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Cutaneous Epithelial Stem Cells
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Cutaneous Epithelial Stem Cells Denise Gay1, Maksim V. Plikus2, Elsa Treffeisen1, Anne Wang1 and George Cotsarelis1 1
Department of Dermatology, Kligman Laboratories, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania 2 Department of Developmental and Cell Biology, Sue and Bill Gross Stem Cell Research Center, University of California, Irvine, Irvine, California
INTRODUCTION The epidermis consists of multiple cell types and layers. The outermost layer, called the stratum corneum is composed of dead ’corneocytes’ that adhere tightly to each other to form a hydrophobic barrier that protects us from the environment and prevents water loss. The innermost layer at the base of the epidermis generates new cells that migrate to the surface while terminally differentiating and eventually forming the stratum corneum. In addition to keratinocytes, which produce intermediate filament proteins called keratins and constitute the majority of the cells, the epidermis also houses melanocytes, fabricators of pigment, and Langerhans cells, sentinels against invaders, whose primary job is that to present foreign antigen to roaming T cells. The epidermal surface is interrupted by orifices arising from adnexal structures, such as the hair follicles and sweat glands. The epidermal basal layer constitutes the outer cell layer of these structures and it has been shown that such basal cells from hair follicles and sweat ducts can move out and repopulate the epidermis after wounding. The adnexal structures possess a greater degree of tissue complexity compared to the epidermis. For example, in contrast to the stratified squamous epithelium of the epidermis, the hair follicle consists of at least eight different concentric layers of epithelia, which undergo degeneration and regeneration with each hair follicle cycle. Because the epidermis and hair follicles continuously generate new cells to replenish dead corneocytes and hairs, which are sloughed into the environment, their homeostasis and repair were thought to depend on epithelial stem cells. Work by many investigators has proved the existence of such cells, and indeed provided a leading example for discovery of stem cells in other regenerative tissues. Epithelial stem cells, found in interfollicular epidermis and the hair follicle, fit a broader definition of adult stem cells, as they are quiescent in nature, with the unique capacity for self-renewal as well as differentiation. In recent years, the concept of interfollicular epithelial stem cells to replenish skin has come into conflict with a new theory. Hair follicle stem cells, once thought to be a single population arising from the bulge region, are now competing with several newly described follicular populations. In this chapter, we will review old and new evidence to integrate historical dogma with newly emerging concepts in the evolving tale of skin regeneration. Principles of Tissue Engineering. http://dx.doi.org/10.1016/B978-0-12-398358-9.00075-6 Copyright Ó 2014 Elsevier Inc. All rights reserved.
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INTERFOLLICULAR STEM CELLS Physiological renewal of epidermis is supported by proliferation of cells in the basal layer. Since epidermal renewal continues throughout a person’s lifetime, it has been postulated that at least a portion of epidermal basal cells behave like stem cells (Fig. 75.1a for location of putative stem cells in interfollicular and follicular epidermis). Bickenbach and Mackenzie, in pioneering work, devised ’label-retaining cell’ methods for detecting quiescent cells in the epidermis [1]. Further, Morris and Potten showed that these cells retained carcinogen and possessed the quiescent characteristic of stem cells (reviewed in [2]). To test the concept that these cells give rise to all skin layers, a replication-deficient retroviral vector carrying the beta galactosidase gene was transduced with low frequency into the skin basal layer in vivo [2a]. Over a period of one month, discreet blue columns of cells could be visualized arising from the basal layer and progressing to the skin surface, thus supporting the existence of clonal epidermal proliferative units (EPUs), at least in the mouse epidermis. To further support this hypothesis, bromodeoxyuridine (BrDU) pulse labeling of the basal layer revealed the presence of a small number of quiescent (label-retaining) cells. Taken together, these data suggested that quiescent stem cells in the basal layer serve to replenish the upper layers during homeostasis and following wounding.
Two models for skin renewal (EPU vs. Committed Progenitor (CP) model) Historically, the favored model has been that basal layer stem cells provide ‘transit amplifying’ progeny which undergo a limited number of divisions to generate the upper strata of the
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FIGURE 75.1 (a) Location of putative epidermal stem cells in the hair follicle and interfollicular epidermis. (b) Two models for interfollicular epidermal homeostasis. EPU model: In this model, rare stem cells (light blue) in the basal layer of the skin give rise to new progeny identical to itself (adjacent dark blue cells) or more differentiated progeny (pink and purple cells in upper layers). Each unit with a single central stem cell is termed an Epithelial Proliferative Unit (EPU). CP model: In this model, all cells in the basal layer have the same potential to make new identical progeny or more differentiated progeny. Therefore all cells in the basal layer are the same color. (c) Two models for hair follicle regeneration during cycling. Historic model (left panel): ‘secondary germ cells,’ (named for their similarity to primary germ cells present during development) were thought to contain the stem cells for the follicle. It was thought that these cells migrate from the base of the telogen follicle to the bulb during anagen onset, and then migrate back up during catagen. Current model (right panel): The secondary germ cells found at the base of the telogen follicle arise from the lowermost portion of the bulge at the end of catagen [15]. These cells migrate down into the secondary germ from their niche region where they alter their gene expression profile, proliferate and ultimately provide all the cells for the new lower half of the anagen follicle [49].
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epidermis [2b,2c]. According to this model, a slower cycling central cell and more rapidly proliferating surrounding cells constitute approximately 10 basal cells and are roughly organized into a hexagonal unit, which lies beneath a single keratinocyte (reviewed in [3], Fig. 75.1b). Based on these proliferative and morphological characteristics, the term ’epidermal proliferative unit’ (EPU) was coined to describe this architecture [2,3]. Without the benefit of direct lineage analysis, it was assumed that the central cell within the EPU generates the rapidly proliferating cells, termed transient or transit amplifying (TA) cells, which move laterally and then differentiate and move upward. Thus, within the epidermis, the main source of cells, i.e., the stem cells, responsible for continual epidermal renewal appear to reside in the center of the EPU. The size of any EPU would be dependent on the total number of mitotic cycles that transiently amplifying progenitors derived from a single stem cell are able to undergo prior to terminal differentiation. The entire epidermal sheet would thus be maintained by a collection of co-existing stable EPUs with one stem cell at the center of each. In recent years, the EPU-based model of epidermal regeneration has been challenged. Using a low frequency cre-inducible genetic model, individual proliferating basal cells were marked and followed for one year in a long-term fate mapping study [4,5]. In contradiction to the canonical EPU model, which predicts the size of each EPU to be finite, it was shown that some epidermal clones continuously expand in size over a period of one year, while others shrink and disappear, and yet others behave like typical EPUs. Mathematical modeling of these clone patterns suggested a stochastic model for epidermal renewal, in which each proliferating basal cell can give rise to two new proliferating basal cells, two differentiated progeny or both [4]. According to this CP model, epidermis is maintained by a uniform population of basal progenitors via stochastically distributed symmetric divisions to maintain the basal layer and asymmetric divisions to generate more differentiated progeny [6]. In support of this model, data showed that a single basal epidermal cell could indeed divide both symmetrically to produce two new basal cells and asymmetrically to generate more differentiated progeny [7]. New data challenge the CP model and reconfirm the existence of at least two populations within interfollicular epidermis; a slow cycling ‘stem cell’ and a rapid cycling ‘progenitor’ pool [8]. It has long been known that heterogeneity in marker expression defines multiple populations within epidermis. Indeed, this has provided a compelling argument against the possibility of a uniform basal population as proposed by the CP model. Mascre et al. [8] have capitalized upon this knowledge to follow the fate of two distinct basal populations, as defined by expression of involucrin. Their work conclusively demonstrates that involucrin-expressing cells divide rapidly (once per week) and quickly contribute to all layers of the epidermis. Thus, these cells are comparable to those described by Jones et al. as ‘committed progenitors’ [6]. However, some cells within the involucrin-negative population divide infrequently (4e6 times/year), have a distinct gene expression profile and can also contribute to skin homeostasis. In contrast to ‘progenitors’, these ‘stem cells’ are the primary contributors to re-epithelialization following wound repair (see below). In reconciling the EPU and CP models, it thus appears that two populations maintain skin homeostasis, a rapid cycling progenitor and a slow cycling stem cell. Tissue damage invokes the aid of the stem cell population only for re-epithelialization. A similar phenomenon has been observed in hematopoiesis in which distinct rapid cycling and slow cycling cells contribute to homeostasis of immune cell lineages but only slow cycling cells are called upon for rapid repair as a stress response [9].
HAIR FOLLICLE STEM CELLS Similar to the epidermis, the hair follicle generates a terminally differentiated keratinized end product, the hair shaft, which is eventually shed. Tracing back a hair shaft cell to its origin in
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adult skin is not straightforward. In contrast to epidermis, the follicle undergoes cyclical regeneration, and has a more complicated proliferative profile and architecture with at least eight different epithelial lineages (Fig. 75.2). Hair is formed by rapidly proliferating matrix keratinocytes in the bulb located at the base of the growing (anagen) follicle. The duration of anagen varies drastically between hairs of differing lengths. For example, mouse hair and human eyebrow hair follicles stay in anagen for only 2e4 weeks while human scalp follicles can remain in anagen for many years. Nevertheless, matrix cells eventually stop proliferating, and hair growth ceases at catagen when the lower follicle regresses to reach a stage of rest (telogen). After telogen, the lower hair-producing portion of the follicle regenerates, marking the new anagen phase (Fig. 75.2). Because the lower portion of the follicle cyclically regenerates, hair follicle stem cells were thought to govern this growth. Historically, hair follicle stem cells were assumed to reside exclusively in the ’secondary germ’, which is located at the base of the telogen hair follicle (Fig. 75.1c). It was felt that the secondary germ moved downward to the hair bulb during
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FIGURE 75.2 Hair cycle and anatomy. The hair follicle cycle consists of stages of rest (telogen), hair growth (anagen), follicle regression (catagen) and hair shedding (exogen). The entire lower epithelial structure is formed during anagen, and regresses during catagen. The transient portion of the follicle consists of matrix cells in the bulb that generate seven different cell lineages, three in the hair shaft and four in the inner root sheath (IRS).
CHAPTER 75 Cutaneous Epithelial Stem Cells
anagen and provided new cells for production of the hair. At the end of anagen, the secondary germ was thought to move upward with the dermal papilla during catagen to come to rest at the base of the telogen follicle. This scenario of stem cell movement during follicle cycling was brought into question when a population of long-lived presumptive stem cells was identified using label-retaining cell methods in an area of the follicle surrounding the telogen club hair, and not in the hair bulb [10]. That the presumptive stem cells localized to a previously defined area called the bulge was not appreciated until this description of the human embryonic follicle by Hermann Pinkus, [11] was rediscovered. ‘This bulge, often the most conspicuous detail of the young germ, is as large as the bulb. The function of the bulge is obscure. While it serves as the point of insertion of the arrector muscle later in life, it develops much earlier than the muscle and the latter seems to originate quite independently in the skin near the sebaceous gland, and in many instances streaks by the bulge before approaching the lower follicle below it. Unna (1876) named the bulge area of the adult follicle the hair bed (Haarbett) believing that the club hair became implanted there and derived additional growth from it. Sto¨hr gave it the neutral name ‘Wulst’ (bulge or swelling). Some texts state that this is an area of marked proliferative activity, but no mitotic figures were observed in the bulge even if other parts of the follicle contained them. Whatever its function, the bulge marks the lower end of the ‘permanent follicle’ later in life. Everything below it is expendable during the hair change [cycle].’ This remarkable description, based purely on morphological observations, portended the characterization of the bulge cells in both human and mouse follicles as an area containing quiescent cells important for hair follicle cycling [10,12,13]. In the mouse pelage follicle, the area analogous to the human bulge becomes morphologically apparent in the postnatal period during the first telogen stage at the site of arrector pili muscle attachment. The shape of the bulge in the mouse follicle results from displacement of the outer root sheath (ORS) by the club hair. In the human follicle, the bulge appears as a true thickening of the ORS, but generally becomes much less apparent with age (Fig. 75.3).
(a)
(b)
(c)
FIGURE 75.3 The bulge is a prominent structure in fetal skin (a), but generally is not morphologically distinct in the adult (b). Immunostaining for keratin 15 in scalp preferentially detects bulge cells (c). apm, arrector pili muscle.
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THE BULGE AS STEM CELL SOURCE Multiple criteria define the bulge as the true stem cell source for hair follicle regrowth. These include quiescence, gene signature and the ability of single cells to regenerate the entire hair follicle in skin reconstitution assays. Quiescence: A salient feature of bulge cells in general is their quiescence. In both adult mouse and human skin grafted onto immunodeficient mice, the administration of nucleoside analogs, such as tritiated thymidine or BrdU, which are taken up by cells in S-phase, does not result in labeling of the bulge cells except at anagen onset [14,15]. Once labeled as either neonates or during anagen onset, when stem cells are proliferating, bulge cells can remain labeled for 14 months in the mouse and at least four months (the longest period examined) in the human [12]. This prolonged quiescence is remarkable given that the surrounding cells proliferate at a much higher rate. These results suggest that bulge cells persist for the lifetime of the organism.
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Molecular signature: Understanding the genes that distinguish bulge cells from proliferating TA cells, as well as the genes that convert resting bulge cells to differentiating cells, provides insights into the precisely orchestrated events of hair follicle formation at anagen onset. With the advent of microarrays, large-scale comparisons of gene expression in bulge cells versus non-bulge basal keratinocytes could be performed [13,49]. These studies found that genes involved in activation of the WNT pathway were generally decreased in the bulge, in line with evidence that WNT activation induces proliferation and cell differentiation and is a hallmark of anagen onset [16]. Microarray analyses of the human hair follicle bulge showed many similarities to the mouse studies, including expression of Keratin15 (K15), thus validating the mouse as a useful model for studying human hair growth [17]. However, one important difference was that CD34, a mouse bulge cell marker, is not expressed by human hair follicle bulge cells. Among other gene candidates important for the stem cell phenotype, functional studies demonstrate that Rac-1 plays an important role in the self-renewal of the epidermis and hair follicle [18,19]. Loss of Rac-1 causes a burst of proliferation in epidermal keratinocytes and then synchronized differentiation and loss of proliferative capability resulting in thinning of the cutaneous epithelium. Thus, this gene suppresses proliferation and differentiation, and seems important for the switch from stem cell to TA cell. Multipotence: If hair follicle stem cells are located in the bulge, then these cells should give rise to all of the epithelial cells in the lower hair follicle. Early evidence supporting the concept that bulge cells generate the lower follicle includes proliferation studies showing that bulge cells preferentially proliferate at anagen onset [15,20]. More convincing evidence suggesting that the lower follicle originates from bulge cells came from in vivo labeling studies and transplantation studies. Taylor used a double-labeling technique to trace the progeny of bulge cells in intact pelage follicles [21]. Faint labeling in a speckled pattern was observed in some cells of the lower follicle suggesting that these cells had indeed originated in the bulge. Similarly, Tumbar et al. used persistence of green fluorescent protein (GFP) label as an indication that lower epithelial cells were progeny of the bulge cells [49]. Neither study provided convincing evidence that all hair matrix keratinocytes in the bulb originated from bulge cells, and both suffered from an inability to permanently mark bulge cells and their progeny. Oshima et al. took a different approach and transplanted bulge regions from vibrissa follicles isolated by dissection from ROSA26 mice into non-ROSA follicles that were then grafted under the kidney capsule of an immunocompromised mouse. ROSA 26 mice express lacZ under the control of the ubiquitous ROSA promoter, thus the fate of the transplanted cells could be followed. After several weeks, they found labeled cells in the lower follicle indicating that bulge cells or their progeny had migrated down the vibrissa follicle. At later time points, some follicles expressed lacZ in all epithelial cell layers of the lower follicle suggesting that bulge
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cells do generate all of the cells of the lower follicle. These elegant studies were limited by several caveats including unclear origin of the marked cell population, the manipulation required for grafting, and the use of vibrissa follicles, which are markedly different than other mouse and human follicles. More definitive evidence for bulge cell multipotency in vivo was reported using the K15 promoter to target these cells with an inducible Cre (CrePR1) construct [13]. CrePR1 is a fusion protein consisting of Cre-recombinase and a truncated progesterone receptor that binds the progesterone antagonist, RU486 [22]. In K15-CrePR1 transgenic mice, CrePR1 remains inactive in the cytoplasm of the K15-positive cells except during RU486 treatment, which permits CrePR1 to enter the nucleus and catalyze recombination. We crossed the K15-CrePR1 mice with Rosa26R (R26R) reporter mice [22] that express LacZ under the control of a ubiquitous promoter after Cre-mediated removal of an inactivating sequence. Transient treatment of adult K15-CrePR1;R26R mice with RU486 results in permanent expression of LacZ in the bulge cells and in all progeny of the labeled bulge cells. Immediately following RU486 induction, only bulge cells showed lacZ expression but after anagen onset, the entire growing lower follicle became lacZþ proving the bulge as the origin of all epithelial cell types in the lower hair follicle.
Multiple bulge populations, some with surprising features Recent data suggest that the bulge contains at least three distinct subpopulations as defined by marker expression. All have been implicated as true hair follicle stem cells (Fig. 75.1a). Keratin 15 (K15) expression in human bulge cells was first described by Lyle et al. [12], (Fig. 75.3). K15 mRNA and protein are reliably expressed at high levels in the bulge, but lower levels of expression can be present in the basal layers of the lower follicle ORS and interfollicular epidermis. A K15 promoter used for generation of transgenic mice possesses a pattern of activity restricted to the bulge in the adult mouse [23]. This proved to be a powerful tool for studying bulge cells, establishing K15þ bulge cells as stem cells responsible for hair follicle growth and cycling (see above). Gli1 expression defines a new population of stem cells in the upper bulge adjacent to perifollicular sensory nerve endings [24]. Remarkably, these nerve endings provide a niche environment by secreting Sonic hedgehog protein (Shh), an essential component for the maintenance of the gli1þ stem cell fate. Gli1þ cells can reestablish the anagen follicle and they appear to contribute to long-term epidermal wound repair which does not appear to be dependent upon Shh signaling (see below [24]). Lgr5, an R-spondin receptor implicated in facilitation of canonical WNT signaling [24a], marks a population of hair follicle progenitors in the lower bulge and secondary hair germ during telogen, and in the lower bulge during anagen [25]. Lgr5 expression is strongest in the secondary germ but overlaps with that of K15. Lineage tracing analyses showed that Lgr5-expressing cells contribute to all lineages during hair follicle cycling. Lgr5þ cells can also reconstitute entire new follicles when injected with dermal cells under the skin of nude mice [25]. The majority of the evidence indicates that Lgr5þ cells derive from a quiescent stem cell pool and thus represent progenitors rather than true stem cells (see below).
OTHER NEWLY DISCOVERED HAIR FOLLICLE STEM CELLS Isthmus: Recently, the junctional zone between infundibulum, sebaceous gland, and bulge, also known as the isthmus, was shown to harbor unexpectedly diverse populations of epithelial cells with stem cell qualities (reviewed in Gordon and Andersen [26]). The top of the isthmus contains stem cells co-expressing transmembrane proteins Lrig1 and Plet1 [27]. These authors place Lrigþ cells in the junctional zone just above the bulge and next to the
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PART 19 Skin sebaceous gland. Lineage tracing experiments have shown that Lrig1þ stem cells tend to be bipotent, physiologically contributing predominantly to infundibulum and sebaceous gland lineages and only occasionally to the interfollicular epidermis. Interestingly, mice lacking Lrig1 develop spontaneous epidermal hyperplasia and exhibit elevated levels of the pro-proliferative cMyc protein. Further analyses indicate that Lrig1 is a downstream target of cMyc and may act to regulate cMyc expression in epidermis in a feedback loop [27]. Lgr6þ stem cells localize immediately below the Lrig1þ compartment [28]. While multipotent during embryonic development, Lgr6þ stem cells undergo progressive developmental fate restriction, and in the adult they participate mainly in epidermal and sebaceous gland maintenance [28]. Interestingly, despite very close physical proximity, Gli1þ stem cells in the upper bulge only marginally overlap with Lgr6þ cells.
Conclusions All of the above described ‘stem cell’ populations have been shown capable of reconstituting hair follicles in cell reconstitution experiments. The lower three populations (Gli1þ, K15þ and Lgr5þ) contribute normally to hair follicle regrowth during cycling. The upper populations (including Gli1þ) generate long-lasting epidermal progeny in the wound epidermis, making their contributions a contrasting feature to that observed by K15þ bulge stem cells [29].
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The obvious question arises: Why are so many different stem cell populations involved in hair follicle cycling? In other well-defined systems, such as hematopoiesis, a single unique stem cell population gives rise to multiple progenitors for establishment of many types of differentiated progeny. Recent work from Petersson et al. [30] and Francis and Niemann [31] suggests this may also be true in the hair follicle. Using lineage tracing to follow K15þ bulge cells, they found that K15þ progeny actually transit through all other putative stem cell compartments, ultimately giving rise to all differentiated hair follicle cell types including sebaceous gland. This implicates the bulge as the true stem cell location and all other regions as harboring more differentiated progenitors (see Horsley review [32]) leaving the K15 high expressing cell as the true stem cell.
STEM CELLS OF OTHER ECTODERMAL APPENDAGES Sebaceous glands Each hair follicle is closely associated with the sebaceous gland and, together, they constitute what is known as the pilosebaceous unit. The predominant cells in the sebaceous gland, sebocytes, secrete lipid-rich products into the infundibular opening of the adjacent hair follicle. Cells expressing the transcriptional repressor Blimp1 comprise unipotent sebocyte progenitors [33]. Horsley et al. [33] found that Blimp1þ progenitors give rise to terminally differentiated Ppargþ sebocytes via transient amplifying progenitors, yet they do not contribute progeny toward interfollicular epidermis or hair follicles. Mechanistically, Blimp1 represses cMyc transcription, likely limiting the input of proliferative progenitors towards the gland from the multipotent stem cell populations of the isthmus [28,31] and the bulge [13,30]. The key rate-limiting role of Blimp1 in sebaceous gland homeostasis was revealed following epithelial Blimp1 deletion, which led to sebaceous gland hypertrophy and oily hair coat phenotype [33].
Sweat glands Acral skin (palms and soles in human and ventral paw in mouse) is hairless, but contains sweat glands, a secretory type of ectodermal appendage. Unlike hair follicles, sweat glands are relatively quiescent and, until recently, very little was known about their regenerative potential. A recent study by Lu C et al. [34] revealed that despite homeostatic quiescence, sweat glands feature several distinct progenitor types, whose regenerative potential can be stimulated by
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injury. Sweat glands have relatively simple organization, consisting of a secretory glandular portion and duct leading to the skin surface. Basal myoepithelial cells and suprabasal luminal cells comprise the glandular portion of the sweat gland. Curiously, sweat-producing luminal cells are maintained by suprabasal unipotent progenitors, largely independent of the basal myoepithelial cells. Neither myoepithelial nor luminal glandular cells contribute progeny toward the duct which, in turn, is maintained by its own basal unipotent progenitors. Unlike glandular cells, ductal progenitor cells become activated upon wounding and help to restore ductal openings onto the skin surface. While ductal progenitors preferentially regenerate the duct itself, they can also regenerate interfollicular epidermis immediately surrounding the sweat gland opening [34]. In this respect, ductal progenitors of the sweat gland share characteristics with the hair follicle isthmus stem cells, which can also generate permanent epidermal progeny following injury [24,27,28]. Rittie recently showed that eccrine ductal cells also contribute to wound healing in human skin [35].
HAIR FOLLICLE STEM CELLS IN SKIN HOMEOSTASIS, WOUND HEALING AND HAIR REGENERATION Homeostasis In addition to the role of bulge cells in hair follicle cycling, their contribution to the maintenance of interfollicular epidermis and sebaceous glands has also been considered. Historically, one view was that bulge cells continuously provide progeny for repopulation of these skin regions [21,36]. Tracking of injected bulge keratinocytes in vivo showed only transient contribution to the interfollicular epidermi [37]. More direct fate mapping studies have shown that K15þ cells do not contribute to homoeostatic maintenance of the epidermis. Using the K15CrePR;R26R transgenic mouse model, bulge cells labeled in three week old mice and followed for 6 months to determine their potential movements out of the hair follicle were never observed in interfollicular epidermis [13]. Furthermore, genetic ablation of K15þ bulge cells does not result in interfollicular epidermal deficiency. Thus, K15þ bulge stem cells can shift their fate towards the epidermal lineage, but only in response to wound-induced signaling (see below [29]). The other bulge populations (Gli1þ and Lgr5þ) also appear to have no role in skin homeostasis [24,25]. Early evidence suggested that follicular cells from the infundibulum (upper follicle) might move into the epidermis. Recent work showed that both Lgr6þ and Lrigþ cells in the hair follicle can migrate into interfollicular epidermis and contribute to all epidermal strata during homeostasis [27,28]. However, their relative importance to skin maintenance compared with that of interfollicular basal cells remains unknown and is likely insignificant.
Wound healing The contribution of the hair follicle to wound healing of the epidermis has been noted for decades by investigators working with mice and rabbits [38]. Clinicians are also well aware that keratinocytes emerge from the follicle to repopulate wounds. However, the role of bulge cells in wound healing has only recently been characterized. Bulge K15þ cell progeny migrate into the epidermis after different types of wounding. Using the K15CrePR;R26R transgenic mouse, K15þ bulge cells were labeled in adult mice [29]. Excisional wounding with a 4 mm (punch) trephine, resulted in the migration of K15þcell progeny into the healing epidermis. At least 25% of the newly formed epidermis originated from these K15þ cells. They were also stimulated to move into the epidermis following incisional wounds and after tape stripping, indicating that K15þ bulge cell activation plays a role in replenishing lost cells from the epidermis after wounding. Surprisingly, however, despite the presence of these cells in the basal layer of the re-epithelialized epidermis, the majority do not persist in the regenerated epidermis (Fig. 75.4).
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FIGURE 75.4 1590
Contribution of epithelial stem cells to wound repair. (a) Various hair follicle and interfollicular stem cells migrate into the new epidermis for apparent contribution to re-epithelialization. (b) Although all three follicular populations are found in new epithelium during early time points, only Gli1þ and Lgr6þ cells exhibit permanent contribution.
Fate mapping studies of Gli1þ and Lgr6þ populations after wounding show that both can make long-term contributions to new epidermis [27,28]. Results suggest however that neither Gli1 nor Lgr6 progeny contributes significantly to re-epithelialization, as labeling studies indicate they make up 16% and 10% respectively of new epidermis. In contrast, interfollicular stem cells contribute substantially to skin regeneration [39]. These combined results challenge the notion that hair follicle stem cells make any significant long-term contribution to re-epithelialization following wounding.
Hair follicle neogenesis Recently we discovered that completely new hair follicles can form in the center of large wounds via a process resembling embryonic development [40]. This hair follicle neogenesis is a relatively late event, contingent upon completion of wound re-epithelialization. Neogenic hair follicles start as bud-like invaginations of the basal layer (aka hair germs or placodes) and soon develop into elongated hair pegs that mature into hair shaft-producing anagen hair follicles within just a few days. Importantly, like normal hair follicles at the wound edge, neogenic hair follicles in the wound center have a prominent K15þ bulge stem cell compartment and are able to undergo multiple hair follicle cycles. Surprisingly, fate mapping approaches have shown that new hair follicles do not arise from existing bulge stem cells which have migrated into the wound during healing [40]. While currently it remains unknown what specific epidermal or isthmus stem cell types, or combination of stem cells, generate neogenic hair follicles, Snippert et al. [28] showed that
CHAPTER 75 Cutaneous Epithelial Stem Cells Lgr6þ isthmus stem cells may be involved. Interestingly, new research suggests that neogenic hair follicles may arise from interfollicular epidermis created during wound re-epithelialization and that induction of these cells to a hair follicle fate depends upon both wound dermal and epidermal WNT activation [28a]. Thus, regenerated hair follicles may have multiple origins.
STEM CELLS AND ALOPECIA Alopecias can be classified into scarring and non-scarring types [41]. The localization of hair follicle stem cells in the bulge may explain why some types of inflammatory alopecias cause permanent follicle loss (such as lichen planopilaris and discoid lupus erythematosis), while others (such as alopecia areata) are reversible [42]. In cicatricial alopecias, inflammation involves the superficial portion of the follicle, including the bulge area, suggesting that the stem cells necessary for follicle regeneration are damaged. The inflammatory injury of alopecia areata, however, especially in early lesions, involves the bulbar region of the hair follicle that is composed of bulge cell progeny. Because this area is immediately responsible for hair shaft production, its destruction leads to hair loss. However, the bulge area remains intact, and a new lower anagen follicle and subsequent hair shaft can be produced. Even patients with alopecia areata for many years can re-grow their hair either spontaneously or in response to immunomodulation. The bulge may be targeted for inflammation in androgenetic alopecia (AGA, common baldness) as well. Jaworsky et al. [43] showed that in patients with early androgenetic alopecia, inflammatory cells localize to the bulge. Over time, this damage could contribute to the irreversible nature of androgenetic alopecia. To examine whether stem or progenitor cells were affected by this disease, we analyzed bald and non-bald regions from AGA patients for the presence of these cells. Facs analyses showed that true non-cycling K15þ stem cells were retained in AGA bald regions. In contrast, larger, proliferative Cd200þ progenitors residing in the lower bulge and secondary germ were markedly depleted. This suggested the lack of stem cell activation in balding scalp [44]. Further studies identified Prostaglandin D2 as an inhibitor of hair growth in bald scalp and a potential inhibitor of hair follicle stem cells [45].
TISSUE ENGINEERING WITH EPIDERMAL STEM CELLS An exciting approach for the use of hair follicle stem cells in the treatment of alopecia includes tissue engineering [46]. In one scenario, isolated hair follicle stem cells could be used for generating new follicles in bald scalp. At least two groups have shown that freshly isolated bulge cells from adult mice, when combined with neonatal dermal cells, form hair follicles after injection into immunodeficient mice [13,39]. These studies provide proof of concept that isolated stem cells may be a part of tissue-engineering approaches for treating alopecia. A goal for treating alopecia with cell-therapy approaches includes increasing the number of follicles, for example, by amplifying the number of stem cells in vitro prior to transplantation. Cultured keratinocytes from neonatal epidermis have been used for many years to generate hair follicles in reconstitution assays. Freshly isolated bulge cells from adult mice were shown to form hair follicles in skin reconstitution assays [13]. Importantly, cultured, individually cloned bulge cells from adult mice also formed hair follicles in skin reconstitution assays [39]. However, the ratio of new follicles formed from the number of donor follicles, and whether non-bulge keratinocytes also possessed these properties, were not analyzed. The use of hair follicle stem cells for tissue-engineering approaches depends on isolation and characterization of human hair follicle stem cells. A major advance in this direction was reported by Ohyama et al. [17] In this work, cell surface markers, including Cd200 were identified on human bulge cells by using laser capture microdissection and microarray analysis for gene expression. A cocktail of antibodies against cell surface proteins was devised allowing for isolation of living hair follicle bulge stem cells, thus setting the stage for isolating human hair follicle stem cells for hair regeneration.
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Human hair regeneration has recently been accomplished [47]. Single cell suspensions of hair bulge stem cells and dermal papilla cells have been layered into ‘organ germs’ in vitro and then transferred onto the backs of nude mice. Such germs create fully functional and pigmented human hair follicles. Analyses of comparable murine bioengineered follicles show that they cycle and maintain appropriate connections with muscle and nerve fibers. These experiments represent the first demonstration of bioengineered human hair and pave the way for exciting new advances in tissue regeneration. Because hair follicle stem and dermal papilla cells exhibit multipotent behavior, ie the capacity to make several different cell types, they are now being exploited for use as pluripotent cells, ie those capable of generating all cell types (Jahoda). Elegant experiments have shown that addition of four defined transcription regulator genes (typically including oct4, nanog, cMyc) can transform fully differentiated fibroblasts into pluripotent cells, however numerous technical difficulties including the length of time required for appropriate dedifferentiation make this technology unfeasible for tissue engineering [48]. In an effort to shorten this time and to reduce the number of introduced genes, investigators have turned to multipotent stem cells for gene transfers, reasoning that these cells are closer in gene expression to pluripotent cells. Recent experiments with dermal papilla cells show that they can indeed undergo dedifferentiation but the timing and the number of transcription factors required remains the same as for fibroblasts [48a]. It remains unknown whether bulge stem cells may prove more useful for this approach.
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Epithelial stem cells make essential contributions to skin and hair maintenance and repair. Balanced contributions by both interfollicular stem cells and more differentiated progenitors provide a constant supply of new cells during normal skin turnover. Interfollicular stem cells are also the primary source for new epidermis in wound healing. The hair follicle cycles throughout the life of the individual. New work suggests that the bulge is the likely source of true stem cells, giving rise to numerous progenitors throughout the follicle, each with distinct roles in hair and skin maintenance and repair. Engineered human hair can now be regenerated in vivo from bulge-derived stem cells, leading the way to future advances for therapeutic treatment of alopecia, chronic wound repair and with important implications for the regeneration of all tissues.
Acknowledgments Some text and figures herein have been reproduced from Plikus MV, Gay DL, Treffeisen E, Wang A, Supapannachart RJ, Cotsarelis G. Epithelial stem cells and implications for wound repair. Semin Cell Dev Biol 2012 Dec;23(9):946e53 and Cotsarelis G.J. Invest Dermatol 2006;126:1459e68.
References [1] Bickenbach J, Mackenzie I. Identification and localisation of label retaining cells in hamster epithelium. J Invest Dermatol 1984;82(6):618e22. [2] Morris RJ. Keratinocyte stem cells: targets for cutaneous carcinogens. J Clin Invest 2000;106(1):3e8. [2a] Mackenzie IC. Retroviral transduction of murine epidermal stem cells demonstrates clonal units of epidermal structure. J Invest Dermatol 1997 Sep;109(3):377e83. [2b] Mackenzie IC. Relationship between mitosis and the ordered structure of the stratum corneum in mouse epidermis. Nature 1970 May 16;226(5246):653e5. [2c] Potten CS. The epidermal proliferative unit: the possible role of the central basal cell. Cell Tissue Kinet 1974 Jan;7(1):77e88. [3] Kaur P. Interfollicular epidermal stem cells: identification, challenges, potential. J Invest Dermatol 2006;126(7):1450e8.
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[4] Clayton E, Doupe DP, Klein AM, Winton DJ, Simons BD, Jones PH. A single type of progenitor cell maintains normal epidermis. Nature 2007;446(7132):185e9. [5] Doupe DP, Klein AM, Simons BD, Jones PH. The ordered architecture of murine ear epidermis is maintained by progenitor cells with random fate. Dev Cell 2010;18(2):317e23. [6] Jones PH, Simons BD, Watt FM. Sic transit gloria: farewell to the epidermal transit amplifying cell? Cell Stem Cell 2007;1(4):371e81. [7] Poulson ND, Lechler T. Asymmetric cell divisions in the epidermis. Int Rev Cell Mol Biol 2012;295:199e232. [8] Mascre G, Dekoninck S, Drogat B, Youssef KK, Brohee´ S, Sotiropoulou PA, et al. Distinct contribution of stem and progenitor cells to epidermal maintenance. Nature 2012;489(7415):257e62. [9] Wilson A, Laurenti E, Oser G, van der Wath RC, Blanco-Bose W, Jaworski M. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 2008;135(6):1118e29. [10] Cotsarelis G, Sun TT, Lavker RM. Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 1990;61(7):1329e37. [11] Pinkus H. Embryology of hair. In: Montagna W, Ellis RA, editors. The Biology of Hair Growth. 1st ed. New York: Academic Press; 1958. [12] Lyle S, Christofidou-Solomidou M, Liu Y, Elder DE, Albelda S, Cotsarelis G. The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells. J Cell Sci 1998;111(Pt 21):3179e88. [13] Morris RJ, Liu Y, Marles L, Yang Z, Trempus C, Li S, et al. Capturing and profiling adult hair follicle stem cells. Nat Biotechnol 2004;22(4):411e7. [14] Ito M, Kizawa K, Toyoda M, Morohashi M. Label-retaining cells in the bulge region are directed to cell death after plucking, followed by healing from the surviving hair germ. J Invest Dermatol 2002;119(6):1310e6. [15] Ito M, Kizawa K, Hamada K, Cotsarelis G. Hair follicle stem cells in the lower bulge form the secondary germ, a biochemically distinct but functionally equivalent progenitor cell population, at the termination of catagen. Differentiation 2004;72(9e10):548e57. [16] Van Mater D, Kolligs FT, Dlugosz AA, Fearon ER. Transient activation of beta-catenin signaling in cutaneous keratinocytes is sufficient to trigger the active growth phase of the hair cycle in mice. Genes Dev 2003;17(10):1219e24. [17] Ohyama M, Terunuma A, Tock CL, Radonovich MF, Pise-Masison CA, Hopping SB, et al. Characterization and isolation of stem cell-enriched human hair follicle bulge cells. J Clin Invest 2006;116(1):249e60. [18] Benitah SA, Frye M, Glogauer M, Watt FM. Stem cell depletion through epidermal deletion of Rac1. Science 2005;309(5736):933e5. [19] Dotto GP, Cotsarelis G. Developmental biology. Rac1 up for epidermal stem cells. Science 2005;309(5736):890e1. [20] Wilson C, Cotsarelis G, Wei ZG, Fryer E, Margolis-Fryer J, Ostead M, et al. Cells within the bulge region of mouse hair follicle transiently proliferate during early anagen: heterogeneity and functional differences of various hair cycles. Differentiation 1994;55(2):127e36. [21] Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM. Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 2000;102(4):451e61. [22] Berton TR, Wang XJ, Zhou Z, Kellendonk C, Schu¨tz G, Tsai S, et al. Characterization of an inducible, epidermal-specific knockout system: differential expression of lacZ in different Cre reporter mouse strains. Genesis 2000;26(2):160e1. [23] Liu Y, Lyle S, Yang Z, Cotsarelis G. Keratin 15 promoter targets putative epithelial stem cells in the hair follicle bulge. J Invest Dermatol 2003;121(5):963e8. [24] Brownell I, Guevara E, Bai CB, Loomis CA, Joyner AL. Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells. Cell Stem Cell 2011;8(5):552e65. [24a] de Lau W, Barker N, Low TY, Koo BK, Li VS, Teunissen H, et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 2011 Jul 4;476(7360):293e7. [25] Jaks V, Barker N, Kasper M, van Es JH, Snippert HJ, Clevers H, et al. Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet 2008;40(11):1291e9. [26] Gordon W, Andersen B. A nervous hedgehog rolls into the hair follicle stem cell scene. Cell Stem Cell 2011;8(5):459e60. [27] Jensen KB, Collins CA, Kasper M, van Es JH, Snippert HJ, Clevers H, et al. Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis. Cell Stem Cell 2009;4(5):427e39. [28] Snippert HJ, Haegebarth A, Kasper M, Jaks V, van Es JH, Barker N, et al. Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science 2010;327(5971):1385e9.
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[28a] Gay D, Kwon O, Zhang Z, Spata M, Plikus MV, Holler PD, et al. Fgf9 from dermal gd T cells induces hair follicle neogenesis after wounding. Nat Med 2013 Jun 2. doi: 10.1038/nm.3181. [Epub ahead of print]. [29] Ito M, Liu Y, Yang Z, Nguyen J, Liang F, Morris RJ, et al. Stem cells in the hair follicle bulge contribute to wound healing but not to homeostasis of the epidermis. Nat Med 2005;11(12):1351e4. [30] Petersson M, Brylka H, Kraus A, John S, Rappl G, Schettina P, et al. TCF/Lef1 activity controls establishment of diverse stem and progenitor cell compartments in mouse epidermis. Embo J 2011;30(15):3004e18. [31] Frances D, Niemann C. Stem cell dynamics in sebaceous gland morphogenesis in mouse skin. Dev Biol 2012;363(1):138e46. [32] Horsley V. Upward bound: follicular stem cell fate decisions. Embo J 2011;30(15):2986e7. [33] Horsley V, O’Carroll D, Tooze R, Ohinata Y, Saitou M, Obukhanych T, et al. Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland. Cell 2006;126(3):597e609. [34] Lu CP, Polak L, Rocha AS, Pasolli HA, Chen SC, Sharma N, et al. Identification of stem cell populations in sweat glands and ducts reveals roles in homeostasis and wound repair. Cell 2012;150(1):136e50. [35] Rittie L, Sachs DL, Orringer JS, Voorhees JJ, Fisher GJ. Eccrine Sweat Glands are Major Contributors to Reepithelialization of Human Wounds. Am J Pathol 2012. [36] Lavker RM, Sun TT. Epidermal stem cells: properties, markers, and location. Proc Natl Acad Sci USA 2000;97(25):13473e5. [37] Claudinot S, Nicolas M, Oshima H, Rochat A, Barrandon Y. Long-term renewal of hair follicles from clonogenic multipotent stem cells. Proc Natl Acad Sci USA 2005;102(41):14677e82. [38] Argyris T. Kinetics of epidermal production during epidermal regeneration following abrasion in mice. Am J Pathol 1976;83(2):329e40. [39] Blanpain C, Lowry WE, Geoghegan A, Polak L, Fuchs E. Self-Renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 2004;118(5):635e48. [40] Ito M, Yang Z, Andl T, Cui C, Kim N, Millar SE, et al. Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding. Nature 2007;447(7142):316e20. [41] Olsen EA, Bergfeld WF, Cotsarelis G, Price VH, Shapiro J, Sinclair R, et al. Summary of North American Hair Research Society (NAHRS)-sponsored Workshop on Cicatricial Alopecia, Duke University Medical Center, February 10 and 11, 2001. J Am Acad Dermatol 2003;48(1):103e10.
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[42] Paus R, Cotsarelis G. The biology of hair follicles. N Engl J Med 1999;341(7):491e7. [43] Jaworsky C, Kligman AM, Murphy GF. Characterization of inflammatory infiltrates in male pattern alopecia: implications for pathogenesis. Br J Dermatol 1992;127(3):239e46. [44] Garza LA, Yang CC, Zhao T, Blatt HB, Lee M, He H. Bald scalp in men with androgenetic alopecia retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle progenitor cells. J Clin Invest 2011;121(2):613e22. [45] Garza LA, Liu Y, Yang Z, Alagesan B, Lawson JA, Norberg SM, et al. Prostaglandin D2 inhibits hair growth and is elevated in bald scalp of men with androgenetic alopecia. Sci Transl Med 2012;4(126):126ra34. [46] Stenn KS, Cotsarelis G. Bioengineering the hair follicle: fringe benefits of stem cell technology. Curr Opin Biotechnol 2005;16(5):493e7. [47] Toyoshima KE, Asakawa K, Ishibashi N, Toki H, Ogawa M, Hasegawa T, et al. Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches. Nat Commun 2012;3:784. [48] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131(5):861e72. [48a] Higgins CA, Itoh M, Inoue K, Richardson GD, Jahoda CA, Christiano AM. Reprogramming of human hair follicle dermal papilla cells into induced pluripotent stem cells. J Invest Dermatol 2012 Jun;132(6):1725e7. [49] Tumbar T, Guasch G, Greco V, Blanpain C, Lowry WE, Rendl M, et al. Defining the epithelial stem cell niche in skin. Science 2004;303(5656):359e63.
75
Cutaneous Epithelial Stem Cells Denise Gay1, Maksim V. Plikus2, Elsa Treffeisen1, Anne Wang1 and George Cotsarelis1 1
Department of Dermatology, Kligman Laboratories, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania 2 Department of Developmental and Cell Biology, Sue and Bill Gross Stem Cell Research Center, University of California, Irvine, Irvine, California
INTRODUCTION The epidermis consists of multiple cell types and layers. The outermost layer, called the stratum corneum is composed of dead ’corneocytes’ that adhere tightly to each other to form a hydrophobic barrier that protects us from the environment and prevents water loss. The innermost layer at the base of the epidermis generates new cells that migrate to the surface while terminally differentiating and eventually forming the stratum corneum. In addition to keratinocytes, which produce intermediate filament proteins called keratins and constitute the majority of the cells, the epidermis also houses melanocytes, fabricators of pigment, and Langerhans cells, sentinels against invaders, whose primary job is that to present foreign antigen to roaming T cells. The epidermal surface is interrupted by orifices arising from adnexal structures, such as the hair follicles and sweat glands. The epidermal basal layer constitutes the outer cell layer of these structures and it has been shown that such basal cells from hair follicles and sweat ducts can move out and repopulate the epidermis after wounding. The adnexal structures possess a greater degree of tissue complexity compared to the epidermis. For example, in contrast to the stratified squamous epithelium of the epidermis, the hair follicle consists of at least eight different concentric layers of epithelia, which undergo degeneration and regeneration with each hair follicle cycle. Because the epidermis and hair follicles continuously generate new cells to replenish dead corneocytes and hairs, which are sloughed into the environment, their homeostasis and repair were thought to depend on epithelial stem cells. Work by many investigators has proved the existence of such cells, and indeed provided a leading example for discovery of stem cells in other regenerative tissues. Epithelial stem cells, found in interfollicular epidermis and the hair follicle, fit a broader definition of adult stem cells, as they are quiescent in nature, with the unique capacity for self-renewal as well as differentiation. In recent years, the concept of interfollicular epithelial stem cells to replenish skin has come into conflict with a new theory. Hair follicle stem cells, once thought to be a single population arising from the bulge region, are now competing with several newly described follicular populations. In this chapter, we will review old and new evidence to integrate historical dogma with newly emerging concepts in the evolving tale of skin regeneration. Principles of Tissue Engineering. http://dx.doi.org/10.1016/B978-0-12-398358-9.00075-6 Copyright Ó 2014 Elsevier Inc. All rights reserved.
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INTERFOLLICULAR STEM CELLS Physiological renewal of epidermis is supported by proliferation of cells in the basal layer. Since epidermal renewal continues throughout a person’s lifetime, it has been postulated that at least a portion of epidermal basal cells behave like stem cells (Fig. 75.1a for location of putative stem cells in interfollicular and follicular epidermis). Bickenbach and Mackenzie, in pioneering work, devised ’label-retaining cell’ methods for detecting quiescent cells in the epidermis [1]. Further, Morris and Potten showed that these cells retained carcinogen and possessed the quiescent characteristic of stem cells (reviewed in [2]). To test the concept that these cells give rise to all skin layers, a replication-deficient retroviral vector carrying the beta galactosidase gene was transduced with low frequency into the skin basal layer in vivo [2a]. Over a period of one month, discreet blue columns of cells could be visualized arising from the basal layer and progressing to the skin surface, thus supporting the existence of clonal epidermal proliferative units (EPUs), at least in the mouse epidermis. To further support this hypothesis, bromodeoxyuridine (BrDU) pulse labeling of the basal layer revealed the presence of a small number of quiescent (label-retaining) cells. Taken together, these data suggested that quiescent stem cells in the basal layer serve to replenish the upper layers during homeostasis and following wounding.
Two models for skin renewal (EPU vs. Committed Progenitor (CP) model) Historically, the favored model has been that basal layer stem cells provide ‘transit amplifying’ progeny which undergo a limited number of divisions to generate the upper strata of the
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FIGURE 75.1 (a) Location of putative epidermal stem cells in the hair follicle and interfollicular epidermis. (b) Two models for interfollicular epidermal homeostasis. EPU model: In this model, rare stem cells (light blue) in the basal layer of the skin give rise to new progeny identical to itself (adjacent dark blue cells) or more differentiated progeny (pink and purple cells in upper layers). Each unit with a single central stem cell is termed an Epithelial Proliferative Unit (EPU). CP model: In this model, all cells in the basal layer have the same potential to make new identical progeny or more differentiated progeny. Therefore all cells in the basal layer are the same color. (c) Two models for hair follicle regeneration during cycling. Historic model (left panel): ‘secondary germ cells,’ (named for their similarity to primary germ cells present during development) were thought to contain the stem cells for the follicle. It was thought that these cells migrate from the base of the telogen follicle to the bulb during anagen onset, and then migrate back up during catagen. Current model (right panel): The secondary germ cells found at the base of the telogen follicle arise from the lowermost portion of the bulge at the end of catagen [15]. These cells migrate down into the secondary germ from their niche region where they alter their gene expression profile, proliferate and ultimately provide all the cells for the new lower half of the anagen follicle [49].
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epidermis [2b,2c]. According to this model, a slower cycling central cell and more rapidly proliferating surrounding cells constitute approximately 10 basal cells and are roughly organized into a hexagonal unit, which lies beneath a single keratinocyte (reviewed in [3], Fig. 75.1b). Based on these proliferative and morphological characteristics, the term ’epidermal proliferative unit’ (EPU) was coined to describe this architecture [2,3]. Without the benefit of direct lineage analysis, it was assumed that the central cell within the EPU generates the rapidly proliferating cells, termed transient or transit amplifying (TA) cells, which move laterally and then differentiate and move upward. Thus, within the epidermis, the main source of cells, i.e., the stem cells, responsible for continual epidermal renewal appear to reside in the center of the EPU. The size of any EPU would be dependent on the total number of mitotic cycles that transiently amplifying progenitors derived from a single stem cell are able to undergo prior to terminal differentiation. The entire epidermal sheet would thus be maintained by a collection of co-existing stable EPUs with one stem cell at the center of each. In recent years, the EPU-based model of epidermal regeneration has been challenged. Using a low frequency cre-inducible genetic model, individual proliferating basal cells were marked and followed for one year in a long-term fate mapping study [4,5]. In contradiction to the canonical EPU model, which predicts the size of each EPU to be finite, it was shown that some epidermal clones continuously expand in size over a period of one year, while others shrink and disappear, and yet others behave like typical EPUs. Mathematical modeling of these clone patterns suggested a stochastic model for epidermal renewal, in which each proliferating basal cell can give rise to two new proliferating basal cells, two differentiated progeny or both [4]. According to this CP model, epidermis is maintained by a uniform population of basal progenitors via stochastically distributed symmetric divisions to maintain the basal layer and asymmetric divisions to generate more differentiated progeny [6]. In support of this model, data showed that a single basal epidermal cell could indeed divide both symmetrically to produce two new basal cells and asymmetrically to generate more differentiated progeny [7]. New data challenge the CP model and reconfirm the existence of at least two populations within interfollicular epidermis; a slow cycling ‘stem cell’ and a rapid cycling ‘progenitor’ pool [8]. It has long been known that heterogeneity in marker expression defines multiple populations within epidermis. Indeed, this has provided a compelling argument against the possibility of a uniform basal population as proposed by the CP model. Mascre et al. [8] have capitalized upon this knowledge to follow the fate of two distinct basal populations, as defined by expression of involucrin. Their work conclusively demonstrates that involucrin-expressing cells divide rapidly (once per week) and quickly contribute to all layers of the epidermis. Thus, these cells are comparable to those described by Jones et al. as ‘committed progenitors’ [6]. However, some cells within the involucrin-negative population divide infrequently (4e6 times/year), have a distinct gene expression profile and can also contribute to skin homeostasis. In contrast to ‘progenitors’, these ‘stem cells’ are the primary contributors to re-epithelialization following wound repair (see below). In reconciling the EPU and CP models, it thus appears that two populations maintain skin homeostasis, a rapid cycling progenitor and a slow cycling stem cell. Tissue damage invokes the aid of the stem cell population only for re-epithelialization. A similar phenomenon has been observed in hematopoiesis in which distinct rapid cycling and slow cycling cells contribute to homeostasis of immune cell lineages but only slow cycling cells are called upon for rapid repair as a stress response [9].
HAIR FOLLICLE STEM CELLS Similar to the epidermis, the hair follicle generates a terminally differentiated keratinized end product, the hair shaft, which is eventually shed. Tracing back a hair shaft cell to its origin in
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adult skin is not straightforward. In contrast to epidermis, the follicle undergoes cyclical regeneration, and has a more complicated proliferative profile and architecture with at least eight different epithelial lineages (Fig. 75.2). Hair is formed by rapidly proliferating matrix keratinocytes in the bulb located at the base of the growing (anagen) follicle. The duration of anagen varies drastically between hairs of differing lengths. For example, mouse hair and human eyebrow hair follicles stay in anagen for only 2e4 weeks while human scalp follicles can remain in anagen for many years. Nevertheless, matrix cells eventually stop proliferating, and hair growth ceases at catagen when the lower follicle regresses to reach a stage of rest (telogen). After telogen, the lower hair-producing portion of the follicle regenerates, marking the new anagen phase (Fig. 75.2). Because the lower portion of the follicle cyclically regenerates, hair follicle stem cells were thought to govern this growth. Historically, hair follicle stem cells were assumed to reside exclusively in the ’secondary germ’, which is located at the base of the telogen hair follicle (Fig. 75.1c). It was felt that the secondary germ moved downward to the hair bulb during
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FIGURE 75.2 Hair cycle and anatomy. The hair follicle cycle consists of stages of rest (telogen), hair growth (anagen), follicle regression (catagen) and hair shedding (exogen). The entire lower epithelial structure is formed during anagen, and regresses during catagen. The transient portion of the follicle consists of matrix cells in the bulb that generate seven different cell lineages, three in the hair shaft and four in the inner root sheath (IRS).
CHAPTER 75 Cutaneous Epithelial Stem Cells
anagen and provided new cells for production of the hair. At the end of anagen, the secondary germ was thought to move upward with the dermal papilla during catagen to come to rest at the base of the telogen follicle. This scenario of stem cell movement during follicle cycling was brought into question when a population of long-lived presumptive stem cells was identified using label-retaining cell methods in an area of the follicle surrounding the telogen club hair, and not in the hair bulb [10]. That the presumptive stem cells localized to a previously defined area called the bulge was not appreciated until this description of the human embryonic follicle by Hermann Pinkus, [11] was rediscovered. ‘This bulge, often the most conspicuous detail of the young germ, is as large as the bulb. The function of the bulge is obscure. While it serves as the point of insertion of the arrector muscle later in life, it develops much earlier than the muscle and the latter seems to originate quite independently in the skin near the sebaceous gland, and in many instances streaks by the bulge before approaching the lower follicle below it. Unna (1876) named the bulge area of the adult follicle the hair bed (Haarbett) believing that the club hair became implanted there and derived additional growth from it. Sto¨hr gave it the neutral name ‘Wulst’ (bulge or swelling). Some texts state that this is an area of marked proliferative activity, but no mitotic figures were observed in the bulge even if other parts of the follicle contained them. Whatever its function, the bulge marks the lower end of the ‘permanent follicle’ later in life. Everything below it is expendable during the hair change [cycle].’ This remarkable description, based purely on morphological observations, portended the characterization of the bulge cells in both human and mouse follicles as an area containing quiescent cells important for hair follicle cycling [10,12,13]. In the mouse pelage follicle, the area analogous to the human bulge becomes morphologically apparent in the postnatal period during the first telogen stage at the site of arrector pili muscle attachment. The shape of the bulge in the mouse follicle results from displacement of the outer root sheath (ORS) by the club hair. In the human follicle, the bulge appears as a true thickening of the ORS, but generally becomes much less apparent with age (Fig. 75.3).
(a)
(b)
(c)
FIGURE 75.3 The bulge is a prominent structure in fetal skin (a), but generally is not morphologically distinct in the adult (b). Immunostaining for keratin 15 in scalp preferentially detects bulge cells (c). apm, arrector pili muscle.
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THE BULGE AS STEM CELL SOURCE Multiple criteria define the bulge as the true stem cell source for hair follicle regrowth. These include quiescence, gene signature and the ability of single cells to regenerate the entire hair follicle in skin reconstitution assays. Quiescence: A salient feature of bulge cells in general is their quiescence. In both adult mouse and human skin grafted onto immunodeficient mice, the administration of nucleoside analogs, such as tritiated thymidine or BrdU, which are taken up by cells in S-phase, does not result in labeling of the bulge cells except at anagen onset [14,15]. Once labeled as either neonates or during anagen onset, when stem cells are proliferating, bulge cells can remain labeled for 14 months in the mouse and at least four months (the longest period examined) in the human [12]. This prolonged quiescence is remarkable given that the surrounding cells proliferate at a much higher rate. These results suggest that bulge cells persist for the lifetime of the organism.
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Molecular signature: Understanding the genes that distinguish bulge cells from proliferating TA cells, as well as the genes that convert resting bulge cells to differentiating cells, provides insights into the precisely orchestrated events of hair follicle formation at anagen onset. With the advent of microarrays, large-scale comparisons of gene expression in bulge cells versus non-bulge basal keratinocytes could be performed [13,49]. These studies found that genes involved in activation of the WNT pathway were generally decreased in the bulge, in line with evidence that WNT activation induces proliferation and cell differentiation and is a hallmark of anagen onset [16]. Microarray analyses of the human hair follicle bulge showed many similarities to the mouse studies, including expression of Keratin15 (K15), thus validating the mouse as a useful model for studying human hair growth [17]. However, one important difference was that CD34, a mouse bulge cell marker, is not expressed by human hair follicle bulge cells. Among other gene candidates important for the stem cell phenotype, functional studies demonstrate that Rac-1 plays an important role in the self-renewal of the epidermis and hair follicle [18,19]. Loss of Rac-1 causes a burst of proliferation in epidermal keratinocytes and then synchronized differentiation and loss of proliferative capability resulting in thinning of the cutaneous epithelium. Thus, this gene suppresses proliferation and differentiation, and seems important for the switch from stem cell to TA cell. Multipotence: If hair follicle stem cells are located in the bulge, then these cells should give rise to all of the epithelial cells in the lower hair follicle. Early evidence supporting the concept that bulge cells generate the lower follicle includes proliferation studies showing that bulge cells preferentially proliferate at anagen onset [15,20]. More convincing evidence suggesting that the lower follicle originates from bulge cells came from in vivo labeling studies and transplantation studies. Taylor used a double-labeling technique to trace the progeny of bulge cells in intact pelage follicles [21]. Faint labeling in a speckled pattern was observed in some cells of the lower follicle suggesting that these cells had indeed originated in the bulge. Similarly, Tumbar et al. used persistence of green fluorescent protein (GFP) label as an indication that lower epithelial cells were progeny of the bulge cells [49]. Neither study provided convincing evidence that all hair matrix keratinocytes in the bulb originated from bulge cells, and both suffered from an inability to permanently mark bulge cells and their progeny. Oshima et al. took a different approach and transplanted bulge regions from vibrissa follicles isolated by dissection from ROSA26 mice into non-ROSA follicles that were then grafted under the kidney capsule of an immunocompromised mouse. ROSA 26 mice express lacZ under the control of the ubiquitous ROSA promoter, thus the fate of the transplanted cells could be followed. After several weeks, they found labeled cells in the lower follicle indicating that bulge cells or their progeny had migrated down the vibrissa follicle. At later time points, some follicles expressed lacZ in all epithelial cell layers of the lower follicle suggesting that bulge
CHAPTER 75 Cutaneous Epithelial Stem Cells
cells do generate all of the cells of the lower follicle. These elegant studies were limited by several caveats including unclear origin of the marked cell population, the manipulation required for grafting, and the use of vibrissa follicles, which are markedly different than other mouse and human follicles. More definitive evidence for bulge cell multipotency in vivo was reported using the K15 promoter to target these cells with an inducible Cre (CrePR1) construct [13]. CrePR1 is a fusion protein consisting of Cre-recombinase and a truncated progesterone receptor that binds the progesterone antagonist, RU486 [22]. In K15-CrePR1 transgenic mice, CrePR1 remains inactive in the cytoplasm of the K15-positive cells except during RU486 treatment, which permits CrePR1 to enter the nucleus and catalyze recombination. We crossed the K15-CrePR1 mice with Rosa26R (R26R) reporter mice [22] that express LacZ under the control of a ubiquitous promoter after Cre-mediated removal of an inactivating sequence. Transient treatment of adult K15-CrePR1;R26R mice with RU486 results in permanent expression of LacZ in the bulge cells and in all progeny of the labeled bulge cells. Immediately following RU486 induction, only bulge cells showed lacZ expression but after anagen onset, the entire growing lower follicle became lacZþ proving the bulge as the origin of all epithelial cell types in the lower hair follicle.
Multiple bulge populations, some with surprising features Recent data suggest that the bulge contains at least three distinct subpopulations as defined by marker expression. All have been implicated as true hair follicle stem cells (Fig. 75.1a). Keratin 15 (K15) expression in human bulge cells was first described by Lyle et al. [12], (Fig. 75.3). K15 mRNA and protein are reliably expressed at high levels in the bulge, but lower levels of expression can be present in the basal layers of the lower follicle ORS and interfollicular epidermis. A K15 promoter used for generation of transgenic mice possesses a pattern of activity restricted to the bulge in the adult mouse [23]. This proved to be a powerful tool for studying bulge cells, establishing K15þ bulge cells as stem cells responsible for hair follicle growth and cycling (see above). Gli1 expression defines a new population of stem cells in the upper bulge adjacent to perifollicular sensory nerve endings [24]. Remarkably, these nerve endings provide a niche environment by secreting Sonic hedgehog protein (Shh), an essential component for the maintenance of the gli1þ stem cell fate. Gli1þ cells can reestablish the anagen follicle and they appear to contribute to long-term epidermal wound repair which does not appear to be dependent upon Shh signaling (see below [24]). Lgr5, an R-spondin receptor implicated in facilitation of canonical WNT signaling [24a], marks a population of hair follicle progenitors in the lower bulge and secondary hair germ during telogen, and in the lower bulge during anagen [25]. Lgr5 expression is strongest in the secondary germ but overlaps with that of K15. Lineage tracing analyses showed that Lgr5-expressing cells contribute to all lineages during hair follicle cycling. Lgr5þ cells can also reconstitute entire new follicles when injected with dermal cells under the skin of nude mice [25]. The majority of the evidence indicates that Lgr5þ cells derive from a quiescent stem cell pool and thus represent progenitors rather than true stem cells (see below).
OTHER NEWLY DISCOVERED HAIR FOLLICLE STEM CELLS Isthmus: Recently, the junctional zone between infundibulum, sebaceous gland, and bulge, also known as the isthmus, was shown to harbor unexpectedly diverse populations of epithelial cells with stem cell qualities (reviewed in Gordon and Andersen [26]). The top of the isthmus contains stem cells co-expressing transmembrane proteins Lrig1 and Plet1 [27]. These authors place Lrigþ cells in the junctional zone just above the bulge and next to the
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PART 19 Skin sebaceous gland. Lineage tracing experiments have shown that Lrig1þ stem cells tend to be bipotent, physiologically contributing predominantly to infundibulum and sebaceous gland lineages and only occasionally to the interfollicular epidermis. Interestingly, mice lacking Lrig1 develop spontaneous epidermal hyperplasia and exhibit elevated levels of the pro-proliferative cMyc protein. Further analyses indicate that Lrig1 is a downstream target of cMyc and may act to regulate cMyc expression in epidermis in a feedback loop [27]. Lgr6þ stem cells localize immediately below the Lrig1þ compartment [28]. While multipotent during embryonic development, Lgr6þ stem cells undergo progressive developmental fate restriction, and in the adult they participate mainly in epidermal and sebaceous gland maintenance [28]. Interestingly, despite very close physical proximity, Gli1þ stem cells in the upper bulge only marginally overlap with Lgr6þ cells.
Conclusions All of the above described ‘stem cell’ populations have been shown capable of reconstituting hair follicles in cell reconstitution experiments. The lower three populations (Gli1þ, K15þ and Lgr5þ) contribute normally to hair follicle regrowth during cycling. The upper populations (including Gli1þ) generate long-lasting epidermal progeny in the wound epidermis, making their contributions a contrasting feature to that observed by K15þ bulge stem cells [29].
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The obvious question arises: Why are so many different stem cell populations involved in hair follicle cycling? In other well-defined systems, such as hematopoiesis, a single unique stem cell population gives rise to multiple progenitors for establishment of many types of differentiated progeny. Recent work from Petersson et al. [30] and Francis and Niemann [31] suggests this may also be true in the hair follicle. Using lineage tracing to follow K15þ bulge cells, they found that K15þ progeny actually transit through all other putative stem cell compartments, ultimately giving rise to all differentiated hair follicle cell types including sebaceous gland. This implicates the bulge as the true stem cell location and all other regions as harboring more differentiated progenitors (see Horsley review [32]) leaving the K15 high expressing cell as the true stem cell.
STEM CELLS OF OTHER ECTODERMAL APPENDAGES Sebaceous glands Each hair follicle is closely associated with the sebaceous gland and, together, they constitute what is known as the pilosebaceous unit. The predominant cells in the sebaceous gland, sebocytes, secrete lipid-rich products into the infundibular opening of the adjacent hair follicle. Cells expressing the transcriptional repressor Blimp1 comprise unipotent sebocyte progenitors [33]. Horsley et al. [33] found that Blimp1þ progenitors give rise to terminally differentiated Ppargþ sebocytes via transient amplifying progenitors, yet they do not contribute progeny toward interfollicular epidermis or hair follicles. Mechanistically, Blimp1 represses cMyc transcription, likely limiting the input of proliferative progenitors towards the gland from the multipotent stem cell populations of the isthmus [28,31] and the bulge [13,30]. The key rate-limiting role of Blimp1 in sebaceous gland homeostasis was revealed following epithelial Blimp1 deletion, which led to sebaceous gland hypertrophy and oily hair coat phenotype [33].
Sweat glands Acral skin (palms and soles in human and ventral paw in mouse) is hairless, but contains sweat glands, a secretory type of ectodermal appendage. Unlike hair follicles, sweat glands are relatively quiescent and, until recently, very little was known about their regenerative potential. A recent study by Lu C et al. [34] revealed that despite homeostatic quiescence, sweat glands feature several distinct progenitor types, whose regenerative potential can be stimulated by
CHAPTER 75 Cutaneous Epithelial Stem Cells
injury. Sweat glands have relatively simple organization, consisting of a secretory glandular portion and duct leading to the skin surface. Basal myoepithelial cells and suprabasal luminal cells comprise the glandular portion of the sweat gland. Curiously, sweat-producing luminal cells are maintained by suprabasal unipotent progenitors, largely independent of the basal myoepithelial cells. Neither myoepithelial nor luminal glandular cells contribute progeny toward the duct which, in turn, is maintained by its own basal unipotent progenitors. Unlike glandular cells, ductal progenitor cells become activated upon wounding and help to restore ductal openings onto the skin surface. While ductal progenitors preferentially regenerate the duct itself, they can also regenerate interfollicular epidermis immediately surrounding the sweat gland opening [34]. In this respect, ductal progenitors of the sweat gland share characteristics with the hair follicle isthmus stem cells, which can also generate permanent epidermal progeny following injury [24,27,28]. Rittie recently showed that eccrine ductal cells also contribute to wound healing in human skin [35].
HAIR FOLLICLE STEM CELLS IN SKIN HOMEOSTASIS, WOUND HEALING AND HAIR REGENERATION Homeostasis In addition to the role of bulge cells in hair follicle cycling, their contribution to the maintenance of interfollicular epidermis and sebaceous glands has also been considered. Historically, one view was that bulge cells continuously provide progeny for repopulation of these skin regions [21,36]. Tracking of injected bulge keratinocytes in vivo showed only transient contribution to the interfollicular epidermi [37]. More direct fate mapping studies have shown that K15þ cells do not contribute to homoeostatic maintenance of the epidermis. Using the K15CrePR;R26R transgenic mouse model, bulge cells labeled in three week old mice and followed for 6 months to determine their potential movements out of the hair follicle were never observed in interfollicular epidermis [13]. Furthermore, genetic ablation of K15þ bulge cells does not result in interfollicular epidermal deficiency. Thus, K15þ bulge stem cells can shift their fate towards the epidermal lineage, but only in response to wound-induced signaling (see below [29]). The other bulge populations (Gli1þ and Lgr5þ) also appear to have no role in skin homeostasis [24,25]. Early evidence suggested that follicular cells from the infundibulum (upper follicle) might move into the epidermis. Recent work showed that both Lgr6þ and Lrigþ cells in the hair follicle can migrate into interfollicular epidermis and contribute to all epidermal strata during homeostasis [27,28]. However, their relative importance to skin maintenance compared with that of interfollicular basal cells remains unknown and is likely insignificant.
Wound healing The contribution of the hair follicle to wound healing of the epidermis has been noted for decades by investigators working with mice and rabbits [38]. Clinicians are also well aware that keratinocytes emerge from the follicle to repopulate wounds. However, the role of bulge cells in wound healing has only recently been characterized. Bulge K15þ cell progeny migrate into the epidermis after different types of wounding. Using the K15CrePR;R26R transgenic mouse, K15þ bulge cells were labeled in adult mice [29]. Excisional wounding with a 4 mm (punch) trephine, resulted in the migration of K15þcell progeny into the healing epidermis. At least 25% of the newly formed epidermis originated from these K15þ cells. They were also stimulated to move into the epidermis following incisional wounds and after tape stripping, indicating that K15þ bulge cell activation plays a role in replenishing lost cells from the epidermis after wounding. Surprisingly, however, despite the presence of these cells in the basal layer of the re-epithelialized epidermis, the majority do not persist in the regenerated epidermis (Fig. 75.4).
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FIGURE 75.4 1590
Contribution of epithelial stem cells to wound repair. (a) Various hair follicle and interfollicular stem cells migrate into the new epidermis for apparent contribution to re-epithelialization. (b) Although all three follicular populations are found in new epithelium during early time points, only Gli1þ and Lgr6þ cells exhibit permanent contribution.
Fate mapping studies of Gli1þ and Lgr6þ populations after wounding show that both can make long-term contributions to new epidermis [27,28]. Results suggest however that neither Gli1 nor Lgr6 progeny contributes significantly to re-epithelialization, as labeling studies indicate they make up 16% and 10% respectively of new epidermis. In contrast, interfollicular stem cells contribute substantially to skin regeneration [39]. These combined results challenge the notion that hair follicle stem cells make any significant long-term contribution to re-epithelialization following wounding.
Hair follicle neogenesis Recently we discovered that completely new hair follicles can form in the center of large wounds via a process resembling embryonic development [40]. This hair follicle neogenesis is a relatively late event, contingent upon completion of wound re-epithelialization. Neogenic hair follicles start as bud-like invaginations of the basal layer (aka hair germs or placodes) and soon develop into elongated hair pegs that mature into hair shaft-producing anagen hair follicles within just a few days. Importantly, like normal hair follicles at the wound edge, neogenic hair follicles in the wound center have a prominent K15þ bulge stem cell compartment and are able to undergo multiple hair follicle cycles. Surprisingly, fate mapping approaches have shown that new hair follicles do not arise from existing bulge stem cells which have migrated into the wound during healing [40]. While currently it remains unknown what specific epidermal or isthmus stem cell types, or combination of stem cells, generate neogenic hair follicles, Snippert et al. [28] showed that
CHAPTER 75 Cutaneous Epithelial Stem Cells Lgr6þ isthmus stem cells may be involved. Interestingly, new research suggests that neogenic hair follicles may arise from interfollicular epidermis created during wound re-epithelialization and that induction of these cells to a hair follicle fate depends upon both wound dermal and epidermal WNT activation [28a]. Thus, regenerated hair follicles may have multiple origins.
STEM CELLS AND ALOPECIA Alopecias can be classified into scarring and non-scarring types [41]. The localization of hair follicle stem cells in the bulge may explain why some types of inflammatory alopecias cause permanent follicle loss (such as lichen planopilaris and discoid lupus erythematosis), while others (such as alopecia areata) are reversible [42]. In cicatricial alopecias, inflammation involves the superficial portion of the follicle, including the bulge area, suggesting that the stem cells necessary for follicle regeneration are damaged. The inflammatory injury of alopecia areata, however, especially in early lesions, involves the bulbar region of the hair follicle that is composed of bulge cell progeny. Because this area is immediately responsible for hair shaft production, its destruction leads to hair loss. However, the bulge area remains intact, and a new lower anagen follicle and subsequent hair shaft can be produced. Even patients with alopecia areata for many years can re-grow their hair either spontaneously or in response to immunomodulation. The bulge may be targeted for inflammation in androgenetic alopecia (AGA, common baldness) as well. Jaworsky et al. [43] showed that in patients with early androgenetic alopecia, inflammatory cells localize to the bulge. Over time, this damage could contribute to the irreversible nature of androgenetic alopecia. To examine whether stem or progenitor cells were affected by this disease, we analyzed bald and non-bald regions from AGA patients for the presence of these cells. Facs analyses showed that true non-cycling K15þ stem cells were retained in AGA bald regions. In contrast, larger, proliferative Cd200þ progenitors residing in the lower bulge and secondary germ were markedly depleted. This suggested the lack of stem cell activation in balding scalp [44]. Further studies identified Prostaglandin D2 as an inhibitor of hair growth in bald scalp and a potential inhibitor of hair follicle stem cells [45].
TISSUE ENGINEERING WITH EPIDERMAL STEM CELLS An exciting approach for the use of hair follicle stem cells in the treatment of alopecia includes tissue engineering [46]. In one scenario, isolated hair follicle stem cells could be used for generating new follicles in bald scalp. At least two groups have shown that freshly isolated bulge cells from adult mice, when combined with neonatal dermal cells, form hair follicles after injection into immunodeficient mice [13,39]. These studies provide proof of concept that isolated stem cells may be a part of tissue-engineering approaches for treating alopecia. A goal for treating alopecia with cell-therapy approaches includes increasing the number of follicles, for example, by amplifying the number of stem cells in vitro prior to transplantation. Cultured keratinocytes from neonatal epidermis have been used for many years to generate hair follicles in reconstitution assays. Freshly isolated bulge cells from adult mice were shown to form hair follicles in skin reconstitution assays [13]. Importantly, cultured, individually cloned bulge cells from adult mice also formed hair follicles in skin reconstitution assays [39]. However, the ratio of new follicles formed from the number of donor follicles, and whether non-bulge keratinocytes also possessed these properties, were not analyzed. The use of hair follicle stem cells for tissue-engineering approaches depends on isolation and characterization of human hair follicle stem cells. A major advance in this direction was reported by Ohyama et al. [17] In this work, cell surface markers, including Cd200 were identified on human bulge cells by using laser capture microdissection and microarray analysis for gene expression. A cocktail of antibodies against cell surface proteins was devised allowing for isolation of living hair follicle bulge stem cells, thus setting the stage for isolating human hair follicle stem cells for hair regeneration.
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Human hair regeneration has recently been accomplished [47]. Single cell suspensions of hair bulge stem cells and dermal papilla cells have been layered into ‘organ germs’ in vitro and then transferred onto the backs of nude mice. Such germs create fully functional and pigmented human hair follicles. Analyses of comparable murine bioengineered follicles show that they cycle and maintain appropriate connections with muscle and nerve fibers. These experiments represent the first demonstration of bioengineered human hair and pave the way for exciting new advances in tissue regeneration. Because hair follicle stem and dermal papilla cells exhibit multipotent behavior, ie the capacity to make several different cell types, they are now being exploited for use as pluripotent cells, ie those capable of generating all cell types (Jahoda). Elegant experiments have shown that addition of four defined transcription regulator genes (typically including oct4, nanog, cMyc) can transform fully differentiated fibroblasts into pluripotent cells, however numerous technical difficulties including the length of time required for appropriate dedifferentiation make this technology unfeasible for tissue engineering [48]. In an effort to shorten this time and to reduce the number of introduced genes, investigators have turned to multipotent stem cells for gene transfers, reasoning that these cells are closer in gene expression to pluripotent cells. Recent experiments with dermal papilla cells show that they can indeed undergo dedifferentiation but the timing and the number of transcription factors required remains the same as for fibroblasts [48a]. It remains unknown whether bulge stem cells may prove more useful for this approach.
CONCLUSION
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Epithelial stem cells make essential contributions to skin and hair maintenance and repair. Balanced contributions by both interfollicular stem cells and more differentiated progenitors provide a constant supply of new cells during normal skin turnover. Interfollicular stem cells are also the primary source for new epidermis in wound healing. The hair follicle cycles throughout the life of the individual. New work suggests that the bulge is the likely source of true stem cells, giving rise to numerous progenitors throughout the follicle, each with distinct roles in hair and skin maintenance and repair. Engineered human hair can now be regenerated in vivo from bulge-derived stem cells, leading the way to future advances for therapeutic treatment of alopecia, chronic wound repair and with important implications for the regeneration of all tissues.
Acknowledgments Some text and figures herein have been reproduced from Plikus MV, Gay DL, Treffeisen E, Wang A, Supapannachart RJ, Cotsarelis G. Epithelial stem cells and implications for wound repair. Semin Cell Dev Biol 2012 Dec;23(9):946e53 and Cotsarelis G.J. Invest Dermatol 2006;126:1459e68.
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[42] Paus R, Cotsarelis G. The biology of hair follicles. N Engl J Med 1999;341(7):491e7. [43] Jaworsky C, Kligman AM, Murphy GF. Characterization of inflammatory infiltrates in male pattern alopecia: implications for pathogenesis. Br J Dermatol 1992;127(3):239e46. [44] Garza LA, Yang CC, Zhao T, Blatt HB, Lee M, He H. Bald scalp in men with androgenetic alopecia retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle progenitor cells. J Clin Invest 2011;121(2):613e22. [45] Garza LA, Liu Y, Yang Z, Alagesan B, Lawson JA, Norberg SM, et al. Prostaglandin D2 inhibits hair growth and is elevated in bald scalp of men with androgenetic alopecia. Sci Transl Med 2012;4(126):126ra34. [46] Stenn KS, Cotsarelis G. Bioengineering the hair follicle: fringe benefits of stem cell technology. Curr Opin Biotechnol 2005;16(5):493e7. [47] Toyoshima KE, Asakawa K, Ishibashi N, Toki H, Ogawa M, Hasegawa T, et al. Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches. Nat Commun 2012;3:784. [48] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131(5):861e72. [48a] Higgins CA, Itoh M, Inoue K, Richardson GD, Jahoda CA, Christiano AM. Reprogramming of human hair follicle dermal papilla cells into induced pluripotent stem cells. J Invest Dermatol 2012 Jun;132(6):1725e7. [49] Tumbar T, Guasch G, Greco V, Blanpain C, Lowry WE, Rendl M, et al. Defining the epithelial stem cell niche in skin. Science 2004;303(5656):359e63.