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The vomeronasal organ (VNO) is a part of the accessory olfactory system, which detects pheromones and chemical factors that trigger a spectrum of sexual and social behaviors. The vomeronasal epithelium (VNE) shares several features with the epithelium of the main olfactory epithelium (MOE). However, it is a distinct neuroepithelium populated by chemosensory neurons that differ from the olfactory sensory neurons in cellular structure, receptor expression, and connectivity. The vomeronasal organ of rodents comprises a sensory epithelium (SE) and a thin non-sensory epithelium (NSE) that morphologically resembles the respiratory epithelium. Sox2-positive cells have been previously identified as the stem cell population that gives rise to neuronal progenitors in MOE and VNE. In addition, the MOE also comprises p63 positive horizontal basal cells, a second pool of quiescent stem cells that become active in response to injury. Immunolabeling against the transcription factor p63, Keratin-5 (Krt5), Krt14, NrCAM, and Krt5Cre tracing experiments highlighted the existence of horizontal basal cells distributed along the basal lamina of SE of the VNO. Single cell sequencing and genetic lineage tracing suggest that the vomeronasal horizontal basal cells arise from basal progenitors at the boundary between the SE and NSE proximal to the marginal zones. Moreover, our experiments revealed that the NSE of rodents is, like the respiratory epithelium, a stratified epithelium where the p63/Krt5+ basal progenitor cells self-replicate and give rise to the apical columnar cells facing the lumen of the VNO., (© 2024 Wiley Periodicals LLC.)

Vertebrate organs are arranged in a stereotypic, species-specific position along the animal body plan. Substantial morphological variation exists between related species, especially so in the vastly diversified teleost clade. It is still unclear how tissues, organs and systems can accommodate such diverse scaffolds. Here, we use the distinctive arrangement of neuromasts in the posterior lateral line (pLL) system of medaka fish to address the tissue-interactions defining a pattern. We show that patterning in this peripheral nervous system is established by autonomous organ precursors independent of neuronal wiring. In addition, we target the keratin 15 gene to generate stuck-in-the-midline (siml) mutants, which display epithelial lesions and a disrupted pLL patterning. By using siml/wt chimeras, we determine that the aberrant siml pLL pattern depends on the mutant epithelium, since a wild type epithelium can rescue the siml phenotype. Inducing epithelial lesions by 2-photon laser ablation during pLL morphogenesis phenocopies siml genetic mutants and reveals that epithelial integrity defines the final position of the embryonic pLL neuromasts. Our results using the medaka pLL disentangle intrinsic from extrinsic properties during the establishment of a sensory system. We speculate that intrinsic programs guarantee proper organ morphogenesis, while instructive interactions from surrounding tissues facilitates the accommodation of sensory organs to the diverse body plans found among teleosts., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Gene knockout of the master regulator of mitochondrial fission, Drp1, prevents neoplastic transformation. Also, mitochondrial fission and its opposing process of mitochondrial fusion are emerging as crucial regulators of stemness. Intriguingly, stem/progenitor cells maintaining repressed mitochondrial fission are primed for self-renewal and proliferation. Using our newly derived carcinogen transformed human cell model, we demonstrate that fine-tuned Drp1 repression primes a slow cycling 'stem/progenitor-like state', which is characterized by small networks of fused mitochondria and a gene-expression profile with elevated functional stem/progenitor markers (Krt15, Sox2 etc) and their regulators (Cyclin E). Fine tuning Drp1 protein by reducing its activating phosphorylation sustains the neoplastic stem/progenitor cell markers. Whereas, fine-tuned reduction of Drp1 protein maintains the characteristic mitochondrial shape and gene-expression of the primed 'stem/progenitor-like state' to accelerate neoplastic transformation, and more complete reduction of Drp1 protein prevents it. Therefore, our data highlights a 'goldilocks' level of Drp1 repression supporting stem/progenitor state dependent neoplastic transformation., Competing Interests: BS, DP, MB, AH, SG, SL, GS, AG, AM, KM No competing interests declared, (© 2021, Spurlock et al.)

The upper gastrointestinal tract, consisting of the esophagus, stomach, and duodenum, controls food transport, digestion, nutrient uptake, and hormone production. By single-cell analysis of healthy epithelia of these human organs, we molecularly define their distinct cell types. We identify a quiescent COL17A1 high KRT15 high stem/progenitor cell population in the most basal cell layer of the esophagus and detect substantial gene expression differences between identical cell types of the human and mouse stomach. Selective expression of BEST4, CFTR, guanylin, and uroguanylin identifies a rare duodenal cell type, referred to as BCHE cell, which likely mediates high-volume fluid secretion because of continual activation of the CFTR channel by guanylin/uroguanylin-mediated autocrine signaling. Serotonin-producing enterochromaffin cells in the antral stomach significantly differ in gene expression from duodenal enterochromaffin cells. We, furthermore, discover that the histamine-producing enterochromaffin-like cells in the oxyntic stomach express the luteinizing hormone, yet another member of the enteroendocrine hormone family., Competing Interests: Declaration of interests H.C. is an inventor on several patents related to organoid technology; his full disclosure is given at https://www.uu.nl/staff/JCClevers/., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Epidermolysis bullosa simplex (EBS) is a rare skin disease usually inherited in an autosomal dominant pattern. EBS is resulting from mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes encoding the keratins 5 and 14 proteins expressed in the keratinocytes of the basal layer of the epidermis. To date, seven pathogenic mutations have been reported to be responsible for EBS in the Canadian population from the province of Quebec: p.Pro25Leu, p.Leu150Pro, p.Met327Thr and p.Arg559X in KRT5; p.Arg125Ser, p.Ile377Thr and p.Ile412Phe in KRT14. Here, we present a novel French-Canadian patient diagnosed with EBS confined to the soles but presenting a severe complication form including blisters, hyperkeratosis, skin erosions and toenail abnormalities. Mutation screening was performed by direct sequencing of the entire coding regions of KRT5 and KRT14 genes and revealed the previously reported missense heterozygous mutation c. 1130T > C in KRT14 (p.Ile377Thr). Furthermore, this patient is carrying a second mutation in KRT5, c.413G > A (p.Gly138Glu), which has been linked to an increased risk of basal cell carcinoma in the literature. We suspect an impact of the p.Gly138Glu variant on the EBS phenotype severity of the studied patient. The pathogenicity and consequences of both genetic variations were simulated by in silico tools., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aim: To investigate the expression and prognostic value of KRT 15 in esophageal carcinoma. Materials & methods: The expression levels of KRT 15 were measured in 128 cases of esophageal carcinoma and matched adjacent normal tissues by immunohistochemistry and Western blot assays. Results & conclusion: Western blot analysis shown the expression levels of KRT 15 in esophageal carcinoma were significantly higher compared with those in matched adjacent normal tissues (p < 0.001). immunohistochemistry result shown the high-expression rate of KRT 15 in esophageal carcinoma were 56.3%, which was significantly higher than those in normal tissues (35.9%; p = 0.002). KRT 15 high-expression correlated with T stage, lymph node metastasis, tumor node metastasis stage and prognosis (p < 0.05). These data indicate KRT 15 as a prognostic biomarker is highly expressed in esophageal carcinoma.

Keratin-15 (KRT15) is a type I keratin lacking a defined type II partner and plays a key role in maintaining cytoplasmic stability. Recently, studies have reported that KRT15 was correlated with tumor formation and progression. However, the clinical significance of KRT15 in colorectal cancer is unclear. In this study, we aimed to investigate the expression of KRT15 and its clinical significance in colorectal cancer. KRT15 expression was examined in 98 cases of colorectal cancer and matched adjacent normal tissues by quantificational real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), respectively. Then, the clinical significance of KRT15 expression was evaluated in colorectal cancer. QRT-PCR results revealed that the mRNA levels of KRT15 in colorectal cancer tissues were significantly higher compared with those in normal tissues (p<0.0001). The rates of KRT15 high-expression in colorectal cancer and normal tissues were 57.1% and 8.9%, respectively, and the difference was statistically significant (p<0.0001). KRT15 high-expression correlated with differentiation, T stage, lymph node metastasis and clinical stage in colorectal cancer (p<0.05). Meanwhile, KRT15 overexpression predicted poor prognosis and could be used as an independent prognostic factor. These data indicate KRT15 is highly expressed in colorectal cancer and may serve as a prognostic biomarker.

Aim: According to the current data, a major factor for phenotypic variation of complex traits and disease susceptibility is the cis -acting effects of noncoding variants on gene expression. Our purpose was to evaluate the association between colorectal cancer (CRC) and six single nucleotide polymorphisms identified using our original bioinformatics approach as regulatory and putatively related to CRC. Materials: One hundred and sixty CRC patients and 185 healthy controls have been genotyped for rs590352, rs2072580, rs78317230, rs3829202, rs11542583 and rs4796672. Results: Genotypes and alleles distributions of rs590352 of ATXN7L3B gene were significantly different between the male CRC subjects and controls. Significant correlation of genotype with CRC is observable for women only for the rs4796672 of KRT15 gene. Analysis of haplotypes reveals that rs2072580 of the ISCU and SART3 genes can be also associated with CRC. Conclusion: We have identified three SNPs associated with CRC risk and demonstrated a gender specificity of rs590352 and rs4796672.

Congenital urinary tract obstruction (UTO) is the leading cause of chronic kidney disease in children; however, current management strategies do not safeguard against progression to end-stage renal disease, highlighting the need for interventions to limit or reverse obstructive nephropathy. Experimental UTO triggers renal urothelial remodeling that culminates in the redistribution of basal keratin 5-positive (Krt5 + ) renal urothelial cells (RUCs) and the generation of uroplakin-positive (Upk) + RUCs that synthesize a protective apical urothelial plaque. The cellular source of Upk + RUCs is currently unknown, limiting the development of strategies to promote renal urothelial remodeling as a therapeutic approach. In the present study, we traced the origins of adult Upk + RUCs during normal development and in response to UTO. Fate mapping analysis demonstrated that adult Upk + RUCs derive from embryonic and neonatal Krt5 + RUCs, whereas Krt5 + RUCs lose this progenitor capacity and become lineage restricted by postnatal day 14 . However, in response to UTO, postnatal day 14 -labeled adult Krt5 + RUCs break their lineage restriction and robustly differentiate into Upk + RUCs. Thus, Krt5 + RUCs drive renal urothelial formation during normal ontogeny and after UTO by differentiating into Upk + RUCs in a temporally restricted manner.

The effective prevention of tumor initiation, especially for potentially inoperable tumors, will be beneficial to obtain an overall higher quality of our health and life. Hence, thorough understanding of the pathophysiological mechanisms of early tumor formation arising from identifiable cellular origins is required to develop efficient preventative and early treatment options for each tumor type. Here, using genetically engineered mouse models, we provide preclinical experimental evidence for a long-standing open question regarding the pathophysiological potential of a microenvironmental and physiological stressor in tumor development, gastric acid-mediated regional microscopic injury in foregut squamous epithelia. This study demonstrates the association of gastric acid stress with Cyclooxygenase-2-dependent tumor formation originating from tumor-competent Krt5 + /Krt15 + foregut basal progenitor cells. Our findings suggest that clinical management of microenvironmental stressor-mediated microscopic injury may be important in delaying tumor initiation from foregut basal progenitor cells expressing pre-existing tumorigenic mutation(s) and genetic alteration(s).

Aim: The present study aims to identify aberrantly methylated and differentially expressed genes (DEGs) in gastric cancer (GC) and explore their potential role in the carcinogenesis and development of GC., Methods: The original RNA-Seq, clinical information and Illumina Human Methylation 27 Chip data associated with GC were downloaded from The Cancer Genome Atlas (TCGA) database using the gdc-client tool. The DEGs and aberrantly methylated genes (AMGs) were screened with edgeR and limma package in R, respectively. The cut-off criteria for DEG identification were P < 0.05 and fold change (FC) >2.0, and for AMG identification were P < 0.05 and |t|>2.0. Genes which were both DEGs and AMGs were considered to be regulated by aberrant DNA methylation in GC. The common genes were used for further functional enrichment analysis in the categories of cellular component, molecular function, biological process and biological pathway., Results: In total 465 genes including 336 down-regulated genes with hyper-methylation (DGs-Hyper) and 129 up-regulated genes with hypo-methylation (UGs-Hypo) were identified. Cellular component analysis showed that these genes were mainly expressed in the cytoplasm and plasma membrane. Molecular function and biological process analysis indicated that the genes primarily participate in cell communication, signal transduction, cell growth/maintenance and function as transcription factors, receptor, cell adhesion molecules, and transmembrane receptor protein tyrosine kinases. Biological pathway analysis revealed that the genes are involved in some crucial pathways including epithelial-to-mesenchymal transition, IL3-mediated signaling, mTOR signaling, VEGF/VEGFR and c-Met signaling. KRT15, INHBA, MATN3, and AGT are significantly associated with the prognosis of GC patients., Conclusion: Our study identified several DEGs regulated by aberrant DNA methylation in GC. The mechanism of DNA methylation in the carcinogenesis and development of GC could be further explored in these genes, especially KRT15, INHBA, MATN3, and AGT., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The emerging variety of signalling roles for ROS in eukaryotic cells and tissues is currently a matter of intense research. Here we make use of ex vivo cultured single human hair follicles as an experimental model to demonstrate that a transient production of non-lethal endogenous ROS levels in these mini-organs promotes efficiently the entry into the growth phase (anagen). The stimulatory process implicates the specific activation of the hair follicle stem cell niche, encompassing the induction of stem cell differentiation markers (Ck15), overall cell proliferation and sustained growth of the tissue associated with expression of gen targets (Ccnd1) concomitant with the inhibition of Wnt signaling antagonists and repressors (Dkk1, Gsk3β) of Wnt signaling. As a whole, this observation indicates that, once activated, ROS signalling is an intrinsic mechanism regulating the hair follicle stem cell niche independently of any external signal.

Milk production is highly dependent on the extensive development of the mammary epithelium, which occurs during puberty. It is therefore essential to distinguish the epithelial cells committed to development from the related epithelial hierarchy. Using cell phenotyping and sorting, we highlighted four cell sub-populations within the bovine mammary gland at puberty. The CD49 f high CD24 neg cells expressing CD10, KRT14, vimentin and PROCR corresponded to cells committed to the basal lineage. The CD49 f low sub-population contained two cell subsets (CD49 f low CD24 neg and CD49 f low CD24 pos ). Both subsets expressed hormone receptors including ER, PR and PRLR, as well as ALDH1 activity but only the CD49 f low CD24 pos subset expressed ELF5. These data indicated that the CD49 f low sub-population is mainly composed of cells displaying a luminal phenotype and that this population comprises two luminal cell subsets, namely the CD24 neg and CD24 pos cells, likely committed to ductal and alveolar lineage, respectively. The putative mammary stem cell (MaSC) fraction was recovered in the CD49 f high CD24 pos sub-population which were shown to form mammospheres in vitro. These cells differentially expressed CD10, KRT14 and KRT7, suggesting the existence of several putative MaSC sub-fractions. In-depth characterization of these epithelial sub-populations provides new insights into the bovine mammary epithelial cell lineage and suggests a common developmental lineage in mammals.

Although trastuzumab-induced cardiotoxicity is an important determinant to limit the use of this drug, the molecular mechanism of risk for this toxicity is not well understood. To identify genetic variants determining the risk of trastuzumab-induced cardiotoxicity, we carried out whole exome sequencing of germline DNA samples from 9 patients with trastuzumab-induced cardiotoxicity, and conducted a case-control association study of 2258 genetic variants between 9 cases (with trastuzumab-induced cardiotoxicity) and general Japanese population controls registered in the Human Genetic Variation Database (HGVD). The top variant which showed the lowest P-value in the screening study was rs139503277 in PHD Finger Protein 3 (P min = .00012, odds ratio [OR] = 51.23). To further validate the result of screening study, we carried out a replication study of 10 variants showing P min < .001 in the screening study using 234 independent patients treated with trastuzumab, including 10 cases and 224 controls (without trastuzumab-induced cardiotoxicity). In the replication study, we observed that three variants had an effect in the same direction as in the screening study (rs78272919 in exon 2 of Keratin 15, rs5762940 in exon 2 of zinc and ring finger 3, and rs139944387 in exon 44 of Eyes shut homologs [EYS]). A combined result of the screening and the replication studies suggested an association of a locus on chromosome 6q12 with trastuzumab-induced cardiotoxicity (rs139944387 in EYS, combined P min = .00056, OR = 13.73). This finding provides new insights into personalized trastuzumab therapy for patients with human epidermal growth factor receptor 2 (HER2)-positive cancer., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The middle ear epithelium is derived from neural crest and endoderm, which line distinct regions of the middle ear cavity. Here, we investigate the distribution of putative stem cell markers in the middle ear, combined with an analysis of the location of label-retaining cells (LRCs) to create a map of the middle ear mucosa. We show that proliferating cells and LRCs were associated with specific regions of the ear epithelium, concentrated in the hypotympanum at the base of the auditory bulla and around the ear drum. Sox2 was widely expressed in the endodermally derived ciliated pseudostratified epithelium of the hypotympanum. This part of the middle ear showed high levels of Wnt activity, as indicated by the expression of Axin2, a readout of Wnt signalling. Keratin 5 showed a more restricted expression within the basal cells of this region, with very little overlap between the Sox2- and keratin 5-positive epithelium, indicating that these genes mark distinct populations. Little expression of Sox2 or keratin 5 was observed in the neural crest-derived middle ear epithelium that lined the promontory, except in cases of otitis media when this epithelium underwent hyperplasia. This study lays the foundation for furthering our understanding of homeostasis and repair in the middle ear., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

miR-184 is a highly evolutionary conserved microRNA (miRNA) from fly to human. The importance of miR-184 was underscored by the discovery that point mutations in miR-184 gene led to corneal/lens blinding disease. However, miR-184-related function in vivo remained unclear. Here, we report that the miR-184 knockout mouse model displayed increased p63 expression in line with epidermal hyperplasia, while forced expression of miR-184 by stem/progenitor cells enhanced the Notch pathway and induced epidermal hypoplasia. In line, miR-184 reduced clonogenicity and accelerated differentiation of human epidermal cells. We showed that by directly repressing cytokeratin 15 (K15) and FIH1, miR-184 induces Notch activation and epidermal differentiation. The disease-causing miR-184 C57U mutant failed to repress K15 and FIH1 and to induce Notch activation, suggesting a loss-of-function mechanism. Altogether, we propose that, by targeting K15 and FIH1, miR-184 regulates the transition from proliferation to early differentiation, while mis-expression or mutation in miR-184 results in impaired homeostasis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: This article concerns the identification of gene pairs or combinations of gene pairs associated with biological phenotype or clinical outcome, allowing for building predictive models that are not only robust to normalization but also easily validated and measured by qPCR techniques. However, given a small number of biological samples yet a large number of genes, this problem suffers from the difficulty of high computational complexity and imposes challenges to the accuracy of identification statistically., Results: In this paper, we propose a parsimonious model representation and develop efficient algorithms for identification. Particularly, we derive an equivalent model subject to a sum-to-zero constraint in penalized linear regression, where the correspondence between nonzero coefficients in these models is established. Most importantly, it reduces the model complexity of the traditional approach from the quadratic order to the linear order in the number of candidate genes, while overcoming the difficulty of model nonidentifiablity. Computationally, we develop an algorithm using the alternating direction method of multipliers (ADMM) to deal with the constraint. Numerically, we demonstrate that the proposed method outperforms the traditional method in terms of the statistical accuracy. Moreover, we demonstrate that our ADMM algorithm is more computationally efficient than a coordinate descent algorithm with a local search. Finally, we illustrate the proposed method on a prostate cancer dataset to identify gene pairs that are associated with pre-operative prostate-specific antigen., Conclusion: Our findings demonstrate the feasibility and utility of using gene pairs as biomarkers.

Background: Retinoic acid (RA), the active metabolite of vitamin A, has been demonstrated to be important for growth and branching morphogenesis of mammalian embryonic salivary gland epithelium. However, it is not known whether RA functions directly within epithelial cells or in associated tissues that influence morphogenesis of salivary epithelium. Moreover, downstream targets of RA regulation have not been identified., Results: Here, we show that canonical RA signaling occurs in multiple tissues of embryonic mouse salivary glands, including epithelium, associated parasympathetic ganglion neurons, and nonneuronal mesenchyme. By culturing epithelium explants in isolation from other tissues, we demonstrate that RA influences epithelium morphogenesis by direct action in that tissue. Moreover, we demonstrate that inhibition of RA signaling represses cell proliferation and expression of FGF10 signaling targets, and upregulates expression of basal epithelial keratins Krt5 and Krt14. Importantly, we show that the stem cell gene Kit is regulated inversely from Krt5/Krt14 by RA signaling., Conclusions: RA regulates Krt5 and Krt14 expression independently of stem cell character in developing salivary epithelium. RA, or chemical inhibitors of RA signaling, could potentially be used for modulating growth and differentiation of epithelial stem cells for the purpose of re-populating damaged glands or generating bioengineered organs. Developmental Dynamics 246:135-147, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Atopic dermatitis is increasing worldwide in correlation with air pollution. Various organic components of pollutants activate the transcription factor AhR (aryl hydrocarbon receptor). Through the use of AhR-CA mice, whose keratinocytes express constitutively active AhR and that develop atopic-dermatitis-like phenotypes, we identified Artn as a keratinocyte-specific AhR target gene whose product (the neurotrophic factor artemin) was responsible for epidermal hyper-innervation that led to hypersensitivity to pruritus. The activation of AhR via air pollutants induced expression of artemin, alloknesis, epidermal hyper-innervation and inflammation. AhR activation and ARTN expression were positively correlated in the epidermis of patients with atopic dermatitis. Thus, AhR in keratinocytes senses environmental stimuli and elicits an atopic-dermatitis pathology. We propose a mechanism of air-pollution-induced atopic dermatitis via activation of AhR.

Hair follicle stem cells (HFSCs) are considered one of the useful donor cell types for skin regenerative medicine owing to their robust proliferative capacity and multipotency. However, methods for easily and effectively obtaining HFSCs from a limited skin biopsy are still lacking. Here we report a novel approach for obtaining a subpopulation of HFSCs from a small skin sample from the rat tail, which uses the sebaceous glands (SGs) to capture the adjacent HFSCs. By means of organ culture, keratinocytes were expanded from the detached SGs, which also included adherent HFSCs from the hair follicle that could be passaged at the single-cell level. These SG-captured keratinocytes strongly expressed the basal layer markers K14, integrin α6, and p63; the bulge stem cell marker K15; and the upper isthmus stem cell marker Plet1. Furthermore, we reconstituted new epidermis, hair follicles, and SGs from the SG-captured keratinocytes using an easily operated, modified skin reconstitution assay based on silicone gel sheeting. This study suggests that the SGs could be an accessible capturer to harvest the adjacent HFSC subpopulation, particularly when the donor tissue is limited.

Background: Keratin 5 (K5) is a cytoskeletal tissue-specific protein expressed in the epithelial cells of skin and esophagus and ectopic K5 expression in lymphocytes has never been reported., Objective: Here we demonstrate an ectopic epidermal self-protein expression in B-1 B cell by fate mapping of K5-expressing cells., Methods: K5-Cre×CAG-CAT-loxP-EGFP double Tg (K5×GFP) mice that express enhanced GFP under the control of the K5 promoter were employed., Results: Unexpectedly, B220(+)GFP(+) cells were found in LN, spleen, peripheral blood and peritoneal cavity. These cells were IgM(+)IgD(low)CD23(-)CD43(+)CD19(+)CD93(-), indicating that they were B-1 B cells. The number of B220(+)GFP(+) cells was significantly larger in spleen than in the other tissues tested. Although GFP(+) B-1 cells did not express K5 in the periphery, Lin(-)CD93(+)B220(low-neg)CD19(+) B-1 B cell progenitors expressed GFP and B220(+)CD93(+) progenitor cells expressed K5 and MHC-class II in BM, indicating that GFP(+) B-1 cells transiently expressed K5 and the progenitor cells were potential APC. GFP(+) B-1 cells in the periphery continued expressing MHC class II and had exogenous antigen-presenting capacity comparable to non-follicular B cells. GFP(+) B-1 cells spontaneously secreted more IgM than GFP(-) B-1 cells in vitro., Conclusion: These results indicate that B-1 B cells transiently and partially express K5 in BM and are potent for both natural antibody production and antigen presentation., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Functional hair follicle (HF) stem cells (SCs) are crucial to maintain the constant recurring growth of hair. In mice and humans, SC subpopulations with different biomarker expression profiles have been identified in discrete anatomic compartments of the HF. The rare studies investigating canine HF SCs have shown similarities in biomarker expression profiles to that of mouse and human SCs. The aim of our study was to broaden the current repertoire of SC-associated markers and their expression patterns in the dog. We combined analyses on the expression levels of CD34, K15, Sox9, CD200, Nestin, LGR5 and LGR6 in canine skin using RT-qPCR, the corresponding proteins in dog skin lysates, and their expression patterns in canine HFs using immunohistochemistry. Using validated antibodies, we were able to define the location of CD34, Sox9, Keratin15, LGR5 and Nestin in canine HFs and confirm that all tested biomarkers are expressed in canine skin. Our results show similarities between the expression profile of canine, human and mouse HF SC markers. This repertoire of biomarkers will allow us to conduct functional studies and investigate alterations in the canine SC compartment of different diseases, like alopecia or skin cancer with the possibility to extend relevant findings to human patients., (© 2016 The Histochemical Society.)

LGR5 is a known marker of embryonic and adult stem cells in several tissues. In a mouse model, Lgr5+ cells have shown tumour-initiating properties, while in human cancers, such as basal cell carcinoma and colon cancer, LGR5 expression levels are increased: however, the effect of increased LGR5 expression is not fully understood. To study the effects of elevated LGR5 expression levels we generated a novel tetracycline-responsive, conditional transgenic mouse line expressing human LGR5, designated TRELGR5. In this transgenic line, LGR5 expression can be induced in any tissue depending on the expression pattern of the chosen transcriptional regulator. For the current study, we used transgenic mice with a tetracycline-regulated transcriptional transactivator linked to the bovine keratin 5 promoter (K5tTA) to drive expression of LGR5 in the epidermis. As expected, expression of human LGR5 was induced in the skin of double transgenic mice (K5tTA;TRELGR5). Inducing LGR5 expression during embryogenesis and early development resulted in macroscopically and microscopically detectable phenotypic changes, including kink tail, sparse fur coat and enlarged sebaceous glands. The fur and sebaceous gland phenotypes were reversible upon discontinued expression of transgenic LGR5, but this was not observed for the kink tail phenotype. There were no apparent phenotypic changes if LGR5 expression was induced at three weeks of age. The results demonstrate that increased expression of LGR5 during embryogenesis and the neonatal period alter skin development and homeostasis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In the murine hair follicle (HF), the transcription factors LHX2 and SOX9 are implicated in epithelial hair follicle stem cell (eHFSC) self-renewal and the maintenance of eHFSC niche characteristics. However, the exact expression patterns of LHX2 and SOX9 in the human HF are unclear. Therefore, we have quantitatively mapped the localisation of known human eHFSC markers keratin 15 (K15) and keratin 19 (K19) in the outer root sheath (ORS) of male occipital scalp anagen HFs and related this to the localisation of LHX2 and SOX9 protein expression. As expected, K15(+) and K19(+) cells represented two distinct progenitor cell populations in the bulge and in the proximal bulb ORS (pbORS). Interestingly, cell fluorescence for K19 was significantly stronger within the pbORS versus the bulge, and vice versa for K15, describing a hitherto unrecognised differential expression pattern. LHX2 and SOX9 expressing cells were distributed throughout the ORS, including the bulge, but were not restricted to it. SOX9 expression was most prominent in the ORS immediately below the human bulge, whereas LHX2(+) cells were similarly distributed between the sub-bulge and pbORS, that is compartments not enriched with quiescent eHFSCs. During catagen development, the intensity of LHX2 and SOX9 protein expression increased in the proximal HF epithelium. Double immunostaining showed that the majority of SOX9(+) cells in the human anagen HF epithelium did not co-express K15, K19 or LHX2. This expression profile suggests that LHX2 and SOX9 highlight distinct epithelial progenitor cell populations, in addition to K15(+) or K19(+) cells, that could play an important role in the maintenance of the human HF epithelium., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKβ, an essential component of the NF-κB pathway, under keratin 5 promoter (K5-Ikkβ). K5-Ikkβ mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkβ mice. The supernumerary incisors in K5-Ikkβ mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions., (© International & American Associations for Dental Research 2014.)

Background: Dermatan sulfate (DS) is a highly sulfated polysaccharide with a variety of biological functions in extracellular matrix organization and processes such as tumorigenesis and wound healing. A distinct feature of DS is the presence of iduronic acid, produced by the two enzymes, DS-epimerase 1 and 2, which are encoded by Dse and Dsel, respectively., Methods: We have previously shown that Dse knockout (KO) mice in a mixed C57BL/6-129/SvJ background have an altered collagen matrix structure in skin. In the current work we studied Dse KO mice in a pure NFR genetic background., Results: Dse KO embryos and newborns had kinked tails and histological staining revealed significantly thicker epidermal layers in Dse KO mice when compared with heterozygote (Het) or wild-type (WT) littermates. Immunochemical analysis of the epidermal layers in newborn pups showed increased expression of keratin 5 in the basal layer and keratin 1 in the spinous layer. In addition, we observed an abdominal wall defect with herniated intestines in 16% of the Dse KO embryos. Other, less frequent, developmental defects were exencephaly and spina bifida., Conclusion: We conclude that the combination of defective collagen structure in the dermis and imbalanced keratinocyte maturation could be responsible for the observed developmental defects in Dse KO mice. In addition, we propose that Dse KO mice could be used as a model in pathogenetic studies of human fetal abdominal wall defects., (© 2014 Wiley Periodicals, Inc.)

We report here senescent changes in the structure and organization of the mucociliary pseudostratified epithelium of the mouse trachea and main stem bronchi. We confirm previous reports of the gradual appearance of age-related, gland-like structures (ARGLS) in the submucosa, especially in the intercartilage regions and carina. Immunohistochemistry shows these structures contain ciliated and secretory cells and Krt5+ basal cells, but not the myoepithelial cells or ciliated ducts typical of normal submucosal glands. Data suggest they arise de novo by budding from the surface epithelium rather than by delayed growth of rudimentary or cryptic submucosal glands. In old mice the surface epithelium contains fewer cells per unit length than in young mice and the proportion of Krt5+, p63+ basal cells is reduced in both males and females. However, there appears to be no significant difference in the ability of basal stem cells isolated from individual young and old mice to form clonal tracheospheres in culture or in the ability of the epithelium to repair after damage by inhaled sulfur dioxide. Gene expression analysis by Affymetrix microarray and quantitative PCR, as well as immunohistochemistry and flow sorting studies, are consistent with low-grade chronic inflammation in the tracheas of old versus young mice and an increase in the number of immune cells. The significance of these changes for ARGL formation are not clear since several treatments that induce acute inflammation in young mice did not result in budding of the surface epithelium.

The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules have been defined which regulate ureteric bud initiation and drive the differentiation of ureteric mesenchyme into peristaltic smooth muscle. Less is known about mechanisms underlying the developmental patterning of the multilayered epithelium characterising the mature ureter. In skin, which also contains a multilayered epithelium, cytokeratin 15 (CK15), an acidic intermediate filament protein, marks cells whose progeny contribute to epidermal regeneration following wounding. Moreover, CK15+ precursor cells in skin can give rise to basal cell carcinomas. In the current study, using transcriptome microarrays of embryonic wild type mouse ureters, Krt15, coding for CK15, was detected. Quantitative polymerase chain reaction analyses confirmed the initial finding and demonstrated that Krt15 levels increased during the fetal period when the ureteric epithelium becomes multilayered. CK15 protein was undetectable in the ureteric bud, the rudiment from which the ureter grows. Nevertheless, later in fetal development, CK15 was immunodetected in a subset of basal urothelial cells in the ureteric stalk. Superficial epithelial cells, including those positive for the differentiation marker uroplakin III, were CK15-. Transformation-related protein 63 (P63) has been implicated in epithelial differentiation in murine fetal urinary bladders. In wild type fetal ureters, CK15+ cells were positive for P63, and p63 homozygous null mutant ureters lacked CK15+ cells. In these mutant ureters, sections of the urothelium were monolayered versus the uniform multilayering found in wild type littermates. Human urothelial cell carcinomas account for considerable morbidity and mortality. CK15 was upregulated in a subset of invasive ureteric and urinary bladder cancers. Thus, in ureter development, the absence of CK15 is associated with a structurally simplified urothelium whereas, postnatally, increased CK15 levels feature in malignant urothelial overgrowth. CK15 may be a novel marker for urinary tract epithelial precursor cells.

Slow cycling is a common feature shared among several stem cells (SCs) identified in adult tissues including hair follicle and cornea. Recently, existence of unipotent SCs in basal and lumenal layers of sweat gland (SG) has been described and label retaining cells (LRCs) have also been localized in SGs; however, whether these LRCs possess SCs characteristic has not been investigated further. Here, we used a H2BGFP LRCs system for in vivo detection of infrequently dividing cells. This system allowed us to specifically localize and isolate SCs with label-retention and myoepithelial characteristics restricted to the SG proximal acinar region. Using an alternative genetic approach, we demonstrated that SG LRCs expressed keratin 15 (K15) in the acinar region and lineage tracing determined that K15 labeled cells contributed long term to the SG structure but not to epidermal homeostasis. Surprisingly, wound healing experiments did not activate proximal acinar SG cells to participate in epidermal healing. Instead, predominantly non-LRCs in the SG duct actively divided, whereas the majority of SG LRCs remained quiescent. However, when we further challenged the system under more favorable isolated wound healing conditions, we were able to trigger normally quiescent acinar LRCs to trans-differentiate into the epidermis and adopt its long term fate. In addition, dissociated SG cells were able to regenerate SGs and, surprisingly, hair follicles demonstrating their in vivo plasticity. By determining the gene expression profile of isolated SG LRCs and non-LRCs in vivo, we identified several Bone Morphogenetic Protein (BMP) pathway genes to be up-regulated and confirmed a functional requirement for BMP receptor 1A (BMPR1A)-mediated signaling in SG formation. Our data highlight the existence of SG stem cells (SGSCs) and their primary importance in SG homeostasis. It also emphasizes SGSCs as an alternative source of cells in wound healing and their plasticity for regenerating different skin appendages.

Background: Although cutaneous stem cells have been implicated in skin tumourigenesis in humans, no studies have been conducted to elucidate the presence and the possible role of stem cells in hair follicle tumours in the dog., Hypothesis: Stem cell markers are expressed in canine epidermal and follicular tumours and can be used to better understand the biology and origin of these tumours., Animals and Methods: In the present study, normal skin sections and 44 follicular tumours were retrospectively investigated for the immunohistochemical expression of keratin 15 (K15) and nestin. In addition, 30 squamous cell carcinomas were evaluated for K15 expression., Results: In normal skin, K15 and nestin were expressed in the outer root sheath cells of the isthmic portion of the hair follicle (bulge region), and K15 expression was also scattered in the basal cell layer of the epidermis. Infundibular keratinizing acanthomas, pilomatricomas and squamous cell carcinomas were mostly negative for K15, trichoblastomas were moderately to strongly positive, tricholemmomas were either negative or strongly positive, and trichoepitheliomas had heterogeneous staining. Nestin expression was generally faint in all follicular tumours., Conclusions and Clinical Importance: Our results show that K15 can be a reliable marker for investigating the role of stem cells in hair follicle tumours of the dog, while nestin was judged to be a nonoptimal marker. Furthermore, our study suggests that hair follicle stem cells are present in the bulge region of hair follicles and could possibly play a role in tumourigenesis of canine tumours originating from this portion of the follicle, namely trichoblastomas, tricholemmomas and trichoepitheliomas. The loss of K15 expression in squamous cell carcinomas compared with normal skin suggests that this event could be important in the malignant transformation., (© 2013 The Authors. Veterinary Dermatology © 2013 ESVD and ACVD.)

Hair follicles facilitate the study of stem cell behavior because stem cells in progressive activation stages, ordered within the follicle architecture, are capable of cyclic regeneration. To study the gene network governing the homeostasis of hair bulge stem cells, we developed a Keratin 15-driven genetic model to directly perturb molecular signaling in the stem cells. We visualize the behavior of these modified stem cells, evaluating their hair-regenerating ability and profile their molecular expression. Bone morphogenetic protein (BMP)-inactivated stem cells exhibit molecular profiles resembling those of hair germs, yet still possess multipotentiality in vivo. These cells also exhibit up-regulation of Wnt7a, Wnt7b, and Wnt16 ligands and Frizzled (Fzd) 10 receptor. We demonstrate direct transcriptional modulation of the Wnt7a promoter. These results highlight a previously unknown intra-stem cell antagonistic competition, between BMP and Wnt signaling, to balance stem cell activity. Reduced BMP signaling and increased Wnt signaling tilts each stem cell toward a hair germ fate and, vice versa, based on a continuous scale dependent on the ratio of BMP/Wnt activity. This work reveals one more hierarchical layer regulating stem cell homeostasis beneath the stem cell-dermal papilla-based epithelial-mesenchymal interaction layer and the hair follicle-intradermal adipocyte-based tissue interaction layer. Although hierarchical layers are all based on BMP/Wnt signaling, the multilayered control ensures that all information is taken into consideration and allows hair stem cells to sum up the total activators/inhibitors involved in making the decision of activation.

Alopecia is known as a symptom of acute radiation, yet little is known concerning the mechanism of this phenomenon and the alteration of hair protein profiles. To examine this, 6-week-old male C57/BL6 mice were exposed to 6 Gy of X-ray irradiation, which caused acute alopecia. Their hair and skin were collected, and hair proteins were analyzed with liquid chromatography/electrospray-ionization mass spectrometry and immunohistochemistry. No change was observed in the composition of major hair keratins, such as Krt81, Krt83 and Krt86. However, cytokeratin Krt15 and CD34, which are known as hair follicle stem cell markers, were decreased in alopecic mice. Cytokeratin Krt5, which is known as a marker for basal and undifferentiated keratinocytes, was increased in the epidermis of alopecic mice. These findings suggest that radiation damages hair stem cells and the differentiation of keratinocytes in the epidermis. For the evaluation of radiation exposure, chromosomal aberration is considered to be the gold standard, yet our results suggest that Krt5 may be a novel biological marker for acute radiation symptoms.

Purpose: The perivascular localization of stem cell (SC) niches suggests the presence of a vascular niche. We aimed to determine the angiogenesis potential of limbal niche cells (NCs)., Methods: Human limbal NCs were isolated and serially passaged on plastic or coated Matrigel in embryonic SC medium containing BFGF and leukemia inhibitory factor before being reseeded in 3D Matrigel. Expression of angiogenesis markers was assessed by RT-qPCR and immunofluorescence staining. Their angiogenesis potential was measured by differentiation into vascular endothelial cells and by supporting vascular tube network formed by human umbilical vein endothelial cells (HUVEC) on Matrigel. Their support of limbal epithelial progenitor cells (LEPC) was examined in sphere growth formed by reunion in 3D Matrigel., Results: On plastic, limbal NC could be cultured only up to four passages before turning into myofibroblasts. In contrast, on coated Matrigel, they could be expanded for up to 12 passages with upregulation of markers suggestive of angiogenesis progenitors when reseeded in 3D Matrigel because they could differentiate into vascular endothelial cells and pericytes stabilizing the tube network formed by HUVEC. Although both expanded limbal NCs and HUVEC rejoined with LEPC to form spheres to upregulate expression of ΔNp63α, CK15, and CEBPδ, the former but not the latter abolished expression of CK12 keratin., Conclusions: Human limbal NCs continuously expanded on the basement membrane differentiate into angiogenesis progenitors that prevent differentiation of LEPC/SCs. They may partake in formation of the vascular niche and contribute to angiogenesis during wound healing.

Acute graft-versus-host disease (GVHD), a major complication of allogeneic stem cell transplantation, involves cytotoxic soluble and cellular effectors that selectively induce apoptosis in normally apoptosis-resistant, cytokeratin 15 (K15)-expressing epithelial stem cells residing at the tips of rete ridges of human epidermis and in analogous rete-like prominences (RLPs) of murine dorsal lingual epithelium. The mechanisms whereby epithelial stem cells are rendered vulnerable to apoptosis during allostimulation are unknown. We hypothesized that GVHD-induced target cell injury may be related to pathways involving the p53 family that are constitutively expressed by epithelial stem cells and designed to trigger physiological apoptosis as a result of environmental danger signals. Among the p53 family members, we found that p73 protein and mRNA were preferentially expressed in K15(+) RLPs of murine lingual squamous epithelium. On in vitro exposure to recombinant TNF-α and IL-1 in an organ culture model previously shown to replicate early GVHD-like target cell injury, apoptosis was selectively induced in K15(+) stem cell regions and was associated with induction of phosphorylated p73, a marker for p73 activation, and apoptosis was abrogated in target tissue obtained from p73-deficient (p73(-/-)) mice. Evaluation of early in vivo lesions in experimental murine GVHD disclosed identical patterns of phosphorylated p73 expression that coincided with the onset of effector T cell infiltration and target cell apoptosis within K15(+) RLPs. This study is the first to suggest that paradoxical apoptosis in GVHD of physiologically protected K15(+) epithelial stem cells is explainable, at least in part, by cytokine-induced activation of suicide pathways designed to eliminate stem cells after exposure to deleterious factors perceived to be harmful to the host., (Copyright © 2012 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Notch is a transmembrane receptor functioning in the determination of cell fate. Abnormal Notch signaling promotes tumor development, showing either oncogenic or tumor suppressive activity. The uncertainty about the exact role of Notch signaling, partially, stems from inconsistencies in descriptions of Notch expression in human cancers. Here, we clarified basal-cell dominant expression of NOTCH1 in squamous epithelium. NOTCH1 was downregulated in squamous neoplasms of oral mucosa, esophagus and uterine cervix, compared with the normal basal cells, although the expression tended to be retained in cervical lesions. NOTCH1 downregulation was observed even in precancers, and there was little difference between cancers and high-grade precancerous lesions, suggesting its minor contribution to cancer-specific events such as invasion. In culture experiments, reduction of NOTCH1 expression resulted in downregulation of keratin 13 and keratin 15, and upregulation of keratin 17, and NOTCH1 knockdown cells formed a dysplastic stratified epithelium mimicking a precancerous lesion. The NOTCH1 downregulation and the concomitant alterations of those keratin expressions were confirmed in the squamous neoplasms both by immunohistochemical and cDNA microarray analyses. Our data indicate that reduction of NOTCH1 expression directs the basal cells to cease terminal differentiation and to form an immature epithelium, thereby playing a major role in the histopathogenesis of epithelial dysplasia. Furthermore, downregulation of NOTCH1 expression seems to be an inherent mechanism for switching the epithelium from a normal and mature state to an activated and immature state, suggesting its essential role in maintaining the epithelial integrity.

Breast phyllodes tumors are rare neoplasms which present challenges for histological classification. Microscopic features are not always predictive of clinical behavior, and scarce data exist on the prognostic role of biological markers. Our study evaluated a series of 145 phyllodes tumors diagnosed at the Department of Pathology, Singapore General Hospital between 2006 and 2009, incorporating 91 (62.8%) benign, 40 (27.6%) borderline, and 14 (9.7%) malignant phyllodes tumors. Antibodies to keratin 15 (KRT15), transcobalamin I (TCN1), and homeobox gene Hox-B13 (HOXB13) were applied to sections cut from tissue microarray blocks. KRT15 and TCN1 positivity was defined when there was reactivity of 1% or more stromal cells, while HOXB13 positivity was defined using a H-score of 100 and above. Positive immunohistochemical expression for KRT15, TCN1, and HOXB13 was seen in 21 (14.5%), 96 (66.2%), and 66 (45.5%) of tumors, respectively. Stromal expression of KRT15, TCN1, and HOXB13 was significantly correlated with tumor grade (P < 0.001, P < 0.001, P = 0.012), stromal hypercellularity (P = 0.005, P < 0.001, P = 0.023), mitotic activity (P < 0.001), and microscopic borders (P = 0.006, P < 0.001, P = 0.011). Co-expression of TCN1 and HOXB13 was seen in 21 of 91 (23.1%) benign, 18 of 40 (45.0%) borderline, and 11 of 14 (78.6%) malignant tumors, suggesting that the dual-marker panels of TCN1 and HOXB13 might be helpful in classifying borderline and malignant tumors. Although expression of TCN1 alone was present in all malignant and 34 of 40 (85.0%) borderline tumors, a combined panel with HOXB13 excluded some benign cases and was a better discriminant for a significant proportion of borderline and malignant tumors.

The intermediate filament keratin 15 (K15) is present in variable amounts in various stratified epithelia, but has also been reported to be a stem cell marker in the hair follicle. Using peptide specific antibodies, we evaluated the temporal and spatial distribution pattern of K15 expression/localization during normal epidermal development and initiation of hair follicle formation, and in the injured mature epidermis (e.g., during acute injury and repair and in tumorigenesis). During development, K15 expression is first localized to a subset of epidermal basal cells and the overlying periderm at E12.5, but its expression is seen throughout the basal layer by E15.5 and beyond. In hair follicle morphogenesis, initial peg formation occurs in a K15-null area at E14.5 and as peg elongation proceeds through to the mature hair follicle, K15 expression follows the leading edge with positive cells restricted to the outer root sheath. In an epidermal injury model, K15 is first up-regulated and associated with both the basal and suprabasal layers of the interfollicular epidermis then expression becomes sporadic and down-regulated before a basal layer-specific association is re-established in the repaired epidermis. During tumorigenesis, K15 is first mis-expressed, and is ultimately down-regulated. Our data suggest that K15 protein expression may reflect not only expression in a stem or progenitor cell subpopulation, but also reflects the activity and responsiveness of basal-like cells to loss of homeostasis of the epidermal differentiation program. Thus, the data suggest caution in using K15 alone to delineate epidermal stem cells, and underscore the need for further investigation of K15 and other markers in epidermal cell subpopulations.

Androgenetic alopecia (AGA), also known as common baldness, is characterized by a marked decrease in hair follicle size, which could be related to the loss of hair follicle stem or progenitor cells. To test this hypothesis, we analyzed bald and non-bald scalp from AGA individuals for the presence of hair follicle stem and progenitor cells. Cells expressing cytokeratin15 (KRT15), CD200, CD34, and integrin, α6 (ITGA6) were quantitated via flow cytometry. High levels of KRT15 expression correlated with stem cell properties of small cell size and quiescence. These KRT15(hi) stem cells were maintained in bald scalp samples. However, CD200(hi)ITGA6(hi) and CD34(hi) cell populations--which both possessed a progenitor phenotype, in that they localized closely to the stem cell-rich bulge area but were larger and more proliferative than the KRT15(hi) stem cells--were markedly diminished. In functional assays, analogous CD200(hi)Itga6(hi) cells from murine hair follicles were multipotent and generated new hair follicles in skin reconstitution assays. These findings support the notion that a defect in conversion of hair follicle stem cells to progenitor cells plays a role in the pathogenesis of AGA.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: Corneal epithelium is maintained by a population of stem cells (SCs) that have not been identified by specific molecular markers. The objective of this study was to find new putative markers for these SCs and to identify associated molecular pathways., Methods: Real time PCR (rt-PCR) was performed in 24 human limbal and central corneal epithelial samples to evaluate the gene expression profile of known corneal epithelial SC-associated markers. A pool of those samples was further analyzed by a rt-PCR array (RT²-PCR-A) for 84 genes related to the identification, growth, maintenance, and differentiation of SCs., Results: Cells from the corneal epithelium SC niche showed significant expression of ATP-binding cassette sub-family G member 2 (ABCG2) and cytokeratin (KRT)15, KRT14, and KRT5 genes. RT²-PCR-A results indicated an increased or decreased expression in 21 and 24 genes, respectively, in cells from the corneal SC niche compared to cells from the central corneal epithelium. Functional analysis by proprietary software found 4 different associated pathways and a novel network with the highest upregulated genes in the corneal SC niche. This led to the identification of specific molecules, chemokine (C-X-C motif) ligand 12 (CXCL12), islet-1 transcription factor LIM/homeodomain (ISL1), collagen-type II alpha 1 (COL2A), neural cell adhesion molecule 1 (NCAM1), aggrecan (ACAN), forkhead box A2 (FOXA2), Gap junction protein beta 1/connexin 32 (GJB1/Cnx32), and Msh homeobox 1 (MSX1), that could be used to recognize putative corneal epithelial SCs grown in culture and intended for transplantation. Other molecules, NCAM1 and GJB1/Cnx32, potentially could be used to positively purify them, and Par-6 partitioning defective 6 homolog alpha (PARD6A) to negatively purify them., Conclusions: Knowledge of these gene and molecular pathways has provided a better understanding of the signaling molecular pathways associated with progenitor-rich limbal epithelium. This knowledge potentially could give support to the design and development of innovative therapies with the potential to reverse corneal blindness arising from ocular surface failure.

Here we demonstrate that physiological concentrations of the thyroid hormones T3 and T4 enhance the KERATIN 15 promoter activity and expression in epithelial stem cells of adult human scalp hair follicles in situ and in vitro. Additionally, T3 and T4 stimulate expression of the immuno-inhibitory surface molecule CD200. Subsequently, T3 and T4 induce apoptosis and differentiation and inhibit clonal growth of these progenitor cells in vitro. These data suggest that human hair follicle bulge-derived epithelial stem cells underlie profound, previously unknown hormonal regulation by thyroid hormones, and show that primary human keratin 15-GFP+ progenitor cells can be exploited to further elucidate fundamental endocrine controls of human epithelial stem cells., (Copyright (c) 2010 Elsevier GmbH. All rights reserved.)

For most types of cancers, the cell at the origin of tumour initiation is still unknown. Here, we used mouse genetics to identify cells at the origin of basal cell carcinoma (BCC), which is one of the most frequently occurring types of cancer in humans, and can result from the activation of the Hedgehog signalling pathway. Using mice conditionally expressing constitutively active Smoothened mutant (SmoM2), we activated Hedgehog signalling in different cellular compartments of the skin epidermis and determined in which compartments Hedgehog activation induces BCC formation. Activation of SmoM2 in hair follicle bulge stem cells and their transient amplifying progenies did not induce cancer formation, demonstrating that BCC does not originate from bulge stem cells, as previously thought. Using clonal analysis, we found that BCC arises from long-term resident progenitor cells of the interfollicular epidermis and the upper infundibulum. Our studies uncover the cells at the origin of BCC in mice and demonstrate that expression of differentiation markers in tumour cells is not necessarily predictive of the cancer initiating cells.

The zebrafish has become a useful model organism for research on development and diseases. However, there has been no zebrafish model system for studying oral carcinogenesis. In the present study, we first characterized the histology of the upper gastrointestinal tract of zebrafish. We found that zebrafish tongue was covered by a non-keratinized stratified squamous epithelium, which was similar to the oro-esophageal epithelium in humans. In situ hybridization showed that keratin 5, a marker of the basal cell layer of mammalian oral epithelium, was expressed in the squamous epithelium of zebrafish tongue. A highly conserved promoter of zebrafish keratin 5 was cloned to drive transgenic expression of GFP. GFP was found to be expressed in the periderm of embryos. In adult fish, GFP was also abundantly expressed in the tongue and fin. GFP expression in transgenic fish recapitulated endogenous zebrafish keratin 5 gene expression as shown by in situ hybridization. This study indicated a high fidelity of GFP reporter gene expression in the tongue under the control of zebrafish keratin 5 promoter. This zebrafish transgenic model system may be used for future studies on oral development and cancer.

In this article we show that cloning of the human K15 promoter before a green fluorescence protein (GFP)/geneticin-resistance cassette and transfection of microdissected, organ-cultured adult human scalp hair follicles generates specific K15 promoter-driven GFP expression in their stem cell-rich bulge region. K15-GFP+ cells can be visualized in situ by GFP fluorescence and 2-photon laser scanning microscopy. Vital K15-GFP+ progenitor cells can then be selected by using the criteria of their green fluorescence, adhesion to collagen type IV and fibronectin, and geneticin resistance. Propagated K15-GFP+ cells express epithelial progenitor markers, show the expected differential gene expression profile of human bulge epithelium, and form holoclones. This application of nonretroviral, K15 promoter-driven, GFP labeling to adult human hair follicles facilitates the characterization and manipulation of human epithelial stem cells, both in situ and in vitro, and should be transferable to other complex human tissues.

Introduction: During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes., Methods: In this study, we investigated the differential expression of 84 stem cell-related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation., Results: Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor beta (TGF-beta)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration., Conclusions: This study has generated an overview of stem cell-related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-beta/BMP, and cadherin signaling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration.

Kindler syndrome is an autosomal recessive disorder characterized by skin atrophy and blistering. It results from loss-of-function mutations in the FERMT1 gene encoding the focal adhesion protein, fermitin family homolog-1. How and why deficiency of fermitin family homolog-1 results in skin atrophy and blistering are unclear. In this study, we investigated the epidermal basement membrane and keratinocyte biology abnormalities in Kindler syndrome. We identified altered distribution of several basement membrane proteins, including types IV, VII, and XVII collagens and laminin-332 in Kindler syndrome skin. In addition, reduced immunolabeling intensity of epidermal cell markers such as beta1 and alpha6 integrins and cytokeratin 15 was noted. At the cellular level, there was loss of beta4 integrin immunolocalization and random distribution of laminin-332 in Kindler syndrome keratinocytes. Of note, active beta1 integrin was reduced but overexpression of fermitin family homolog-1 restored integrin activation and partially rescued the Kindler syndrome cellular phenotype. This study provides evidence that fermitin family homolog-1 is implicated in integrin activation and demonstrates that lack of this protein leads to pathological changes beyond focal adhesions, with disruption of several hemidesmosomal components and reduced expression of keratinocyte stem cell markers. These findings collectively provide novel data on the role of fermitin family homolog-1 in skin and further insight into the pathophysiology of Kindler syndrome.