Bifunctional ectodermal stem cells around the nail display dual fate homeostasis and adaptive wounding response toward nail regeneration Yvonne Leunga,b,1, Eve Kandybaa,b,1, Yi-Bu Chenc, Seth Ruffinsa, Cheng-Ming Chuongb,d, and Krzysztof Kobielaka,b,2 aEli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research, bDepartment of Pathology, and cNorris Medical Library, University of Southern California, Los Angeles, CA 90033; and dInternational Research Center of Wound Repair and Regeneration, Institute of Clinical Medicine, Cheng Kung University, Tainan City 70101, Taiwan Edited by Mina J. Bissell, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA, and approved August 28, 2014 (received for review October 7, 2013) Regulation of adult stem cells (SCs) is fundamental for organ fingertip (Fig. 1A). Found at the “root” of the NP is the actively maintenance and tissue regeneration. On the body surface, different proliferating nail matrix (Mx) with a keratogenous zone (KZ) ectodermal organs exhibit distinctive modes of regeneration and the directly above where Mx cells differentiate into the overlying NP dynamics of their SC homeostasis remain to be unraveled. A slow (Fig. 1A). Pulse–chase studies have confirmed that Mx cells move cycling characteristic has been used to identify SCs in hair follicles and superficially into the NP and also distally toward the nail bed (4). sweat glands; however, whether a quiescent population exists in The proximal end of the NP is covered by the proximal fold (PF), continuously growing nails remains unknown. Using an in vivo label which is a continuation of the epidermis that folds inward (Fig. retaining cells (LRCs) system, we detected an unreported population 1A). Interestingly, epidermal differentiation is “switched off” just of quiescent cells within the basal layer of the nail proximal fold, after the PF invaginates inward at the eponychium, where cells organized in a ring-like configuration around the nail root. These nail continuing beyond this point (including adjacent proximal fold LRCs express the hair stem cell marker, keratin 15 (K15), and lineage A tracing show that these K15-derived cells can contribute to both the cells, Mx, KZ, and nail bed) differentiate to form a NP (Fig. 1 ). nail structure and peri-nail epidermis, and more toward the latter. Previously, the slow cycling, label-retaining cells (LRCs) charac- CELL BIOLOGY Thus, this stem cell population is bifunctional. Upon nail plucking teristic has been successfully used to identify SCs in various organs injury, the homeostasis is tilted with these SCs dominantly delivering including the hair follicle, cornea, and sweat gland; however, exis- progeny to the nail matrix and differentiated nail plate, demonstrat- tence of a quiescent SC population in continuously growing nails ing their plasticity to adapt to wounding stimuli. Moreover, in vivo has not been revealed so far (2, 3, 5–7). Here, we aim to identify the engraftment experiments established that transplanted nail LRCs can location of LRCs in mouse nails, characterize their molecular fea- actively participate in functional nail regeneration. Transcriptional tures, and evaluate their stem cell potential in vivo. This study is profiling of isolated nail LRCs revealed bone morphogenetic protein important in the light of recent discoveries that the tips of mam- signaling favors nail differentiation over epidermal fate. Taken malian digits may regenerate after amputation, depending on its together, we have found a previously unidentified ring-configured interaction with the nail appendage (8). Therefore, defining the population of bifunctional SCs, located at the interface between the quiescent reservoir of nail SCs and their regenerative potential nail appendage organ and adjacent epidermis, which physiologically might help fill in this newly opened research area with novel un- display coordinated homeostatic dynamics but are capable of redi- derstanding of nail organ biology and provide novel forms of verting stem cell flow in response to injury. BMP signaling | nail proximal fold stem cells | nail stem cells | skin stem cells Significance Skin appendages including hair follicles, sweat glands, and nails dult stem cells are capable of self-renewal and are respon- offer a source of regenerative support following injury. Here, sible for the maintenance of their respective organs during A we focus on the regenerative potential of the peri-nail region homeostasis and repair. The skin contains a number of associated and reveal a population of K15-positive, label retaining cells appendages such as hair follicles, sebaceous glands, sweat glands, (LRCs) within the nail proximal fold with self-renewal capa- and nails that display different modes of renewal, regeneration, bilities. Physiologically, these cells display bifunctional stem cell and stem cell (SC) homeostatic dynamics during normal wear and qualities and contribute to both the nail structure and peri-nail tear (1). For example, the epidermis undergoes continual re- epidermis long term; however, upon injury, the homeostatic newal, hair follicles undergo episodic regeneration, and sweat balance is tilted toward nail regeneration. Molecularly, reduced glands display little turnover as demonstrated by the presence of bone morphogenetic protein signaling tilts nail keratinocytes quiescent label-retaining stem cells in vivo (2, 3). Moreover, nails toward an epidermal fate. Collectively, we demonstrate the exhibit continuous growth under physiological conditions and plasticity of these stem cells: bifunctional under normal ho- can fully regenerate upon removal. During development, these meostasis, but adaptive in response to wounding. Such princi- different skin appendages are derived from a common ectodermal ples may exist in the interface between other ectodermal organs origin and share similar characteristics but ultimately adopt alter- and skin. native, individual homeostatic mechanisms to fulfill the different functions of each distinct miniorgan. Therefore, dissecting the Author contributions: Y.L. and K.K. designed research; Y.L., E.K., and K.K. performed underlying similarities and differences that occur during distinct research; Y.-B.C. and S.R. contributed new reagents/analytic tools; Y.L., E.K., Y.-B.C., skin appendage renewal may prove highly beneficial in unrav- C.-M.C., and K.K. analyzed data; and Y.L., E.K., and K.K. wrote the paper. eling the fundamental principles that govern ectodermal organ re- The authors declare no conflict of interest. generation in vivo. This article is a PNAS Direct Submission. The nail contains a hard keratinized structure called the nail 1Y.L. and E.K. contributed equally to this work. plate (NP), which acts as a protective cover to prevent trauma to 2To whom correspondence must be addressed. Email: [email protected]. the digit tips. Beneath the distal end of the NP lies the nail bed This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with both regions sealed together at the hyponychium of the 1073/pnas.1318848111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1318848111 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 near conical structure, dorsal-ventral sections revealed two clus- ters of nail LRCs: one in the upper proximal fold (U-PF) above the NP (upper LRCs) (Fig. 1F) and one in the lower proximal fold (L-PF) localized below the NP (lower LRCs) (Figs. 1H and 2C), indicating the LRCs population encircles the proximal end of the NP. Topological localization was emphasized by using hematoxylin and eosin staining (H&E) on the same sections (Fig. S1 C–F). Furthermore, in vivo confocal microscopy (with serial Z- stack 3D reconstruction) revealed multiple rows of LRCs or- ganized in a concave fashion around the outer surface of the proximal NP after a 3-wk chase (Fig. S2A and Movie S1). Characterization of Nail Proximal Fold LRCs. To specifically localize the LRCs within the nail epithelium, we stained for the basal marker, K5, in 4-wk chased nails and confirmed nail LRCs lo- calized to the β4-integrin–positive basal layer (Fig. 2A) and at- tached to the basement membrane (Fig. 2B). Although the epidermis contains a stratum granulosum that expressed loricrin, this layer fades away at both upper and lower LRCs positions in the nail PF (Fig. 2C and Fig. S2B). Proliferating Mx progenitor cells are found directly adjacent to the nail PF region and differentiate into cells of the KZ to form the external NP. Previous reports have described the presence of LRCs within the nail Mx (11); however, we observed a clear distinction between slow-cycling, nail LRCs in the PF and the actively dividing Mx cells after only 1 wk of chase (Fig. 2D). Indeed, nuclear Ki67 immunolabeling primarily localized to pro- + liferating Mx cells and a minority of weakly H2BGFP cells + immediately adjacent to Ki67 negative, strongly H2BGFP PF LRCs (Fig. 2D, arrows), indicating increased proliferation within the Mx in comparison with the adjacent PF region. We observed + + Fig. 1. Localization of LRCs in the nail proximal fold. (A) Components of the that the area containing weakly H2BGFP and Ki67 cells mouse nail. Top view, horizontal sections of the fingertip before (C) and or “intermediate zone,” arising from H2BGFP label dilution after (B and D) 4 wk of chase with Dox identifying a population of H2BGFP after cell division, decreased in size with increasing periods of marked LRCs surrounding the nail structure. Side view,