8 - NAIL Flashcards
The nail appendage is composed of several layers that organize the nail organ:
■ The eponychium creates a border between skin epidermis and nail organ at the dorsal limit of the nail proximal fold (NPF), forming a protective seal.
■ The NPF forms after skin epidermis bends inward ventrally at the eponychium’s border and becomes the nail epidermis, creating the NPF, which localizes slow-cycling bifunctional nail proximal fold stem cells (NPFSCs). NPFSCs actively deliver progeny to the perinail epidermis and nail matrix along with differentiated nail plate upon nail regeneration.
■ The matrix, a ventral continuation of the proximal fold after it bends dorsally and distally, is composed of actively proliferating cells called onychocytes. In the proximal nail matrix, fastproliferating nail stem cells are located. Their differentiation is coupled directly with the ability to orchestrate digit regeneration.
■ Nail matrix differentiates, forming the keratogenous zone, which finally deposit cells into the overlying nail plate.
■ Hyponychium is the most distal part of the nail epithelium located peripherally to the nail bed, and beneath the nail plate at the junction between the free edge and the skin epidermis of the fingertip, it forms a seal that protects the nail bed.
creates a border between skin epidermis and nail organ at the dorsal limit of the nail proximal fold (NPF), forming a protective seal
eponychium
forms after skin epidermis bends inward ventrally at the eponychium’s border and becomes the nail epidermis, creating the NPF, which localizes slow-cycling bifunctional nail proximal fold stem cells (NPFSCs). NPFSCs actively deliver progeny to the perinail epidermis and nail matrix along with differentiated nail plate upon nail regeneration
nail proximal fold (NPF)
a ventral continuation of the proximal fold after it bends dorsally and distally, is composed of actively proliferating cells called onychocytes. In the proximal nail matrix, fastproliferating nail stem cells are located. Their differentiation is coupled directly with the ability to orchestrate digit regeneration.
matrix
differentiates, forming the keratogenous zone, which finally deposit cells into the overlying nail plate.
Nail matrix
the most distal part of the nail epithelium located peripherally to the nail bed, and beneath the nail plate at the junction between the free edge and the skin epidermis of the fingertip, it forms a seal that protects the nail bed.
Hyponychium
NAIL ORGAN STRUCTURE ANATOMY AND FUNCTION
Nails belong to one of the skin appendages and are located on the distal phalanx of each finger and toe in the human body (Fig. 8-1). The nail appendage begins as an extension of the distal phalanx skin epidermis at the border called the eponychium (Fig. 8-2). At that point, the skin epidermis bends inward ventrally and becomes the nail epidermis, forming the nail proximal fold (NPF) (see Fig. 8-2). Interestingly, normal epidermal differentiation ceases just after the proximal fold folds inward at the eponychium border; thus, nail layers continuing beyond this point do not form the granular layer typically observed in normal skin epidermis. As a ventral continuation of the proximal fold after it bends dorsally and distally is the nail matrix composed of actively proliferating nail cells called onychocytes (see Figs. 8-2 and 8-3). 1 Above the matrix lies the keratogenous zone (KZ) where matrix cells differentiate, flatten out, die, and deposit into the overlying nail plate (see Figs. 8-2 and 8-3). The nail plate is a hard structure that serves as a protective covering by preventing trauma to the tips of toes and fingers; it is also used as a tool to pick up small objects, which is important for fine manipulations and subtle finger functions. Through the nail plate, the whitish crescent-shaped base is called the lunula (“small moon”), the visible part of the matrix (see Fig. 8-1). The lunula can best be seen in the thumb and may not be visible in the little finger.
Within the nail organ, the nail plate contains a hard keratinized structure composed of flattened and anucleated cells called corneocytes overlying the distal phalange (see Figs. 8-1 and 8-2). Corneocytes are formed during nail differentiation from nail cells, onychocytes. The nail plate exerts counterpressure at the fingertips for protection and enhances sensitivity. Distal to the nail matrix is the nail bed, composed of a basal layer and one or two layers of suprabasal postmitotic keratinocytes, contributing a few horn cells to the undersurface of the distal nail plate (see Fig. 8-2).2 The nail plate is attached to the finger by interdigiting with the underlying nail bed (see Fig. 8-2). The nail cuticle extends from the edge of the proximal fold onto the nail plate, sealing the proximal end of the nail and protecting it from toxins and foreign substances (see Fig. 8-1). Laterally, the nail plate is surrounded by lateral nail grooves and lateral nail folds (see Fig. 8-1). Similarly, at the distal end border of the nail unit, the hyponychium seals the nail plate to the nail bed under the onychodermal band to prevent infection (see Figs. 8-1 and 8-2).
HISTOLOGY OF THE NAIL APPARATUS
Comparing human nails with those of other mammals (eg, mouse nail in this example), it is apparent that the shape is very different, likely owing to differences in evolution and function. The human nail is flatter (see Figs. 8-1 and 8-3); a mammal’s claw is a near conical structure with greater curvature. This shape is influenced by the underlying distal phalanx and the distribution of the matrix.1,3
NAIL GROWTH AND DIFFERENTIATION
So far, it is believed that the matrix is largely responsible for nail plate production. However, previously, it was unclear whether the matrix is the sole source of nail differentiation or if other parts of the nail unit, such as the nail bed, also contribute to the nail plate. In the past, Lewis observed that the nail plate is composed of three different layers and proposed that they arise from three different parts of the nail unit. It was theorized that the superficial layer of the dorsal nail originates from the proximal nail fold, the intermediate nail arises from the matrix, and the deep ventral nail is produced by the nail bed.4 About a decade later, Zaias and Alvarez used radioactive tritiated glycine to mark and follow nail cells in squirrel monkeys, demonstrating that the uptake in the label moves from the matrix into the nail plate over time. 5 Only small amounts of radioactive tritiated glycine were incorporated in the nail bed and were therefore dismissed as a source of nail production because of its inactivity. Taken together, the nail matrix was proposed to be solely responsible for nail plate formation. 5 Similar studies were also conducted on human volunteers using both tritiated thymidine (3 H-thymidine) and glycine in which matrix cells were proposed to migrate into the nail bed in addition to the nail plate. 6 The hypothesis that the nail is produced only by the matrix was further supported by Berker and Angus, who demonstrated that the matrix cells were highly proliferative compared with the relatively inactive nail bed. 7 In other studies, it has been proposed that although the matrix produces the bulk of the nail plate, the nail bed also contributes to the nail plate based on nail thickness and mass. 8,9 It is argued that the contribution of the nail bed to the ventral nail plate allows distal movement of the nail plate as it grows. Thus, in conclusion, there appear to be a general consensus that the nail plate is produced by matrix cells.
TWO POOLS OF STEM CELLS EXIST IN NAIL ORGAN HOMEOSTASIS AND REGENERATION
Whether the matrix cells are the only source of progenitor cells required for nail production or if the other layers including NPF also contribute to portion of the nail during the lifetime, meeting the criteria of being stem cells per se is evaluated in this section.
Adult stem cells found in various organs throughout the body are responsible for maintaining the normal turnover of organs as well as tissue repair in the event of an injury. 10 These are made possible by its ability to self-renew and differentiate into the different specialized cell types in its respective organs.10 Because stem cells are required for tissue homeostasis and regeneration throughout life, they must have long-term self-renewal capacity. Moreover, it has been proposed that stem cells divide infrequently to avoid incorporation of mutations associated with cell division. In each organ, stem cells reside in specialized microenvironments called niches, which helps maintain quiescence or regulate proliferation and differentiation, which are important for tissue homeostasis and repair.10-13
Upon activation, these stem cells divide and leave the niche to form more differentiated transitamplifying (TA) progenitor cells. In contrast to the relatively quiescent stem cells, TA cells rapidly proliferate and differentiate into cells needed for regeneration or repair. 10 Adult stem cells are theoretically present in all regenerative tissues, but their localization and characterization are often difficult because little is known about these organs. Therefore, scientists have used the slow-cycling property to label and identify putative stem cells in various organs. In early pulse-chase studies, animals were injected with 3 H-thymidine, which gets incorporated into the newly synthesized DNA of dividing cells. After efficient labeling, the animal undergoes a period of chase when dividing cells dilute out the 3 H-thymidine label and slow-cycling cells retain this radioactive label. This method allowed for the identification of slow-cycling label-retaining cells (LRCs).
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In 1990, Cotsarelis and coworkers identified a unique population of slow-cycling LRCs in the bulge region of the hair follicle using 3 H-thymidine pulse chase experiments. They demonstrated that these bulge LRCs can be stimulated to proliferate upon treatment with a tumor promoter (12-O-tetradecanoylphorbol13-acetate [TPA]) and went on to hypothesize that these LRCs are stem cells of the hair follicle. 14 Before identifying LRCs in the hair follicle bulge, Cotsarelis and coworkers also reported on LRCs in the cornea limbus using similar pulse chase experiments. 15 These limbal LRCs were also preferentially activated in response to injury and can also be stimulated by TPA.
These data support the theory that stem cells persist throughout the lifetime of an animal to contribute to the tissue during regeneration and wound repair.
Using radioactive labeling, however, researchers were unable to probe for colocalization of potential markers through immunological histology. Soon after, scientists began to use a new synthetic thymidine analog, bromodeoxyuridine (BrdU). 16 In contrast to 3 H-thymidine, BrdU is not radioactive. In addition, antibodies against BrdU allow for immunohistochemistry to probe for markers that colocalize with BrdU-marked LRCs. 17 Pulse-chase studies with BrdU have been widely used to identify LRCs in numerous tissues.
More recently, a new method for identifying LRCs using transgenic mice with tetracycline-induced histone 2B-green fluorescent protein (H2B-GFP) has been described. 18 With this system, a specific or ubiquitous promoter can be used to drive inducible H2B-GFP expression in the tissues of interest. One advantage of this system is that it can more efficiently label the cells before chase, but the BrdU method can only label cells that are actively dividing in the synthesis phase during pulse. Moreover, this H2B-GFP transgenic label-retaining system allows for the isolation of live LRCs for further characterization and analysis. For the first time, this method allowed the isolation of live hair follicle label-retaining stem cells for gene expression profiling to determine the molecular characteristics of these stem cells independent of any other markers such as keratin 15 (K15) or CD34. 18 Over the years, this method allowed understanding of how hair follicles and their stem cells are regulated.
SLOWCYCLING BIFUNCTIONAL NAIL PROXIMAL FOLD STEM CELLS IN THE NAIL ORGAN
Subsequently, in addition to the hair follicle in many skin appendages, such as the cornea and sweat glands, slow-cycling stem cell characteristics were demonstrated14,15,19-21 ; however, little was known about other skin appendages such as nails.
Therefore, recent studies tried to address whether such a quiescent stem cell population also exists in continuously growing nails. First notice of existence of LRCs in the nail after BrdU pulse-chase experiments suggested the presence of LRCs in the basal layer of the nail matrix adjacent to the nail bed in the mouse nail; however, further characterization has not been performed. 22 In contrast to this localization, using immunohistochemical staining, Sellheyer and coworkers showed that the ventral proximal fold in human nails expresses hair follicle stem cell (hfSC) markers (eg, K15; keratin 19 [K19]; and pleckstrin homologylike domain, family A, member 1 protein [PHLDA1])
during embryogenesis and contains very few Ki67+ cells (a nuclear protein associated with cell proliferation), making them more quiescent similar to hfSCs, suggesting that the proximal fold may represent the human NSC niche.23
These discrepancies have been addressed in recent studies using H2B-GFP LRCs system in transgenic mice for the in vivo detection of quiescent, slowcycling skin cells. 24 This method allows identifying a previously unreported population of nail LRCs within the basal layer of the NPF (Fig. 8-4). 24 Interestingly, in mice, the nail exists as a three-dimensional near-conical structure; therefore, LRCs are organized in a ringlike configuration. 24 Nail LRCs express the hfSC marker K15, and in vivo lineage tracing experiments show that K15-labeled cells, originating in the NPF, contribute to both the nail structure (see Fig. 8-4, black arrow #2, long term) and more predominantly to the perinail epidermis (see Fig. 8-4, green arrow #1), thus possessing a bifunctional stem cell characteristic. Upon nail regeneration, these K15-derived NPFSCs actively deliver progeny to the nail matrix and differentiate into the nail plate (see Fig. 8-4, black arrow #2). Similarly, in vivo engraftment experiments demonstrate that the nail LRCs can actively participate in functional nail regeneration. Transcriptional profiling of isolated nail LRCs revealed a requirement for BMP signaling in proper nail formation and differentiation (Fig. 8-5). Thus, BMP signaling guides NPFSCs toward nail differentiation, and without this pathway, matrix cells proliferate less, and a KZ is not observed above the matrix region (see Fig. 8-5). 24 Also, proper nail plate differentiation is compromised, and without BMP signaling, the nail adopts an epidermal fate in vivo (see Fig. 8-5). It manifests the extension of the skin epidermis granular layer throughout the nail with typical epidermal markers such as keratin 1 (K1) and loricrin expression observed in the nail plate (see Fig. 8-5). In contrast, typical differentiation nail plate marker AE13 (keratin expressed in both the hair cortex and nail plate) was undetectable (see Fig. 8-5). 24 Interestingly, a similar role of BMP signaling was observed in proper hair follicle differentiation. 25 This observation was also consistent, and the phenotype was even more severe than a phenotype of the Msx2 and Foxn1 double mutant; both genes previously have been showed as downstream targets of BMP signaling regulating normal nail differentiation. 26 It was also similar to Foxn1 and Hoxc13 single mutants, with aberrant extension of the epidermal stratum granulosum within the nail structure (see Fig. 8-5). 2,27 In addition, the role of BMP signaling is also required for maintenance of the LRC characteristic of NPFSCs (see Fig. 8-5), as previously observed in hfSCs. 28 Taken together, a novel population of bifunctional stem cells within the NPF region displays plastic homeostatic dynamics capable of responding to injury and suggests a common, coordinated mechanism of protective barrier formation that could occur between the nail and adjacent epidermis.24
RAPIDLY CYCLING UNIPOTENT NAIL STEM CELLS IN THE NAIL ORGAN
The nail matrix is a morphological continuation of the NPF cells that attach to each other through the IZ wrapping around the proximal end of the nail plate (see Fig. 8-4). Thus, proliferating matrix progenitor cells are found in the near vicinity of the NPF region and differentiate into cells of the KZ to form the external nail plate.
In terms of the cell proliferation dynamic, a clear distinction was observed between slow-cycling nail LRCs in the NPF and the actively dividing matrix cells after only 1 week of chase. 24 At that time point, Ki67 immunolabeling primarily is localized to proliferating
matrix cells and a minority of weakly H2B-GFP+ cells immediately adjacent to Ki67 negative strongly H2BGFP+ proximal fold (PF) LRCs, indicating increased proliferation within the matrix in comparison with the adjacent PF region (see Fig. 8-4).
Thus, the area between both regions—the proliferating matrix and the quiescent PF IZ—has been described as containing weakly H2B-GFP+ and Ki67+ cells, which arise from H2B-GFP label dilution after cell division. The IZ gradually decreases in size with increasing periods of chase, being minimal just after a 2-week chase to completely disappearing after a 4-week chase. This indicates the existence of a gradient in cell division expanding from relatively quiescent slow-cycling LRCs in the NPF (through the IZ of more active cells) to rapidly dividing matrix cells (see Fig. 8-4).24
Thus, it was demonstrated that the nail matrix contains rapidly dividing progenitor cells responsible for nail differentiation. However, whether matrix cells alone possess stem cell characteristics and can generate the whole nail appendage was investigated and is addressed further later in this chapter.
To locate stem cells in nails, Takeo and coworkers used a transgenic mouse lineage tracing system. Under control of keratin 14 (K14) promoter-driven Cre recombinase–mutated estrogen receptor (Cre-ER) (K14–Cre-ER), they marked keratinocytes in the basal layer of the skin epidermis and the nail epidermis by activating LacZ expression. 29 In that system, LacZ expression was driven by the Rosa26 promoter, and its activation followed Cre-mediated removal of the floxed stop cassette. Thus, it allowed labeling genetically a small subset of K14 nail basal epidermal cells, including nail matrix cells and bed cells, using a single injection of tamoxifen and then assessing participation of LacZ-labeled cells in nail regeneration over an extended period of time. Over 5 months, the number of LacZ+ descendants of the labeled K14 nail epithelial cells extended linearly and distally, persisting as streaks that emerged only from the proximal matrix but not from the distal matrix. Proximal matrix cells possessed characteristics of undifferentiated epidermal cells expressing keratin 17 (K17) in addition to K14 with highly proliferative Ki67 marker (see Fig. 8-4). The isolated proximal matrix cells had the highest colony-forming ability in vitro, a general characteristic of epithelial stem cells. Thus, prolonged labeling results along with in vitro culture data demonstrated that the rapidly dividing proximal matrix contains self-renewing cells, meeting the criteria to be per se NSCs that sustain nail growth (see Fig. 8-4).29
Interestingly, a gene expression analysis between proximal matrix versus distal matrix revealed that proximal matrix cells were enriched with NSCs with downregulated Wnt signaling pathway. 29 Indeed, analyses using two different Wnt reporter mice confirmed that although the Wnt signal started from the distal part of the K17-positive NSC region and persisted into the distal matrix of K17-negative distal matrix, both signals were absent in the proximal end of the nail matrix. Consistently, Wnt signaling pathway components such
as Tcf1 (also known as hepatocyte nuclear factor 1a), a nuclear mediator of Wnt signaling, and Wls (Wntless homologue) were not expressed in the proximal end of NSCs, but several keratins that contained a TCF1 and LEF1 consensus binding site were upregulated in the distal matrix, thus suggesting direct involvement of Wnt signaling in nail differentiation. This was verified by deletion of Wnt signaling activation in the nail epithelium by removing β-catenin (an essential mediator of Wnt signaling) in adult epithelium revealed by the lack of AE13, a marker for keratinized nail cells. Interestingly, without β-catenin or Wntless, the entire nail epithelium showed characteristics of the NSC region with K17-positive and highly proliferating Ki67positive cells, thus confirming the essential role of Wnt signaling in nail differentiation.
Moreover, in a recent study, scientists identified a cell population expressing a mediator of Wnt signaling, Lgr6 (leucine-rich repeat containing G proteincoupled receptor 6), as key stem cells marker for the nail, which was localized within the nail proximal matrix, thus being consistent and confirming previous discoveries by Takeo and coworkers. 30 Indeed, Lgr6 is a molecular marker specific to the NSCs, giving rise to the nail plate and contributing to the growth of nail over time (see Fig. 8-4). Thus, these data confirmed that canonical Wnt signaling (which results in β-catenin localization to the nucleus of the nail matrix) broadly correlates with the expression of Lgr6-expressing nail matrix cells.
ROLE OF NAIL EPITHELIUM IN DIGIT REGENERATION
The tips of the digits of humans, similar to those of some mammals, including mice, are capable of complete regeneration after injury like those of amphibians. 31,32 Interestingly, this capacity is limited to the area associated with the nail, and previous studies showed that nail transplantation after amputation at the middle phalanx can induce ectopic digit bone differentiation. 31-33 Thus, this observation suggests that the nail epithelium has a special function, creating a permissive regenerative environment for digit regeneration. Recently, some cellular and molecular components of the digit regeneration process have been discovered by Takeo and associates. 29 Indeed, the process is orchestrated and depends on the presence of the overlaying nail organ and its reciprocal interaction with underlying blastema. It has been shown that nail epithelium, particularly NSCs located in the proximal nail matrix and their differentiation, is coupled directly with ability to orchestrate digit regeneration. On a molecular level in the mouse model, Wnt/β-catenin signaling from nail epithelium has been shown to be necessary for digit regeneration. The current proposed model is that NSCs in the proximal matrix give rise to distal matrix cells with Wnt activation (through Wntless), and, simultaneously, NSCs and distal matrix cells differentiate into the nail plate (Fig. 8-6). After distal
amputation at the level of the distal matrix expressing Wntless (but NSCs in proximal matrix remain intact), the wound site is covered by regenerating nail epithelial cells, which in turn activate Wnt signaling (Wntless) to differentiate into distal matrix cells and the nail plate (see Fig. 8-6). The Wnt pathway activation promotes blastema innervations through semaphorin 5a (Sema5a, an axon-guidance molecule), which is upregulated in control nail epithelium 3 weeks after amputation. Conversely, innervations are necessary for fibroblast growth factor 2 (FGF2) expression in the regenerating nail epithelium (see Fig. 8-6). Then FGF2 promotes proliferation of either Runx2-positive progenitors or Sp7 osteoblasts, ultimately leading to coupled digit regeneration (see Fig. 8-6). Interestingly, receptor for FGF2 ligand FGFR1 is expressed in mesenchymal blastema (a Runx2 progenitor); thus, the ERK pathway could be recruited to activate and maintain their proliferation (see Fig. 8-6).
Thus, nail differentiation is regulated by a WNTdependent mechanism that is linked to digit regeneration. Indeed, Wnt deletion (in β-catenin conditional knockout [KO] mice) results in a lack of nail differentiation that manifests by absence of TopGal expression in the nail matrix (a gene reporter for WNT pathway) and AE13 expression (a marker for keratinized nail plate cells), and bone regeneration is blocked. Moreover, Runx2 progenitor cells, mesenchymal cells, and Sp7 osteoblasts underneath the nail matrix epithelium in β-catenin conditional KO mice were not stimulated to proliferate or produce BMP4. Moreover, Wntdependent innervation can promote digit regeneration because Sema5a is downregulated in the nail epithelium 3 weeks after amputation in β-catenin conditional KO mice. These data are in agreement that denervation (nerves removed surgically before amputation)
suppresses blastema growth similar to that observed in conditional KO mice without FGF2 expression in the nail epithelium after denervation. 34,35 Interestingly, although after proximal amputation of nail matrix cells, stabilization of β-catenin induces TCF1 expression and regeneration of the distal matrix and formation of well-innervated blastema cells, β-catenin stabilization in the nail epithelium does not promote digit regeneration after amputation proximal to the NSC niche. Therefore, the remaining proximal matrix epidermis is not able to respond to β-catenin stabilization, and digit regeneration cannot be restored, which is in contrast to distal matrix function.
This results are consistent with inhibition of proper digit-tip regeneration after ablation of Lgr6, an important agonist of the Wnt pathway that marks NSCs and gives rise to the nail plate. 30 In addition, Lgr6 expression is not only limited to the nail matrix but is more broadly expressed within a subset of cells in the digit tip, namely, in the bone osteoblasts and eccrine sweat glands. Therefore, Lgr6-expressing cells not only mark NSC epithelium but also contribute to the blastema, which suggest not only direct but also a potential indirect role for Lgr6-expressing cells during digit-tip regeneration. 30 Indeed, this role has been confirmed by analysis of Lgr6-deficient mice, which have both a nail and bone regeneration defect, but it remains unclear whether the Lgr6-positive osteoblasts contribute to this phenotype. Thus, the role of these cells in normal skeletal homeostasis and digit regeneration remains to be determined in the future.
Collectively, the dual function of Wnt signaling in the NSC lineage that directs nail formation and differentiation and digit regeneration appears to be a key mechanism that coordinates the regeneration of epithelial and mesenchymal tissues in mammalian digit-tip regeneration (see Fig. 8-6). Indeed, interestingly, some nail disorders in humans that may affect only nails include inherited anonychia and isolated congenital nail dysplasia. Affected families with inherited anonychia have severe hypoplasia of the nails in which mutations in R-spondin 4 (Rspo4), which is implicated in the Wnt signaling pathway, were identified, suggesting an important role of Rspo4 in nail development.36
Stem cells have long term self-renewal potential and are capable of differentiating into a variety of different cell types. This persistence and multipotency are crucial for the maintenance of tissue during homeostasis and repair. Because of its extraordinary regenerative potential, understanding its maintenance and regulation will prove its usefulness in tissue regeneration and treatment of various disorders.
Current discoveries unveiled the model in which stem cells in nail organs exist in two different activation mode—slow-cycling and actively proliferating ones—to fulfill the demands of continuously growing nails. 24,29 These data demonstrated that the nail organ contains a gradient of slow- to fast-cycling stem cells, with slow-cycling NPFSCs in the proximal fold, more active cells in the IZ, and finally rapidly proliferative NCSs in the nail proximal matrix regions.
Collectively, this model supports the current view about adult stem cell dynamics and potency in several organs and tissues during homeostasis and regeneration. For example, the label-retaining, NPFSCs, and rapidly proliferative NCSs present a dynamic hierarchy similar to that described in hfSCs in which some hfSCs participate in the new hair cycle and other hfSCs of the upper outer root sheath (ORS) of growing follicles reestablish the new bulge with quiescent hfSCs.37 During this bulge activation, a gradient of slow- to fast-cycling cells is observed within the ORS extending from the bulge region toward the matrix, respectively.
Why does the nail organ possess two populations of stem cells? One answer might come from observation of similar system of two stem cell populations with different cell cycling dynamics in the intestine. In the intestine, slow-cycling LRCs stem cells have been identified in the +4 position of intestinal crypts that expressed Bmi1. 38,39 In contrast, fast-cycling stem cells were also found at the bottom of the crypts expressing Lgr5, 40 and both stem cell populations are capable of differentiating into all intestinal lineages. 39 Interestingly, recently, in the absence of rapidly proliferating Lgr5+ stem cells, Bmi1 stem cells can maintain the normal turnover of the intestines while repopulating the Lgr5+ stem cell population. 41 This suggests that Bmi1+ cells, localized where LRCs are found, act as a reserve stem cell population. Thus, a similar backup system between two populations of stem cells in nails, namely NPFSCs and NSCs, might exist to protect overall nail organ maintenance. Thus, it will be interesting to further address this in the future. Overall, this
suggest that NSCs in the proximal matrix 29 integrate the adjacent neighboring proximal fold as a location of bifunctional NPFSCs. 24 Collectively, these examples underscore an inherent plasticity of the adult stem cell populations within nail epithelia, and slow-cycling NPFSCs in these highly proliferative tissues might represent a backup system for NSCs during times of stress, injury, or depletion of the stem cell population.
All these exciting discoveries about stem cell populations in the nail organ and their mutual relationships as well as reciprocal interactions between stem cells and stem cell niches help unravel new concepts in stem cell biology and have great translational potential that could considerably impact regenerative medicine. Indeed, in mouse digit-tip regeneration, it has been demonstrated that although nail epithelium with its stem cells is critical to orchestrate this process, crosstalk with other germ-layer and lineagerestricted stem/progenitor cells is essential to successfully complete this regeneration. 42 Thus, in the future, this research might potentially restore anatomical and functional structures by orchestrating full-digit regeneration, including bone, innervations, and vasculature with skin and skin appendages along with nail, therefore benefiting individuals with digit amputations.
These results have established a direct relationship between NSC differentiation and digit regeneration and suggest that NSCs may have therapeutic potential to contribute toward the development of novel treatments for amputees. This is a very important problem because many people are affected by accidental amputation of fingers or toes worldwide. Recent nail and digit regeneration data have begun to pave the way toward unveiling the underlying mechanisms required to expose this regenerative potential. Therefore, new discoveries in nail biology will very likely enhance our understanding of the fundamental mechanisms required to orchestrate digit regeneration with the potential to unlock the broad regenerative potential to reconstitute the whole limb beyond digit regeneration.
Although more studies are needed for further characterization of these stem cell population in the nail organ, these data may shed some light on putative stem cells markers for human nails in the future. Indeed, interestingly, initial observation in human of the ventral proximal fold was proposed to be the NSCs in the developing embryonic human nail based on the expression of bulge stem cell markers, K15, K19, and PHLDA1 with quiescent characteristics that might represent NPFSCs in the mouse model. 23 In contrast, the human nail matrix contains highly proliferative cells marked by Ki67, similar to the mouse nail matrix, which might be similar as NSCs highly proliferating stem cells in the proximal matrix in mice. 23 Thus, further identification, characterization, and isolation of those two distinct populations of stem cells in nail organ in humans at the ventral proximal fold and nail matrix might provide crucial understanding of nail organ biology at the molecular level, which can potentially provide insight into designing translational therapies for regrowing greater portions of the limbs and other nonregenerative tissues in the future. Therefore, defining the regenerative potential of these NSCs and NPFSCs and the molecular mechanisms by which they are regulated in vivo (with the intent that novel therapies) may emerge toward the successful nail organ regeneration and wound healing, including treatment of amputee patients.
