course of surgical procedures. Punch biopsies of hair-bearing skin from forearm and thigh were taken as controls for an experimental analysis of wound healing following suction blisters, to be published elsewhere;s 96-h specimens from this suction blister series were examined and were used to obtain FIG. 1. All samples were snap-frozen in liquid nitrogen, cryosectioned, and kept at -70 “C until use. They were then air dried and stained by 1. standard immunoperoxidase procedures6 using the antibodies listed in TABLE ~~

~~~

~

~~

FIGURE 1. (a) Montage of light micrographs of resin sections of a plucked human hair follicle to illustrate the multiple concentric layers of differentiated cells. The Wulst or bulge of the outer root sheath begins at the top of this section; below this, the outer root sheath is thin. Arrows demarcate transitions as the Henle and Huxley layers (lower single arrow) and cuticle (upper single arrow) and the inner root sheath (double arrow) become cornified. Vertical alignment of keratin filament bundles within the early hair matrix cells is apparent as the hair bulb begins to narrow. Hu = Huxley’s layer, He = Henle’s layer, M = hair matrix,

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D = dermal papilla, CI = cuticle, Co = hair cortex. (b) Outer root sheath cells at the level of the Wulst in a resin-embedded follicle; a mitotic figure is visible (arrow) in the basal cell layer. (c) Expansion of this Wulst zone may be a feature of regenerating epidermis: 96 hours after suction blister injury to the epidermis, the outer root sheath below the sebaceous duct frequently shows irregular expansion, where basal cells react with the monoclonal antibody LP2K to keratin 19.

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TABLE 1. Monoclonal Antibodies Used in This Study mAb M20 CAM5.2 NCL-5D3 LE61 RCK106 DA7

Keratin Specificity Type I1 K8 Type I1 K8 Type I1 K8 K18 x K8 Type I K18 Type I K18

Source Reference 45 46 47 48 49 50

LPlK US68

Type I1 K7 Type I1 K7

13 14

LLm1 LLoo2

Type I K14 Type I K14

51 51

LH8 LH6

Basal marker Basal marker

51 51

C46

52

E3

Type 11 K7 Type I K17 Type I K17

LLO25 LM17 LHP2

Type I K16 Type I1 K1 Type I K10

54 54

1C7 6B10

Type I K13 Type I1 K4

11 11

LP2K

Type I K19

6

53

5

RESULTS The immunohistochemical staining patterns obtained were fundamentally the same in hair follicles from all body sites tested, except for the fact that the scalp follicles were much larger, due to attenuation of the deep follicle, with the result that the staining characteristics of deep follicle zones were similarly attenuated.

Simple Epithelium Kerntins

M20,NCL-5D3, and CAM5.2 to Keratin 8; LE61, DA7, and RCK106 to Keratin 18 Reactivity with these monoclonal antibodies to keratins 8 and 18 is not significant in hair follicle epithelia, although it is strong in the lumind secretory cells of the sweat gland body (providing an integral positive control) (see FIG.2). Weak traces of basal cell staining in the deep outer root sheath have sometimes been seen within scalp follicles when using CAMS.2, which is a very strong antibody to keratin 8, although even this was in a minority of follicles.

FIGURE 2. (a) Multiple scalp follicles and associated glands with scalp epidermis showing continuity of basal staining with basal cell marker LH8 between basal epidermis and all cells in outer root sheath at the follicular isthmus and throughout the deep outer root sheath, including the basal cells around the hair bulb. (b) Extensive but interrupted staining in basal cells of deep outer root sheath with LP2K to keratin 19. The sebaceous lobules show no reaction, nor does the sebaceous duct leading into the follicular lumen. The sweat gland acini provide a good positive internal control. (c) Equivalent oblique longitudinal section of deep follicle showing no evidence of keratin 18 (LE61) in the outer root sheath, with adjacent sweat gland acini providing a strong positive internal control. Light counterstain with Mayer’s hematoxylin. 201

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LPlK, L a 6 8 to Keratin 7

Inner root sheath reactivity is seen above the hair bulb region, defining a cylindrical column of cells that terminally differentiate to form the inner root sheath and the cuticle of the hair (FIG. 3). These are the cell layers of Huxley and Henle, and the staining is seen from the lowest appearance of trichohyalin granules in these cells to the point at which they become cornified. The lobular secretory epithelium of most sebaceous glands is also positive, but not the sebaceous ducts. The very thin layer of basal cells surrounding the gland appear to be negative. Cells of the sebaceous gland and of the inner root sheath at the base of the follicle thus express keratin 7 in the absence of keratin 8, which is a rare phenotype in the body. Sweat gland luminal secretory cells are also positive.

Strah3ed Epithelium Keratins LLOO1, LL002 to Keratin 14

Full-thickness epidermis was strongly stained, apart from the negative stratum corneum, where proteolysis of keratins occurs. Within the hair follicle, staining extended down into all cell layers of the outer root sheath, sebaceous duct, and sebaceous gland. In the deep outer root sheath, the staining extended right around the full length of the outer root sheath, including the thin layer of flattened cells around the hair bulb and up over the hair papilla (FIG.4). No inner root sheath cells were stained. In the sweat gland secretory portion the basal cells reacted strongly, but the luminal cells were negative. Basal Cell Marker Antibodies LH8, LH6

Basal cell reactivity of normal interfollicular epidermis with these nonblotting antibodies is continuous with basal cell reactivity of upper outer root sheath (infundibulum) (FIG. 2). The stained zone expands to include the full thickness of the outer root sheath epithelium on passing through the isthmus and throughout the deep outer root sheath, down to and including the basal cells of the hair bulb. LL025 to Keratin 16

Weak staining of suprabasal normal interfollicular epidermis is seen in scalp skin (FIG. 4), but not in body skin. Where the hair shaft leaves the epidermis there is a funnel of positive suprabasal cells. The upper outer root sheath showed strong reactivity in the suprabasal cells, which extended downward to a variable degree below the isthmus; staining remained suprabasal but then decreased progressively, and is no longer seen in the deep outer root sheath and hair bulb (FIG. 4). There is no reactivity with sebaceous glandular secretory or basal epithelium, but strong staining of suprabasal sweat gland ducts is seen. Studies to be published elsewhere show that keratin 16 staining is seen in suprabasal cells in involved lesions from psoriatic patients’ and in regenerating blister epidermis? However, it was not detectable in basal cell carcinomas.8

FIGURE 3. (a) Section showing scalp interfollicular epidermis and mid portion of follicle, including sebaceous gland lobules and sebaceous duct. The reaction of suprabasal cells in the epidermis, sebaceous duct, and upper outer root sheath with LM17 to keratin 1 is accompanied by a definite reaction of glandular sebaceous secretory cells but not basal sebaceous keratinocytes. (b) Longitudinal section through deep hair bulb shows focal reaction of inner root sheath cells with LPlK to keratin 7 but not of the outer root sheath cells. (c) Upper outer root sheath is seen where entry of sebaceous duct interconnects lobules of sebaceous cells. There is no reaction of sebaceous duct, outer root sheath, or attenuated basal layer of sebaceous gland with LPlK (keratin 7), but the glandular secretory cells of the sebaceous lobules are all strongly reactive.

203

FIGURE 4. (a) Longitudinal section of hair follicle from normal scalp showing the extent of keratin 16 expression in the upper and lower outer root sheath: LW25 reacts with all cells at the isthmus and with a decreasing number of suprabasal cell layers down into the deep outer root sheath. (b) Tangential longitudinal section through hair bulb of scalp follicle to show extension of keratin 17 (C46) to the end of the hair bulb, including a narrow cell layer encircling the bulb; staining is, therefore, more extensive in the deep follicle than for keratin 16.An adjacent follicle shows further basal layer staining around the bulb and (c) over the dermal papilla. 204

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C4b to Keratins 7 and 17; E3 to Keratin 17

Weak heterogeneous areas of epidermal basal cell reactivity were seen in some specimens. Intense basal cell staining was seen at the exit point of the sweat gland, together with reaction of sweat gland ducts and acini. The normal suprabasal epidermis and upper outer root sheath showed no reaction down as far as, and including, the sebaceous gland and its duct. From the isthmus down to the hair bulb, fullthickness staining of he outer root sheath (and inner root sheath with C46) was seen (FIG.4). Our other studies have shown that keratin 17 staining is seen in suprabasal cells in involved lesions from psoriatic patients' and in regenerating blister epidermis: and is extensive in basal cell carcinomas.8

i

LL017 to Keratin 1; LHP2 to Keratin 10 Strong uniform staining of the suprabasal cells in interfollicular epidermis was seen with the antibody LL017 to K1 (FIG.3). Rare scattered cells in the basal layer were also positive (see Ref. 9). The suprabasal cells in the upper outer root sheath were strongly reactive with LL017, as far down as the suprabasal sebaceous duct and gland cells. No reaction of suprabasal cells below the sebaceous duct in the deep outer root sheath was seen. A few suprabasal cells were positive in larger sweat gland ducts. An identical pattern of staining was seen with LHP2 (to KlO), except that fewer basal cells were stained within the epidermis. The ratio between basal cells expressing detectable keratin 1 and those expressing detectable keratin 10 was approximately 1O:l. Strong suprabasal epidermal staining was seen as far as the upper outer root sheath and sebaceous duct. Some sebaceous glandular epithelium reacted with LHP2. Staining was not seen below the infundibulum.

IC7 to Keratin 13; bBI0 to Keratin 4 No reaction with either antibody was seen anywhere in hair follicle or epidermis. This was not unexpected, since these keratins are associated with noncornifying mucosal type differentiation.lO,ll LP2K to Keratin 19

Although no reaction was seen in interfollicular epidermis or upper outer root sheath, sebaceous duct, or sebaceous gland, intense but heterogeneous basal cell staining was found in the deep outer root sheath, which was maximal just below the isthmus where the follicle expands in diameter-that is, the Wulst region4 (FIGS.1, 2, and 5). The extent of K19 staining in this location is subject to body site variation and depends on the hair cycle. In telogen the retracted follicle retains basal cell keratin 19 (FLG.5). During anagen the keratin 19 staining is discrete and restricted in small trunk-skin follicles, but in the large follicles of the scalp it extends down towards the hair bulb along a variable proportion of the outer root sheath (see Ref. 12 for very extensive staining) and can be seen to persist irregularly in some scattered suprabasal cells. Strongly positive luminal cells of sweat gland epithelium provided a good positive control (FIG.2). When all these results on hair follicles are compared with our results on other

FIGURE 5. In the hair follicle growth cycle, when the follicle reaches the end of telogen and is going into anagen 1(T/Al), the follicle has retracted up to the level of the Wulst, just below the sebaceous gland (SG). This telogen follicle has a smoother outline than the regenerating follicle (FIG.1); but many of the basal (B) keratinocytes still retain keratin 19 expression, although this varies in intensity, and small numbers of negative basal cells are interspersed.

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proliferative and static epidermal states, it becomes apparent that there are three interesting facts about the keratin distribution within the pilosebaceous duct that deserve emphasis: (i) the discrete staining of Wulst basal cells by keratin 19 in body skin follicles and (less discrete) scalp; (ii) an overlap, in this region, of aspects of upper and lower outer root sheath types of differentiation-notably, the staining indicative of keratin 16 and keratin 17; and (iii) the appearance of keratin 7 in the hair follicles. Although preliminary indications of all of these features has been obtained from previous studies, their significance has not been fully discussed.

DISCUSSION This study illustrates the sensitivity and precision with which the keratin expression of distinct cell layers and subpopulations within the pilosebaceous unit can be examined, using a well-characterized panel of monospecific monoclonal antibodies to keratins. In particular, two subpopulations of cells are highlighted. One population consists of the cells in the deep follicle in Henle and Huxley layers in the inner root sheath, and of sebaceous gland cells, which are all recognized by antibodies to keratin 7. The inner root sheath staining was first observed in fetal and adult hair follicles with LPlK and LP5K,I3 it was later guardedly recorded as positive staining with a third anti-keratin 7 antibody CK7, but negative with a fourth, RCK105.12 Here, we report that the inner root sheath cuticle cells are additionally positive with a fifth K7 antibody, MS68.I4Keratin 7 is also expressed in sebaceous gland cells, and both these locations are unusual because there are apparently no other simple epithelial keratins 8 and 18 present, although keratin 7 is usually expressed, like K8 as a simple epithelial keratin. The expression of keratin 7 unlinked to keratin 8 seems, therefore, to be characteristic of the pilosebaceous tract. The appearance of K7 may be the earliest keratin change that marks the beginning of development of hair germs from epidermis, around the end of the first trimester of fetal de~elopment.'~ Even at this early stage, asymmetry of keratin 7 staining can be seen within the hair peg, marking the incipient sebaceous gland and Wulst developmental buds (E.B.L., unpublished observations). The second highlighted population is the zone of basal cells in the upper portion of the deep outer root sheath, below the insertion of the sebaceous duct, earlier referred to as the Wulst area; these cells are keratin 19 positive, and this area has features of a pluripotential cell compartment. It is well known that the progenitor cells that give rise to trichocytes and the formation of the hair shaft are located in the hair bulb region of the follicle, above and around the dermal papilla.I5 These cells maintain a rapid rate of proliferation, with a cycle time of 18-24 h,26throughout an anagen phase of approximately 3 years. This would imply a lifespan of over 1,000 cell divisions. In spite of this substantial capacity for proliferation, it is unlikely that these cells in the hair bulb are the sole follicle stem cells, since this whole cell population appears to involute with every hair cycle. Within interfollicular epidermis cell turnover is much lower, with cycle times estimated to be from 50 hl7 to 457 h.I8 Cultures of adult human keratinocytes, which probably exclude deep hair follicle cells, since isolation methods usually aim to avoid dermal fibroblast contamination, have an average cycle time of around 22 hi9.*0and a lifespan of 50-60 population doublings. Thus, proliferative capacity of the order of that shown by the trichocyte progenitor cells is clearly not required to maintain the interfollicular keratinocyte population. Wherever the progenitor cells are within the hair follicle, they must give rise to

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a substantial variety of differentiated phenotypes within the pilosebaceous complex. LLOOl and LLoo2 (to K14) stain outer root sheath epithelium only; trichocytespecific antisera will not stain outer root sheath. Although keratins 16 and 17 are both suprabasal keratins in hyperproliferative conditions such as psoriasis and regenerating epidermis, in the hair follicle they are expressed over two distinct but overlapping ranges: keratin 16 is predominantly in the upper follicle, and keratin 17 is more extensive in the deep follicle (and in basal cell carcinomas).* The Wulst zone is apparent very early in embryonic skin development. The presumptive hair follicle first appears as a swelling (hair germ) at the epidermal interface with the dermis, following induction by mesodermal cells. This cluster of cells grows downwards (the hair peg) at an angle from the epidermal surface, and later develops two buds or bulges on its upper aspect. These will give rise to the (upper) sebaceous gland and (lower) to the top end of the deep outer root sheath, lying just below the isthmus. This lower swelling or bulge of the hair follicle was described as a Wulst by StOi~r.~ Based on observations on cell morphology, differentiation, and division, there are several lines of evidence to indicate the existence of multipotent progenitor cells higher up in the outer root sheath of the hair follicle. Such a population is situated in the deep outer root sheath just below the isthmus. This position is the transition zone from which the hair growth cycle is reinitiated. Staining of hair follicles at late telogen with antibodies to keratin 19 confirms that a keratin 19-positive population of cells is persistent even at the point of minimal hair follicle structure (FIG. 5). The evidence can be summarized as follows. (i) Immunohistochemistry with keratin antibodies indicates flexibility of differentiation across the Wulst region. In addition to the overlap between keratin 16 and keratin 17 ranges that occurs in this region, the Wulst cells are distinct from other epidermal keratinocytes in that the basal cells express keratin 19. Although the Wulst is not as morphologically well defined in the fully developed adult follicle as it is in fetal tissues, it retains its identity into adult follicles by this expression of keratin 19.6 Expression of keratin 19 has previously been suggested to indicate a flexible state of differentiation6 and is thought by others to be associated with regions containing stem cells*' or associated with a risk of malignant The structure of this keratin, lacking the carboxy-terminal nonhelical domain,6ja suggests that it may have a function unrelated to its filament-forming proper tie^.^^ It may function as a switch or buffer keratin, which may be important in stabilizing unpaired keratins during alterations in keratin expression, in response to different local requirements.6.u Thus the association of keratin 19 with a restricted subset of hair follicle epithelial cells is of interest, since it may indicate the proximity of a multipotential cell population. (ii) The morphology of this region of the hair follicle can be very variable. It was observed recently that during epidermal regeneration there was a high incidence of irregularity, suggestive of locally increased cell proliferation.s There was also a marked increase in the number of basal keratin 19-positive cells (FIG.1). Related morphological irregularities in this region of the hair follicle have been observed in psoriatic epidermal hyperpr~liferation.~ (iii) The regenerative essence of the cycling follicle probably resides much higher up than the hair bulb. Early experiments demonstrated that regeneration of the (large) vibrissa follicle depended upon approximately the lower third of the folIn the course of the hair growth cycle, the involuting catagen follicle recedes back up to, but not beyond, the Wulst. The keratin 19-expressing cells thus mark the upper limit of retraction of the hair follicle during its growth cycle and the

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transition zone from which the next cycle of downward growth is initiated. With follicle retraction all detectable trichocyte-specific differentiation markers are progressively lost, and they do not appear again until an anagen follicle has developed. From samples taken during this interval we observe that staining for keratin 14 is still present (LLOO1, LL002 are positive), but keratin 7 is absent (LPlK, US68 are negative). Regrowth in anagen begins as a peg of epithelial cells growing down from this zone (FIG.5), and the keratin 19-positive cells are retained throughout the cycle. (iv) Other aspects of hair follicle morphology can also be interpreted as supporting the importance of the retention of the Wulst region throughout the hair cycle. The arrector pili muscle is attached to this site of keratin 19-positive cells. This is consistent with this point being the lowest persistent part of the follicle, since it provides the deepest anchorage point for the muscle, and thus gives the most efficient muscle leverage on the hair shaft. Protection of the muscle attachment point may be reciprocated in that the muscle attachment may also anchor the follicle and protect the Wulst cells from being stripped out with a plucked hair. Hair follicles play an important role in touch perception, and sensory innervation of the hair follicles is also focused on this structural domain of the Wulst.z8 This is reflected in the location of intraepidermal Merkel cells in this region, as well as the complex perifollicular innervation characteristic of eyelash and whisker follicles. Proximity to the arrector pili will give maximum displacement of follicle-associated cells following deflection of the hair shaft, and thus greatest stimulation of touch receptors, consistent with these specializations occurring at the lowest protected point. (v) In comparison with the rest of the outer root sheath, the Wulst is a region where cell proliferation is in evidence; mitotic figures are readily seen in this part of the hair follicle (FIG.l),and cycling cells can be detected by labeling with BrdU and antibody Ki67 (Wilson, Leigh, and Lane, unpublished observations). This proliferation may vary with different stages of the hair ~ y c l e . * ~ T hisi s in addition to the well-documented proliferative and differentiative activity in the hair bulb region.30 (vi) Finally, recent studies in rodent have obtained evidence for the presence of label-retaining cells within the Wulst of the hair follicle,3’ which suggests that the label-retaining cell population and the keratin 19-positive population could be linked; they would appear to be at least spatially overlapping. Retention of autoradiographic label is thought to be a characteristic of stem c e l l ~ , 3since ~ it indicates that mitotic cycling is a rare event with a long periodicity, as would be expected to protect the precious resource of a stem cell population.

LOCATION OF STEM CELLS IN EPIDERMIS Numerous studies have attempted to identify the location of stem cells in epid e r m i ~ . Some ~ ~ - ~aspects ~ of morphological heterogeneity have been interpreted as ~ ~ greatest , ~ ~ range of morphological heterogeneity has indicative of stem ~ e l l s .The been observed in basal cells of glabrous skin, where the basal cells at the bottom of the rete pegs look quite different from the “dark, serrated” basal cells along the dermal ridges. In hairy skin, however, there is very little variation in the morphology of interfollicular basal cells, and basal cell heterogeneity is more easily correlated with relative states of differentiation and impending progression into the suprabasal c ~ m p a r t m e n tthan ~ * ~with ~ proliferative capacity. Even certain antibodies to cell cycle-restricted antigens have given homogeneous staining in epidermal interfol-

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licular basal cells.39This, together with the known capacity of hair follicle-derived keratinocytes to generate a full-thickness, normally differentiating epidermis after partial thickness excision,'.2 raises the question of whether or not true stem cells, rather than slow-cycling amplifying cells, reside in interfollicular epidermis. Predicted locations for stem cells should incorporate structural features to protect the cells, both chemically and physically. The advantages of bone marrow for hematopoietic stem cells are obvious,40as are those of a deep cryptal location below a mucous barrier within the constantly abraded intestinal e p i t h e l i ~ m ~ and * . ~ 2a sheltered location down between the filiform papillae on the tongue.43In the eye, limbal epithelium is well anchored in a way that clear corneal epithelium cannot be, and the stem cells are restricted in location to the l i m b ~ s ; ~Cotsarelis 5 et ~ 1discuss . ~ such ~ stem cell locations in various tissues. The epidermis in particular is a highly abraded organ, the last barrier between the vertebrate organism and its outside world, and a wide range of structural strategies to protect progenitor cells have been adopted during the evolution of vertebrate epidermal tissues. Where the epidermis is completely flat, some fish have evolved giant pillarlike supporting cells filled with skeins of keratin filaments, which almost certainly serve to physically shelter the basal cells.4 In mammalian hairless skin, deep rete pegs help anchor the tissue and provide a harbor for stem cells.35.36 In hairy skin, we feel that accumulated observations point to the Wulst region of the deep outer root sheath as an ideal, and very probable, location of follicle progenitor cells for the hair follicle structure (as opposed to the hair-producing trichocytes, specifically), and possibly even as contributing to the surrounding epidermis. The possibility that this cell population may be locally highlighted by a particular keratin expression phenotype may prove very useful in future analysis of both progenitor cell behavior and the biological functions of the keratin cytoskeleton.

ACKNOWLEDGMENTS The authors acknowledge gratefully the technical assistance of D. Deane of the Slade Hospital, Oxford and the helpful assistance of Dr. R. Cerio with photomicrography. We should like to thank Drs. Wojnarowska and Dawber for their encouragement to perform this study. REFERENCES 1. LENOIR, M.-C., B. A. BERNARD, G . PAUTRAT, M. DARMON & B. SHROOT. 1988. Outer root sheath cells of human hair follicle are able to regenerate a fully differentiated epidermis in vitro. Dev. Biol. 1 3 0 610-620. H. R. 1967. Recovery and repopulation in vivo by the mouse skin epithelial cells 2. WITHERS, during fractionated irradiation. Radiat. Res. 32: 227-239. 3. AL-BARWARI, s.E. & c.s. POTTEN.1976. Regenerationand dose-response characterization of irradiated mouse dorsal epidermal cells. Int. J. Radiat. Biol. 30: 201-216. 4. STOHR, P. 1904. Entwicklungsgeschichte des menschlichen Wollhaares. h a t . Hefte Abt. 1. 23: 1-66. 5. LANE,E. B., J. B. STEEL,P. E. PURKIS, N. TIDMAN, M. KASPER&I. M. LEIGH. 1991. Regeneration of human epidermis following suction blister injury: Identification of changes in keratin expression. Manuscript in preparation. 6. STASIAK, P. C., P. E. PURKIS, I. M. LEIGH & E. B. LANE.1989. Keratin 19: Predicted amino acid sequence and broad tissue distribution suggest it evolved from keratinocyte keratins. J. Invest. Dermatol. 92: 707-716.

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DISCUSSION OF THE PAPER T.-T. SUN(New York University Medical School, New York, N.Y.): I’m curious about the distribution of K19 in other tissues. For example, you mentioned that it is actually very difficult to see K19 in the epidermis. Are the few cells that you do see in the epidermis at the bottom of the rete ridges where people assume the stem cells are, or is there a relationship to the so-called EPU? How about the corneal epithelium? Do you see them selectively in the limbal region? How about the intestine? What is the relationship between K19 expression and the location of positive stem cells? 1. M. LEIGH: It is difficult to convince oneself that there are K19 positive cells in the interfollicular epidermis, except for Merkel cells. If we look at oral mucosa, the keratin 19 positivity is extremely heterogeneous. It does not localize specifically to the rete pegs. It may be along the sides of the rete pegs. We’ve only recently started to look at cornea, but you do find quite widespread basal expression of keratin 19. SUN:Relating to basal cell carcinoma, when we were looking at the BCC keratin pattern, when we analyzed the gel pattern, we thought we saw K6-Kl6. But you mentioned that actually there is no K6 and 16, which is consistent with what Roland Moll found. Is your conclusion based on your antibody staining pattern or 2D gel pattern? LEIGH: It is based mainly on the antibody studies. One feature that I didn’t mention was that there is strong expression of keratin 6 and 16 in the normal epidermis overlying basal cell carcinoma and in the adjacent hair follicles. By biochemical examination, there is K6 and K16 in the vicinity, but not actually in the tumor islands. I was interested to see recently that in correlation with the 6 and 16 expression, it has been found that the normal epidermis over basal cell carcinoma also shows increased labeling indices, which suggests that the carcinoma is producing growth factors that are stimulating the epidermal hyperproliferation, much as was found in the epidermis overlying histiocytomas.