Lgr6-expressing functional nail stem-like cells differentiated from human-induced pluripotent stem cells.

The nail matrix containing stem cell populations produces nails and may contribute to fingertip regeneration. Nails are important tissues that maintain the functions of the hand and foot for handling objects and locomotion. Tumor chemotherapy impairs nail growth and, in many cases, loses them, although not permanently. In this report, we have achieved the successful differentiation of nail stem (NS)-like cells from human-induced pluripotent stem cells (iPSCs) via digit organoids by stepwise stimulation, tracing the molecular processes involved in limb development. Comprehensive mRNA sequencing analysis revealed that the digit organoid global gene expression profile fits human finger development. The NS-like cells expressed Lgr6 mRNA and protein and produced type-I keratin, KRT17, and type-II keratin, KRT81, which are abundant in nails. Furthermore, we succeeded in producing functional Lgr6-reporter human iPSCs. The reporter iPSC-derived Lgr6-positive cells also produced KRT17 and KRT81 proteins in the percutaneously transplanted region. To the best of our knowledge, this is the first report of NS-like cell differentiation from human iPSCs. Our differentiation method and reporter construct enable the discovery of drugs for nail repair and possibly fingertip-regenerative therapy.

Single-cell RNA sequencing of human nail unit defines RSPO4 onychofibroblasts and SPINK6 nail epithelium.

Research on human nail tissue has been limited by the restricted access to fresh specimen. Here, we studied transcriptome profiles of human nail units using polydactyly specimens. Single-cell RNAseq with 11,541 cells from 4 extra digits revealed nail-specific mesenchymal and epithelial cell populations, characterized by RSPO4 (major gene in congenital anonychia) and SPINK6, respectively. In situ RNA hybridization demonstrated the localization of RSPO4, MSX1 and WIF1 in onychofibroblasts suggesting the activation of WNT signaling. BMP-5 was also expressed in onychofibroblasts implicating the contribution of BMP signaling. SPINK6 expression distinguished the nail-specific keratinocytes from epidermal keratinocytes. RSPO4+ onychofibroblasts were distributed at close proximity with LGR6+ nail matrix, leading to WNT/ß-catenin activation. In addition, we demonstrated RSPO4 was overexpressed in the fibroblasts of onychomatricoma and LGR6 was highly expressed at the basal layer of the overlying epithelial component, suggesting that onychofibroblasts may play an important role in the pathogenesis of onychomatricoma.

The Potential of Nail Mini-Organ Stem Cells in Skin, Nail and Digit Tips Regeneration.

Nails are highly keratinized skin appendages that exhibit continuous growth under physiological conditions and full regeneration upon removal. These mini-organs are maintained by two autonomous populations of skin stem cells. The fast-cycling, highly proliferative stem cells of the nail matrix (nail stem cells (NSCs)) predominantly replenish the nail plate. Furthermore, the slow-cycling population of the nail proximal fold (nail proximal fold stem cells (NPFSCs)) displays bifunctional properties by contributing to the peri-nail epidermis under the normal homeostasis and the nail structure upon injury. Here, we discuss nail mini-organ stem cells' location and their role in skin and nail homeostasis and regeneration, emphasizing their importance to orchestrate the whole digit tip regeneration. Such endogenous regeneration capabilities are observed in rodents and primates. However, they are limited to the region adjacent to the nail's proximal area, indicating the crucial role of nail mini-organ stem cells in digit restoration. Further, we explore the molecular characteristics of nail mini-organ stem cells and the critical role of the bone morphogenetic protein (BMP) and Wnt signaling pathways in homeostatic nail growth and digit restoration. Finally, we investigate the latest accomplishments in stimulating regenerative responses in regeneration-incompetent injuries. These pioneer results might open up new opportunities to overcome amputated mammalian digits and limbs' regenerative failures in the future.

Nail matrix regenerative engineering: in vitro evaluation of poly(lactide-co-glycolide)/gelatin fibrous substrates.

Acute traumatic nail injury treatment repair procedures are commonly conducted in emergency departments and primary care offices. Current repair methods use nail splints that are inserted within the nail root to prevent the fusion of the proximal nail fold and the matrix tissue. Splints provide a protective barrier overlying the nail bed soft tissue during recovery periods, but uncertain prognoses (i.e., aesthetic and functional disadvantages) reveal a need for improved nail repair techniques. Nail splints are not specifically designed for nail organ restoration via biological mechanisms, thus, a clinical application that utilizes regenerative engineering techniques can prove useful in improving the nail injury prognoses. Using the coaxial electrospinning method, hybrid poly(lactide-co-glycolide) (PLGA) (85:15) and gelatin fibrous scaffolds (Hybrid1: PLGA shell, gelatin core and Hybrid2 : gelatin shell, PLGA core) with average fiber diameters of 540 ± 118 and 2,215 ± 1,135 nm, respectively, were produced and successful encapsulation of core fibers was observed. Furthermore, nail stem cells expressing stem cell characteristic markers CD90, CD29, and Lgr6 showed preferred attachment to Hybrid2 scaffolds after 24 hr. Overall, an in vitro regenerative engineered nail matrix may aid to improve the cosmetic appearance and function of injured nail organs post-traumatic injury.

The genetic profile of bone marrow transplant patients in different samples of forensic interest.

Humans constantly lose epithelial cells, and these biological traces are frequently studied in the context of criminal investigations. The objective of this work was to examine the genetic profile in samples of forensic interest (nail and skin epithelial cells) of bone marrow transplant patients and discuss its forensic and clinical implications. The genetic profile of nail, epidermal cells and blood samples of patients receiving HSCT was analyzed by the amplification and sequencing of 38 insertion/deletion polymorphisms and 15 short tandem repeat polymorphisms. In this analysis, the age of patients and donors, the time elapsed from the transplant, the type of conditioning prior to the transplant and whether the patient suffered graft-versus-host disease were considered. Donor chimerism can be detected in the DNA extracted from nail and skin epithelial cells of transplant patients. No statistically significant correlation was found between the type of conditioning and the percentage of donor DNA in nail (p > 0.05). A positive correlation, without statistical significance, was encountered when we analyzed the relationship between the time elapsed from the transplant with the percent donor chimerism found in epithelial cells of the epidermis and in nails. We conclude that within a judicial context (e.g. when testifying as an expert witness) it is necessary to consider whether we are facing a possible transplant patient or a person who has been a bone marrow donor.

Human nail stem cells are retained but hypofunctional during aging.

The nail is a continuous skin appendage. Cells located around the nails, which display coordinated homeostatic dynamics and release a flow of stem cells in response to regeneration, have been identified in mice. However, very few studies regarding human nail stem cells exist in the literature. Using specimens isolated from humans, we detected an unreported population of cells within the basal layer of postnatal human nail proximal folds (NPFs) and the nail matrix around the nail root. These cells were multi-expressing and expressed stem cell markers, such as keratin 15 (K15), keratin 14 (K14), keratin 19 (K19), CD29, CD34, and leucine-rich repeat-containing G protein-coupled receptor 6 (Lgr6). These cells were very similar to mouse nail stem cells in terms of cell marker expression and their location within the nail. We also found that the putative nail stem cells maintained their abundance with advancing age, but cell proliferation and nail growth rate were decreased on comparison of young and aged specimens. To summarize, we found a putative population of stem cells in postnatal human nails located at NPFs and the nail matrix. These cells may have potential for cell differentiation and be capable of responding to injury, and were retained, but may be hypofunctional during aging.

Immunohistochemical identification of nail matrix melanocytes.

BACKGROUND: No previous studies have been conducted to determine the normal number of nail matrix melanocytes in Latin American individuals. The objective of this work was to determine the number of melanocytes per linear millimetre present in the nail matrix and the nail bed in samples obtained from Colombian individuals. METHODS: Twenty-six unilateral biopsies were taken from 19 cadavers subjected to clinical and medico-legal autopsies. These biopsy samples were processed with conventional histotechnology and immunohistochemistry (IHC) with anti-HMB-45 and anti-MiTF. Three sets of photographs (HE, HMB-45 and MiTF) were taken of each biopsy sample and independently assessed by three pathologists. Each observer counted the number of melanocytes present in 1 linear mm of the nail matrix or bed. RESULTS: We found an average of 4.6 melanocytes x linear mm with H & E staining, 9.8 with HMB-45 and 12.4 with MiTF. CONCLUSIONS: The use of IHC significantly increases and facilitates the identification of melanocytes in unilateral biopsies. Our IHC counts exceed the averages found in the literature. This finding warrants new studies to verify whether the Colombian population presents higher numbers of melanocytes in the nail matrix than other populations or whether the observed increase is a result of the use of MiTF.

Melanocytes Pattern in the Normal Nail, With Special Reference to Nail Bed Melanocytes.

Previous studies of the density of melanocytes in the normal nail bed have had conflicting results. This is unfortunate because knowing the normal values might help the difficult distinction between a benign subungual melanotic macule and an early melanoma in situ. Five specimens of normal nail unit were analyzed. On hematoxylin and eosin-stained sections the melanocytes were undetectable. We defined the melanocyte count (MC) as the number of melanocytes per 1-mm stretch of nail epithelium. The mean MC for nail matrix was 6.86 with a range of 4-14. The melanocytes were irregularly scattered in the basal and suprabasilar layer of the matrix epithelium. Abundant and uneven cytoplasmic dendrites were focally observed in the matrix. The MC for the nail bed ranged from 0 to 5 with a mean of 0.43. The melanocytes were restricted to the basal layer with thin cytoplasmic dendrites. Two cases showed a complete absence of melanocytes in the nail bed. In the ventral portion of the proximal nail fold, called the eponychium, the MC ranged between 0 and 5/mm with a mean of 2.27/mm. In conclusion, we discovered foci in normal nail beds, in which the melanocytic density can be relatively high and reach the level seen in the matrix. HMB45 is more sensitive than Microphtalmia-associated transcription factor (MITF) for the evaluation of intraepithelial melanocytic density of the nail unit. If MITF is used alone in the nail bed, its weak sensitivity may result in a false-negative interpretation and may be wrongly reassuring in the evaluation of early melanomas. On hematoxylin and eosin sections, basal and suprabasal nail keratinocytes are sometimes crowded, showing oval or elongated dark-staining nucleus and a clear cytoplasm and mimics a melanocytic proliferation. On HMB45 or Melan A staining, the morphology and the distribution of the dendrites of matrical melanocytes can mimic the dendritic pattern usually described in acral melanoma. Therefore, the interpretation of nail melanocytic atypia must be prudent.

CD13 is a marker for onychofibroblasts within nail matrix onychodermis: Comparison of its expression patterns in the nail unit and in the hair follicle.

BACKGROUND: We previously demonstrated the presence of onychodermis, a specialized mesenchymal cell population beneath the nail matrix and proximal nail bed demonstrating CD10 expression. We hypothesize that the onychodermis could be the nail analog of the follicular dermal papilla, which is known to express CD13. We compare CD13 expression patterns between specialized mesenchymes of nail and hair, and compare these findings with CD10 expression patterns. METHODS: CD10 and CD13 immunohistochemistry was performed on polydactyly and adult cadaveric nail units, and on hair follicles in scalp nevus sebaceus excision specimens. RESULTS: CD10 and CD13 were expressed in the mesenchyme below the nail matrix and nail bed. Stronger CD13 expression was observed in the mesenchyme containing onychofibroblasts below the nail matrix compared with that below the nail bed. CD10 was expressed in the dermal sheath of terminal hair follicles, but it was expressed in the dermal sheath and follicular dermal papilla of primitive hair follicles within nevus sebaceus lesions. CD13 was expressed in the dermal sheath and dermal papilla of terminal and primitive hair follicles. CONCLUSION: CD13 may be a marker for onychofibroblasts within nail matrix onychodermis. We demonstrate CD13 expression in the specialized mesenchymes of both nail and hair.

DNA from nails for genetic analyses in large-scale epidemiologic studies.

BACKGROUND: Nails contain genomic DNA that can be used for genetic analyses, which is attractive for large epidemiologic studies that have collected or are planning to collect nail clippings. Study participants will more readily participate in a study when asked to provide nail samples than when asked to provide a blood sample. In addition, nails are easy and cheap to obtain and store compared with other tissues. METHODS: We describe our findings on toenail DNA in terms of yield, quality, genotyping a limited set of SNPs with the Sequenom MassARRAY iPLEX platform and high-density genotyping with the Illumina HumanCytoSNP_FFPE-12 DNA array (>262,000 markers). We discuss our findings together with other studies on nail DNA and we compare nails and other frequently used tissue samples as DNA sources. RESULTS: Although nail DNA is considerably degraded, genotyping a limited set of SNPs with the Sequenom MassARRAY iPLEX platform (average sample call rate, 97.1%) and high-density genotyping with the Illumina HumanCytoSNP_FFPE chip (average sample call rate, 93.8%) were successful. CONCLUSIONS: Nails are a suitable source of DNA for genotyping in large-scale epidemiologic studies, provided that methods are used that are suitable or optimized for degraded DNA. For genotyping through (next generation) sequencing where DNA degradation is less of an issue, nails may be an even more attractive DNA source, because it surpasses other sources in terms of ease and costs of obtaining and storing the samples. IMPACT: It is worthwhile to consider nails as a source of DNA for genotyping in large-scale epidemiologic studies. See all the articles in this CEBP Focus section, "Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology." Cancer Epidemiol Biomarkers Prev; 23(12); 2703-12. ©2014 AACR.

Morphopathological aspects of healthy nails and nails affected by onychomycosis.

Patients of onychomycosis are common in the dermatology practice. Contemporary morphology creates opportunities to study the functional units of the nail when such infections occur from morphopathological point of view. There were 22 nails biopsies from onychomycosis patients taken for the research of morphopathological changes in the thickened nail plate affected by onychomycosis. Samples of cadaverous' nails were used as a control material. The material was stained with haematoxylin and eosin and immunohistochemical methods. Terminal deoxynucleotidyl transferase dUTP nick end labelling reaction and periodic acid-Schiff reaction were also performed. We found patchy hypertrophy in the granulose layer of the epidermis, with focal acanthosis. In the horn layer, we identified nests of parakeratosis of various sizes, with incorporations of homogenous and eosinophil masses. We found high levels of interleukin 6 and interleukin 10 positive cells in the nail bed and in the bloodstream. Interleukin 1, however, was not a part of any of the functional units of any of the nails. Significant amount of fibres containing human beta defensin-2 were found in the bed and plate of the nail. Therefore one can conclude that as regards the nails affected by onychomycosis, the most effective morphopathogenical processes include cytokine and defensin excretion occurrence in the nail bed.

Confocal microscopy for healthy and pathological nail.

Nail diseases are often annoying for the patient and diagnostically challenging for dermatologists. New imaging techniques are of high interest in the diagnosis of nail disorders to reduce the number of nail biopsies. Confocal microscopy is a high-resolution emerging imaging technique that can be used to explore the entire body surface, including skin, mucosa, hair and nails. A systematic review of the literature concerning the use of confocal microscopy for the study of either healthy or pathological nail has been performed to evaluate the current use of this technique and possible future applications. Confocal microscopy is particularly suitable for nails because it allows a non-invasive in vivo examination of this sensitive body area, and nail plate transparency permits to image up to the nail bed with an easy identification of corneocytes. Confocal microscopy can play a role in the diagnosis of onychomycosis and melanonichia, and in the study of drug penetration through the nail plate. It could be used in the future as a non-invasive procedure for the investigation of different nail diseases, such as psoriasis and lichen planus. Further application could be the intra-operative ex vivo examination of nail specimens to outline tumour margins to assist surgery.

Nail stem cells.

Our knowledge on stem cells of the hair follicle has increased exponentially after the bulge was characterized as the stem cell niche two decades ago. In contrast, little is known about stem cells in the nail unit. Whereas hair follicles are plentiful and easy to access, the human body has only twenty nails and they are rarely biopsied. Therefore, examining fetal material offers unique advantages. In the following mini-review, our current knowledge on nail stem cells is summarized and analogies to the hair follicle stem cells are drawn.

The concept of the onychodermis (specialized nail mesenchyme): an embryological assessment and a comparative analysis with the hair follicle.

BACKGROUND: Recently, an intriguing concept was introduced into the literature that defines the area underlying the nail bed as a specific mesenchymal substructure unique to the nail organ. It has been termed onychodermis. The onychodermis expresses CD10 with remarkable specificity. Herein, we compare adult and fetal human hair follicles with fetal nail organs in an attempt to draw analogies for the mesenchyme associated with both adnexal structures. METHODS: We examined immunohistochemically samples from adult and fetal hair follicles for the expression of CD10, CD34 and the mesenchymal stem cell marker nestin and compared the antigen profile with that of the fetal nail organ. RESULTS: The CD10-positive/CD34-negative onychodermis is prominently visible at the end of the second trimester. A corresponding follicular structure was not identified, either in the adult or in the developing hair follicle. Nestin staining does not define the onychodermis. CONCLUSIONS: The concept of the onychodermis is equally valid in the developing nail organ where it is also defined by its expression for CD10. Its function may be related to the anchorage of the overlying nail bed but may also involve a more dynamic role in the induction of hard keratins in the latter, contributing to the formation of the nail plate.

The ventral proximal nail fold: stem cell niche of the nail and equivalent to the follicular bulge--a study on developing human skin.

BACKGROUND: Although the bulge is well characterized as a stem cell niche of the hair follicle, comparatively little is known about the location of stem cells in the nail. Herein, we describe the spatiotemporal expression pattern of six stem cell markers in the developing human nail and compared it with the embryonic and fetal human hair follicle. The areas of proliferative activity were additionally examined using labeling with Ki-67. METHODS: We examined immunohistochemically samples from embryonic and fetal human nail, hair and skin for the expression of cytokeratin 15 (CK15, two clones), cytokeratin 19 (CK19), PHLDA1, CD200, nestin and Ki-67 using standard techniques. RESULTS: CK15 (clone LHK15), CK19 and PHLDA1 are negative in the nail and hair matrix but positive in the ventral proximal nail fold and in the follicular bulge. Over the course of embryogenesis they display a highly specific spatiotemporal expression pattern both in the nail and in the hair follicle. CONCLUSIONS: We propose that at least during embryogenesis the proximal ventral nail fold represents the niche for the nail stem cells. In contrast to animal experiments, autoradiographic pulse-chasing studies cannot be performed in human, and immunohistochemical studies are a valid alternative although they have their limitations. Further studies on adult human nail units are suggested.

The human nail--barrier characterisation and permeation enhancement.

The human nail remains one of the most challenging membranes for formulation scientists to target and for clinicians to heal. Its formidable barrier properties are the primary reason that oral therapy remains the primary approach to manage ungual infections. This article considers the major structural properties underlying the excellent barrier function of the nail, with particular emphasis on the role of biophysical methods in advancing our knowledge of this appendage. Formulations currently available for management of ungual disease are discussed and their therapeutic efficacy is assessed. Finally, experimental strategies to enhance ungual permeation are reviewed and prospects for future developments in the field are considered.

The presence and localization of onychodermis (specialized nail mesenchyme) containing onychofibroblasts in the nail unit: a morphological and immunohistochemical study.

AIMS: Due to its limited availability there has been very little research on the mesenchyme of the nail unit. Recently, we discovered specialized mesenchymal cells beneath the nail matrix and proposed to call them onychofibroblasts. The purpose of this study was to further delineate more precisely by histology and immunohistochemistry the specialized nail mesenchyme. METHODS AND RESULTS: Thirty supernumerary digits were obtained during operations to correct polydactyly. Longitudinal and transverse sections were obtained from formalin-fixed paraffin-embedded blocks. In sections stained with haematoxylin and eosin, a mesenchymal area that showed much more cellularity and less eosinophilic, loose connective tissue was identified beneath the nail matrix and nail bed. Using Alcian blue staining, mucin was detected in this mesenchymal area below the nail matrix and nail bed. Immnunohistochemically, CD10 and versican were expressed strongly in the mesenchyme containing onychofibroblasts under the nail matrix and nail bed. CONCLUSIONS: These results demonstrate the presence and localization of a specialized nail mesenchyme containing onychofibroblasts in a well-defined area beneath the nail matrix and nail bed. Thus, we propose the term onychodermis for this specialized nail mesenchyme that is histologically and immunohistochemically distinct from the dermis of other parts of the nail unit.

Immunohistochemical study of specialized nail mesenchyme containing onychofibroblasts in transverse sections of the nail unit.

In the frozen longitudinal section of the nail unit, CD10 was previously found in nail mesenchymal cells beneath nail matrix, and we proposed calling the nail mesenchymal cells onychofibroblasts. In this study, to further characterize nail mesenchyme containing onychofibroblasts, we examined the expression of several mesenchymal markers immunohistochemically in transverse paraffin sections of the nail unit. CD10 was strongly expressed in the nail mesenchyme containing onychofibroblasts beneath the nail matrix. However, CD10 was not observed in dermal fibroblasts and surrounding extracellular matrix of the lateral nail fold (LNF), except around blood vessels and eccrine structures. In addition, versican was expressed diffusely in the nail mesenchyme containing onychofibroblasts in contrast to the dermis of LNF. Fibrillin, which is a major component of elastic fiber in the dermis, was expressed very weakly on the nail mesenchyme below the nail matrix but was expressed strongly in the dermis of LNF. These findings support the existence of specialized nail mesenchyme containing onychofibroblasts that is distinguished from the dermis of LNF.