The esophageal lumen is lined by a stratified squamous epithelium comprised of proliferative basal cells that differentiate while migrating toward the luminal surface and eventually desquamate. Rapid epithelial renewal occurs, but the specific cell of origin that supports this high proliferative demand remains unknown. Herein, we have described a long-lived progenitor cell population in the mouse esophageal epithelium that is characterized by expression of keratin 15 (Krt15). Genetic in vivo lineage tracing revealed that the Krt15 promoter marks a long-lived basal cell population able to self-renew, proliferate, and generate differentiated cells, consistent with a progenitor/stem cell population. Transcriptional profiling demonstrated that Krt15+ basal cells are molecularly distinct from Krt15- basal cells. Depletion of Krt15-derived cells resulted in decreased proliferation, thereby leading to atrophy of the esophageal epithelium. Further, Krt15+ cells were radioresistant and contributed to esophageal epithelial regeneration following radiation-induced injury. These results establish the presence of a long-lived and indispensable Krt15+ progenitor cell population that provides additional perspective on esophageal epithelial biology and the widely prevalent diseases that afflict this epithelium.

Chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease, is a leading cause of morbidity and mortality worldwide. Prolonged cigarette smoking (CS) that causes irreversible airway remodeling and significantly reduces lung function is a major risk factor for COPD. Keratin15+ (Krt15+) cells with the potential of self-renewal and differentiation properties have been implicated in the maintenance, proliferation, and differentiation of airway basal cells; however, the role of Krt15 in COPD is not clear.

The epidermis is continuously renewed by stem cell proliferation and differentiation. Basal keratinocytes append the dermal-epidermal junction, a cell surface-associated, extracellular matrix that provides structural support and influences their behaviour. It consists of laminins, type IV collagen, nidogens, and perlecan, which are necessary for tissue organization and structural integrity. Perlecan is a heparan sulfate proteoglycan known to be involved in keratinocyte survival and differentiation. Aging affects the dermal epidermal junction resulting in decreased contact with keratinocytes, thus impacting epidermal renewal and homeostasis. We found that perlecan expression decreased during chronological skin aging. Our in vitro studies revealed reduced perlecan transcript levels in aged keratinocytes. The production of in vitro skin models revealed that aged keratinocytes formed a thin and poorly organized epidermis. Supplementing these models with purified perlecan reversed the phenomenon allowing restoration of a well-differentiated multi-layered epithelium. Perlecan down-regulation in cultured keratinocytes caused depletion of the cell population that expressed keratin 15. This phenomenon depended on the perlecan heparan sulphate moieties, which suggested the involvement of a growth factor. Finally, we found defects in keratin 15 expression in the epidermis of aging skin. This study highlighted a new role for perlecan in maintaining the self-renewal capacity of basal keratinocytes.

Squamous cell carcinoma (SCC) is the second commonest type of skin cancer, and SCCs make up about 90% of head and neck cancers (HNSCCs). HNSCCs harbor two frequent molecular alterations, namely, gain-of-function alterations of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and loss-of-function mutations of tumor protein p53 (TP53). However, it remains poorly understood whether HNSCCs harboring different genetic alterations exhibit differential immune tumor microenvironments (TME). It also remains unknown whether PIK3CA hyperactivation and TP53 deletion can lead to SCC development spontaneously. Here, we analyzed the Cancer Genome Atlas (TCGA) datasets of HNSCCs and found that patients with both PIK3CA and TP53 alterations exhibited worse survival, significantly lower CD8 tumor infiltrating lymphocytes (TILs) and higher M0 macrophages than other controls. To better model human tumorigenesis, we deleted TP53 and constitutively activated PIK3CA in mouse keratin-15-expressing stem cells, which leads to the spontaneous development of multilineage tumors including SCCs, termed Keratin-15-p53-PIK3CA (KPPA) tumors. KPPA tumors were heavily infiltrated with myeloid-derived suppressor cells (MDSCs), with a drastically increased ratio of polymorphonuclear-MDSC (PMN-MDSC) versus monocytic-MDSC (M-MDSC). CD8 TILs expressed more PD-1 and reduced their polyfunctionality. Overall, we established a genetic model to mimic human HNSCC pathogenesis, manifested with an immunosuppressive TME, which may help further elucidate immune evasion mechanisms and develop more effective immunotherapies for HNSCCs.

Keratin 15 (KRT15) overexpression links with tumor initiation, metastasis, and poor survival in several solid carcinomas. While its clinical relevance is scarcely reported in endometrial cancer (EC). Therefore, the current study aimed to investigate the abnormal expression of KRT15 and its correlation with clinical characteristics, survival in EC patients. Totally, 135 surgical EC patients were enrolled. KRT15 protein expression in formalin-fixed and paraffin-embedded tumor and adjuvant tissues was detected by immunohistochemical staining; meanwhile, KRT15 mRNA expression in fresh-frozen tumor and adjacent tissues was detected by reverse transcription-quantitative polymerase chain reaction. KRT15 protein and mRNA expressions were higher in tumor tissue compared with adjacent tissue (both P < .001). Elevated KRT15 protein expression was correlated with the occurrence of lymphovascular invasion (P = .010) and more advanced International Federation of Gynecology and Obstetrics stage (P = .018); meanwhile, elevated KRT15 mRNA expression was linked with more advanced International Federation of Gynecology and Obstetrics stage (P = .038) and marginally associated with the occurrence of stromal cervical invasion (P = .052). Besides, KRT15 protein and mRNA expressions were not correlated with other clinical features (all P > .05). KRT15 protein high was marginally correlated with poor accumulating disease-free survival (DFS) (P = .091) and overall survival (OS) (P = .059); meanwhile, the correlation of KRT15 mRNA expression with accumulating DFS (P = .212) and OS (P = .092) was even weaker. However, multivariate Cox's regressions showed that tumor KRT15 protein (high vs low) was independently correlated with poor DFS (P = .045) and OS (P = .043). KRT15 is abnormally increased in EC tissue, meanwhile, its upregulation links to the occurrence of lymphovascular invasion, stromal cervical invasion, and poor prognosis in EC patients.

Keratin 15 (K15) is a type I keratin that is used as a marker of stem cells. Its expression is restricted to the basal layer of stratified epithelia, and the bulge in hair follicles. However, in certain clinical situations including oral lichen planus, K15 is induced in suprabasal layers, which is inconsistent with the role of a stem cell marker. This study provides insights into the mechanisms of K15 expression in the basal and differentiating keratinocytes.

Keratin 15 (K15), a type I keratin, which pairs with K5 in epidermis, has been used extensively as a biomarker for stem cells. Two commercial antibodies, LHK15, a mouse monoclonal and EPR1614Y, a rabbit monoclonal, have been widely employed to study K15 expression. Here we report differential reactivity of these antibodies on epithelial cells and tissue sections. Although the two antibodies specifically recognised K15 on western blot, they reacted differently on skin sections and cell lines. LHK15 reacted in patches, whereas EPR1614Y reacted homogenously with the basal keratinocytes in skin sections. In cultured cells, LHK15 did not react with K15 deficient NEB-1, KEB-11, MCF-7 and SW13 cells expressing only exogenous K8 and K18 but reacted when these cells were transduced with K15. On the other hand, EPR1614Y reacted with these cells even though they were devoid of K15. Taken together these results suggest that EPR1614Y recognises a conformational epitope on keratin filaments which can be reconstituted by other keratins as well as by K15. In conclusion, this report highlights that all commercially available antibodies may not be equally specific in identifying the K15 positive stem cell.

The first point of our body's contact with tactile stimuli (innocuous and noxious) is the epidermis, the outermost layer of skin that is largely composed of keratinocytes. Here, we sought to define the role that keratinocytes play in touch sensation in vivo and ex vivo. We show that optogenetic inhibition of keratinocytes decreases behavioral and cellular mechanosensitivity. These processes are inherently mediated by ATP signaling, as demonstrated by complementary cutaneous ATP release and degradation experiments. Specific deletion of P2X4 receptors in sensory neurons markedly decreases behavioral and primary afferent mechanical sensitivity, thus positioning keratinocyte-released ATP to sensory neuron P2X4 signaling as a critical component of baseline mammalian tactile sensation. These experiments lay a vital foundation for subsequent studies into the dysfunctional signaling that occurs in cutaneous pain and itch disorders, and ultimately, the development of novel topical therapeutics for these conditions.

The large Forkhead (Fox) transcription factor family has essential roles in development, and mutations cause a wide range of ocular and nonocular disease. One member, Foxc2 is expressed in neural crest (NC)-derived periocular mesenchymal cells of the developing murine eye; however, its precise role in the development, establishment, and maintenance of the ocular surface has yet to be investigated.

Sox13, a group D member of the Sry-related high-mobility group box (Sox) transcription factor family, is expressed in various tissues including the hair follicle. However, its spatiotemporal expression patterns in the hair follicle and its role in hair development remain to be elucidated. To address these questions, we generated Sox13-LacZ-knock-in mice (Sox13LacZ/+), in which the LacZ reporter gene was inserted in-frame into exon 2, which contains the translation initiation codon. X-gal staining in Sox13LacZ/+ embryos revealed that Sox13 is initially expressed in the epithelial portion of the placode, and subsequently in the hair germ and the hair peg during early hair follicle development. In postnatal catagen and anagen, Sox13 was detected in the epithelial sheath, whereas in telogen, Sox13 was localized in the bulge region, where hair follicle stem cells reside. Immunohistochemistry with an anti-β-galactosidase antibody and anti-hair keratin antibodies that specifically mark the different layers of the hair follicle revealed that Sox13 was predominantly expressed in the outer root sheath in anagen. However, the integumentary structures of Sox13LacZ/LacZ mice were grossly and histologically indistinguishable from those of wild type mice. These results suggest that although Sox13 is dispensable for epidermal and adnexal development, Sox13 is a useful marker for early hair follicle development.

β1 integrin regulates multiple epithelial cell functions by connecting cells with the extracellular matrix (ECM). While β1 integrin-mediated signaling in murine epithelial stem cells is well-studied, its role in human adult epithelial progenitor cells (ePCs) in situ remains to be defined. Using microdissected, organ-cultured human scalp hair follicles (HFs) as a clinically relevant model for studying human ePCs within their natural topobiological habitat, β1 integrin-mediated signaling in ePC biology was explored by β1 integrin siRNA silencing, specific β1 integrin-binding antibodies and pharmacological inhibition of integrin-linked kinase (ILK), a key component of the integrin-induced signaling cascade. β1 integrin knock down reduced keratin 15 (K15) expression as well as the proliferation of outer root sheath keratinocytes (ORSKs). Embedding of HF epithelium into an ECM rich in β1 integrin ligands that mimic the HF mesenchyme significantly enhanced proliferation and migration of ORSKs, while K15 and CD200 gene and protein expression were inhibited. Employing ECM-embedded β1 integrin-activating or -inhibiting antibodies allowed to identify functionally distinct human ePC subpopulations in different compartments of the HF epithelium. The β1 integrin-inhibitory antibody reduced β1 integrin expression in situ and selectively enhanced proliferation of bulge ePCs, while the β1 integrin-stimulating antibody decreased hair matrix keratinocyte apoptosis and enhanced transferrin receptor (CD71) immunoreactivity, a marker of transit amplifying cells, but did not affect bulge ePC proliferation. That the putative ILK inhibitor QLT0267 significantly reduced ORSK migration and proliferation and induced massive ORSK apoptosis suggests a key role for ILK in mediating the ß1 integrin effects. Taken together, these findings demonstrate that ePCs in human HFs require β1 integrin-mediated signaling for survival, adhesion, and migration, and that different human HF ePC subpopulations differ in their response to β1 integrin signaling. These insights may be exploited for cell-based regenerative medicine strategies that employ human HF-derived ePCs.

Basal cell carcinomas (BCCs) are hedgehog-driven tumors that resemble follicular and interfollicular epidermal basal keratinocytes and hence long have been thought to arise from these cells. However, the actual cell of origin is unknown. Using cell fate tracking of X-ray induced BCCs in Ptch1(+/-) mice, we found their essentially exclusive origin to be keratin 15-expressing stem cells of the follicular bulge. However, conditional loss of p53 not only enhanced BCC carcinogenesis from the bulge but also produced BCCs from the interfollicular epidermis, at least in part by enhancing Smo expression. This latter finding is consistent with the lack of visible tumors on ears and tail, sites lacking Smo expression, in Ptch1(+/-) mice.

Engineered skin substitutes (ESS), prepared using primary human fibroblasts and keratinocytes with a biopolymer scaffold, were shown to provide stable closure of excised burns, but relatively little is known about innervation of ESS after grafting. This study investigated innervation of ESS and, specifically, whether Merkel cells are present in healed grafts. Merkel cells are specialized neuroendocrine cells required for fine touch sensation in skin. We discovered cells positive for keratin 20 (KRT20), a general marker for Merkel cells, in the basal epidermis of ESS after transplantation to mice, suggesting the presence of Merkel cells. Cells expressing KRT20 were not observed in ESS in vitro. However, widely separated KRT20-positive cells were observed in basal epidermis of ESS by 2 weeks after grafting. By 4 weeks, these cells increased in number and expressed keratins 18 and 19, additional Merkel cells markers. Putative Merkel cell numbers increased further between weeks 6 and 14; their densities varied widely and no specific pattern of organization was observed, similar to Merkel cell localization in human skin. KRT20-positive cells co-expressed epidermal markers E-cadherin and keratin 15, suggesting derivation from the epidermal lineage, and neuroendocrine markers synaptophysin and chromogranin A, consistent with their identification as Merkel cells. By 4 weeks after grafting, some Merkel cells in engineered skin were associated with immature afferents expressing neurofilament-medium. By 8 weeks, Merkel cells were complexed with more mature neurons expressing neurofilament-heavy. Positive staining for human leukocyte antigen demonstrated that the Merkel cells in ESS were derived from grafted human cells. The results identify, for the first time, Merkel cell-neurite complexes in engineered skin in vivo. This suggests that fine touch sensation may be restored in ESS after grafting, although this must be confirmed with future functional studies.

The p53 homolog p63 is essential for development, yet its role in cancer is not clear. We discovered that p63 deficiency evokes the tumor-suppressive mechanism of cellular senescence, causing a striking absence of stratified epithelia such as the skin. Here we identify the predominant p63 isoform, ΔNp63α, as a protein that bypasses oncogene-induced senescence to drive tumorigenesis in vivo. Interestingly, bypass of senescence promotes stem-like proliferation and maintains survival of the keratin 15-positive stem cell population. Furthermore, we identify the chromatin-remodeling protein Lsh as a new target of ΔNp63α that is an essential mediator of senescence bypass. These findings indicate that ΔNp63α is an oncogene that cooperates with Ras to promote tumor-initiating stem-like proliferation and suggest that Lsh-mediated chromatin-remodeling events are critical to this process.

Previous studies have shown that Meis1 plays an important role in blood development and vascular homeostasis, and can induce blood cancers, such as leukemia. However, its role in epithelia remains largely unknown. Here, we uncover two roles for Meis1 in the epidermis: as a critical regulator of epidermal homeostasis in normal tissues and as a proto-oncogenic factor in neoplastic tissues. In normal epidermis, we show that Meis1 is predominantly expressed in the bulge region of the hair follicles where multipotent adult stem cells reside, and that the number of these stem cells is reduced when Meis1 is deleted in the epidermal tissue of mice. Mice with epidermal deletion of Meis1 developed significantly fewer DMBA/TPA-induced benign and malignant tumors compared with wild-type mice, suggesting that Meis1 plays a role in both tumor development and malignant progression. This is consistent with the observation that Meis1 expression increases as tumors progress from benign papillomas to malignant carcinomas. Interestingly, we found that Meis1 localization was altered to neoplasia development. Instead of being localized to the stem cell region, Meis1 is localized to more differentiated cells in tumor tissues. These findings suggest that, during the transformation from normal to neoplastic tissues, a functional switch occurs in Meis1.

We have investigated how Wnt and vitamin D receptor signals regulate epidermal differentiation. Many epidermal genes induced by beta-catenin, including the stem cell marker keratin 15, contain vitamin D response elements (VDREs) and several are induced independently of TCF/Lef. The VDR is required for beta-catenin induced hair follicle formation in adult epidermis, and the vitamin D analog EB1089 synergizes with beta-catenin to stimulate hair differentiation. Human trichofolliculomas (hair follicle tumours) are characterized by high nuclear beta-catenin and VDR, whereas infiltrative basal cell carcinomas (BCCs) have high beta-catenin and low VDR levels. In mice, EB1089 prevents beta-catenin induced trichofolliculomas, while in the absence of VDR beta-catenin induces tumours resembling BCCs. We conclude that VDR is a TCF/Lef-independent transcriptional effector of the Wnt pathway and that vitamin D analogues have therapeutic potential in tumors with inappropriate activation of Wnt signalling.

A specialized population of cells residing in the hair follicle is quiescent but shows pluripotency for differentiating into epithelial-mesenchymal lineage cells. Therefore, such cells are hoped to be useful as implantable donor cells for regenerative therapy. Recently, it was reported that intracellular delivery of TAT-VHL peptide induces neuronal differentiation of skin-derived precursors. In the present study, we successfully isolated multipotent stem cells derived from the epidermis of elderly humans, characterized these cells as being capable of sphere formation and strong expression of nestin, fibronectin, and CD34 but not of keratin 15, and identified the niche of these cells as being the outer root sheath of the hair follicles. In addition, we showed that TAT-VHL peptide induced their neuronal differentiation in vitro, and confirmed by fluorescence immunohistochemistry the neuronal differentiation of such peptide-treated cells implanted into rodent brains. These multipotent nestin-expressing stem cells derived from human epidermis are easily accessible and should be useful as donor cells for neuronal regenerative cell therapy.

Two principal stem cell pools orchestrate the rapid cell turnover in the intestinal epithelium. Rapidly cycling Lgr5+ stem cells are intercalated between the Paneth cells at the crypt base (CBCs) and injury-resistant reserve stem cells reside above the crypt base. The intermediate filament Keratin 15 (Krt15) marks either stem cells or long-lived progenitor cells that contribute to tissue repair in the hair follicle or the esophageal epithelium. Herein, we demonstrate that Krt15 labels long-lived and multipotent cells in the small intestinal crypt by lineage tracing. Krt15+ crypt cells display self-renewal potential in vivo and in 3D organoid cultures. Krt15+ crypt cells are resistant to high-dose radiation and contribute to epithelial regeneration following injury. Notably, loss of the tumor suppressor Apc in Krt15+ cells leads to adenoma and adenocarcinoma formation. These results indicate that Krt15 marks long-lived, multipotent, and injury-resistant crypt cells that may function as a cell of origin in intestinal cancer.

Hair-follicle-associated pluripotent (HAP) stem cells reside in the upper part of the bulge area of the the hair follicle. HAP stem cells are nestin-positive and keratin 15-negative and have the capacity to differentiate into various types of cells in vitro. HAP stem cells are also involved in nerve and spinal cord regeneration in mouse models. Recently, it was shown that the DNA-damage response in non-HAP hair follicle stem cells induces proteolysis of type-XVII collagen (COL17A1/BP180), which is involved in hair-follicle stem-cell maintenance. COL17A1 proteolysis stimulated hair-follicle stem-cell aging, characterized by the loss of stemness signatures and hair-follicle miniaturization associated with androgenic alopecia. In the present study, we demonstrate that HAP stem cells co-express nestin and COL17A1 in vitro and in vivo. The expression of HAP stem cell markers (nestin and SSEA1) increased after HAP stem-cell colonies were formed, then decreased after differentiation to epidermal keratinocytes. In contrast COL17A1 increased after differentiation to epidermal keratinocytes. These results suggest that COL17A1 is important in differentiation of HAP stem cells.

The esophagus is protected from the hostile environment by a stratified epithelium, which renews rapidly. Homeostasis of this epithelium is ensured by a rare population of stem cells in the basal layer: Keratin 15+ (Krt15+) cells. However, little is known about the molecular mechanisms regulating their distinct features, namely self-renewal, potency and epithelial regeneration. Achaete-scute family BHLH transcription factor 2 (ASCL2) is strongly upregulated in Krt15+ stem cells and is known to contribute to stem cell maintenance in other tissues. Herein, we investigated the role of ASCL2 in maintaining homeostasis under normal and stress conditions in the esophageal epithelium. ASCL2 overexpression severely dysregulated cell differentiation and cell fate. Proliferation was also reduced due potentially to a blockage in the G1 phase of the cell cycle or an induction of quiescence. Mass spectrometry analysis confirmed alterations in several proteins associated with differentiation and the cell cycle. In addition, overexpression of ASCL2 enhanced resistance to radiation and chemotherapeutic drugs. Overall, these results denote the role of ASCL2 as a key regulator of the proliferation-differentiation equilibrium in the esophageal epithelium.