The aryl hydrocarbon receptor AhR links atopic dermatitis and air pollution via induction of the neurotrophic factor artemin.

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.

Following the p63/Keratin5 basal cells in the sensory and non-sensory epithelia of the vomeronasal organ.

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.

Local tissue interactions govern pLL patterning in medaka.

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.

Keratin 15 protects against cigarette smoke-induced epithelial mesenchymal transformation by MMP-9.

BACKGROUND: 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. METHODS: Krt15 knockout (Krt15-/-) and wild-type (WT) mice of C57BL/6 background were exposed to CS for six months to establish COPD models. Krt15-CrePGR;Rosa26-LSL-tdTomato mice were used to trace the fate of the Krt15+ cells. Hematoxylin and eosin (H&E) and Masson stainings were performed to assess histopathology and fibrosis, respectively. Furthermore, lentivirus-delivered short hairpin RNA (shRNA) was used to knock down KRT15 in human bronchial epithelial (HBE) cells stimulated with cigarette smoke extract (CSE). The protein expression was assessed using western blot, immunohistochemistry, and enzyme-linked immunosorbent assay. RESULTS: Krt15-/- CS mice developed severe inflammatory cell infiltration, airway remodeling, and emphysema. Moreover, Krt15 knockout aggravated CS-induced secretion of matrix metalloproteinase-9 (MMP-9) and epithelial-mesenchymal transformation (EMT), which was reversed by SB-3CT, an MMP-9 inhibitor. Consistent with this finding, KRT15 knockdown promoted MMP-9 expression and EMT progression in vitro. Furthermore, Krt15+ cells gradually increased in the bronchial epithelial cells and were transformed into alveolar type II (AT2) cells. CONCLUSION: Krt15 regulates the EMT process by promoting MMP-9 expression and protects the lung tissue from CS-induced injury, inflammatory infiltration, and apoptosis. Furthermore, Krt15+ cells transformed into AT2 cells to protect alveoli. These results suggest Krt15 as a potential therapeutic target for COPD.

Mapping the distribution of stem/progenitor cells across the mouse middle ear during homeostasis and inflammation.

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.

Accelerated complete human skin architecture restoration after wounding by nanogenerator-driven electrostimulation.

BACKGROUND: Electrostimulation (ES) therapy for wound healing is limited in clinical use due to barriers such as cumbersome equipment and intermittent delivery of therapy. METHODS: We adapted a human skin xenograft model that can be used to directly examine the nanogenerator-driven ES (NG-ES) effects on human skin in vivo-an essential translational step toward clinical application of the NG-ES technique for wound healing. RESULTS: We show that NG-ES leads to rapid wound closure with complete restoration of normal skin architecture within 7 days compared to more than 30 days in the literature. NG-ES accelerates the inflammatory phase of wound healing with more rapid resolution of neutrophils and macrophages and enhances wound bed perfusion with more robust neovascularization. CONCLUSION: Our results support the translational evaluation and optimization of the NG-ES technology to deliver convenient, efficient wound healing therapy for use in human wounds.

Desmoplastic Trichoepithelioma With Pseudocarcinomatous Hyperplasia: A Folliculosebaceous Neoplasm in Young Persons.

ABSTRACT: Pseudocarcinomatous desmoplastic trichoepithelioma (PDTE) features verrucous squamous epidermal hyperplasia with a jagged undersurface overlying cords of follicular germinative cells in a fibrotic stroma. To date, only 5 cases have been reported. We identified 7 new PDTEs from 2 institutions and reviewed their clinical manifestations and immunohistochemical profile. The median age was 14 years (range 8-34 years). New findings included vacuolization of the basal layer of the pseudocarcinomatous surface epithelium, and the frequent presence of singly distributed sebocytes within the cords of basaloid cells. The immunohistochemical profile resembles desmoplastic trichoepithelioma, with expression of TDAG51, CK15, and Ber-Ep4. Colonizing CK20+ Merkel cells were present in all cases. PDTE needs to be differentiated from malignant neoplasms such as squamous cell carcinoma, morphoeic basal cell carcinoma, and microcystic adnexal carcinoma. Recognizing the features of this sclerosing folliculosebaceous neoplasm facilitates accurate diagnosis and avoids overtreatment.

Conditional Deletion of AP-2ß in the Periocular Mesenchyme of Mice Alters Corneal Epithelial Cell Fate and Stratification.

The cornea is an anterior eye structure specialized for vision. The corneal endothelium and stroma are derived from the periocular mesenchyme (POM), which originates from neural crest cells (NCCs), while the stratified corneal epithelium develops from the surface ectoderm. Activating protein-2ß (AP-2ß) is highly expressed in the POM and important for anterior segment development. Using a mouse model in which AP-2ß is conditionally deleted in the NCCs (AP-2ß NCC KO), we investigated resulting corneal epithelial abnormalities. Through PAS and IHC staining, we observed structural and phenotypic changes to the epithelium associated with AP-2ß deletion. In addition to failure of the mutant epithelium to stratify, we also observed that Keratin-12, a marker of the differentiated epithelium, was absent, and Keratin-15, a limbal and conjunctival marker, was expanded across the central epithelium. Transcription factors PAX6 and P63 were not observed to be differentially expressed between WT and mutant. However, growth factor BMP4 was suppressed in the mutant epithelium. Given the non-NCC origin of the epithelium, we hypothesize that the abnormalities in the AP-2ß NCC KO mouse result from changes to regulatory signaling from the POM-derived stroma. Our findings suggest that stromal pathways such as Wnt/ß-Catenin signaling may regulate BMP4 expression, which influences cell fate and stratification.

Dynamic spatiotemporal expression pattern of limbal stem cell putative biomarkers during mouse development.

Limbal stem cells (LSCs), a subpopulation of limbal epithelial basal cells, are crucial to the homeostasis and wound healing of corneal epithelium. The identification and isolation of LSCs remains a challenge due to lack of specific LSCs biomarkers. In this study, Haematoxylin-eosin (HE), 4', 6-diamidino-2-phenylindole (DAPI), and immunohistochemistry (IHC) stains were performed on the pre- and post-natal limbus tissues of mice which has the advantage of more controllable in term of sampling age relative to human origin. By morphological analysis, we supported that there is an absence of the Palisades of Vogt (POV) in the mouse. The development of prenatal and neonatal cornea was dominated by its stroma, whereas after eyelids opened at P14, the corneal epithelial cells (CECs) quickly go stratification in response to the liquid-air interface. Based on IHC staining, we found that the expression of LSCs putative biomarkers in limbal epithelial basal cells appeared in chronological order as follows: Vim = p63 > CK14 > CK15 (where = represents same time; > represents earlier), and in corneal epithelial basal cells were weakened in chronological order as follows: Vim > p63 > CK15 > CK14, which might also represent the stemness degree. Furthermore, the dynamic spatial expression of the examined LSCs putative biomarkers during mouse development also implied a temporal restriction. The expression of Vim in epithelial cells of mouse ocular surface occurred during E12-E19 only. The expression of CK15 was completely undetectable in CECs after P14, whereas the others putative molecular markers of LSCs, such as p63 and CK14, still remained weak expression, suggesting that CK15 was suitable to serve as the mouse LSCs biomarkers after P14. In this study, our data demonstrated the dynamic spatiotemporal expression pattern of LSCs putative biomarkers in mouse was age-related and revealed the time spectrum of the expression of LSCs in mouse, which adds in our knowledge by understanding the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness.

KRT 15 as a prognostic biomarker is highly expressed in esophageal carcinoma.

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.

Deletion of p53 and Hyper-Activation of PIK3CA in Keratin-15+ Stem Cells Lead to the Development of Spontaneous Squamous Cell Carcinoma.

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.

Krt5+ urothelial cells are developmental and tissue repair progenitors in the kidney.

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.

Differential distribution of the epigenetic marker 5-hydroxymethylcytosine occurs in hair follicle stem cells during bulge activation.

BACKGROUND: Hair follicle (HF) cycling is dependent upon activation and differentiation of an epithelial subpopulation of cells with stem-like characteristics. These cells express cytokeratin 15 (CK15) and are sequestered within a specialized niche termed the follicular bulge. The pathways that mediate bulge activation are poorly understood, although growing evidence suggests a role for epigenetic events. METHODS: Here we investigated murine and human HFs to determine whether a recently described epigenetic hydroxymethylation marker, 5-hmC, known to mediate cell growth and differentiation, may play a role in bulge activation. RESULTS: We found the bulge region of murine HFs to show variable 5-hmC distribution within the nuclei of CK15-positive stem cells during early anagen, a pattern that was not associated with resting stem cells of telogen follicles, which did not express 5-hmC. Moreover, during phases of early anagen that were induced in an organ culture model, spatial alterations in bulge stem cell 5-hmC reactivity, as assessed by dual labeling, were noted. CONCLUSIONS: These preliminary findings suggest that 5-hmC may play a dynamic role in bulge activation during anagen growth, and provide a foundation for further experimental inquiry into epigenomic regulation of HF stem cells.

[Immunohistochemical study of Ki-67, PHH3, and CK15 protein expression in oral epithelial malignancy]. / Immunogistokhimicheskoe issledovanie ékspressii belkov Ki-67, PHH3 i CK15 pri malignizatsii épiteliia slizistoi obolochki rta.

OBJECTIVE: To investigate the expression of Ki-67, phosphohistone H-3 (PHH3), and cytokeratin 15 (CK15) proteins in the cells of the oral mucosa (OM) according to the degree of its malignant transformation. MATERIAL AND METHODS: OM biopsy specimens from 69 patients diagnosed with focal epithelial hyperplasia, intraepithelial squamous cell neoplasia, cancer in situ, and squamous cell carcinoma were examined. Tissue antigens were determined using mouse Ki-67 monoclonal antibodies, rabbit PHH3 polyclonal antibodies, and mouse CK15 monoclonal antibodies. RESULTS: There was an increase in epithelial proliferative and mitotic activities in squamous cell carcinoma and a sharp decrease in the expression of CK15 in the cytoplasm in cancer in situ and squamous cell carcinoma of the OM. CONCLUSION: The protein CK15 can be used for the differential diagnosis between high-grade dysplasia and OM epithelial malignancy at the stage of carcinoma in situ and squamous cell carcinoma.

microRNA-203 Modulates Wound Healing and Scar Formation via Suppressing Hes1 Expression in Epidermal Stem Cells.

BACKGROUND/AIMS: Little is known how miR-203 is involved in epidermal stem cells (ESCs) differentiation and scar formation. METHODS: We first used luciferase assay to determine the interaction of miR-203 with the 3'-UTR in regulation of Hes1 expression. We then used flow cytometry to analyze the effects of miR-203 expression on the differentiation of ESCs to MFB by determination of CK15 ratio and α-SMA. To confirm the results of flow cytometry analysis, we used Western blot to examine the expression of α-SMA, Collagen I (Col I), and Collagen III (Col III), as well as the expression of Notch1, Jagged1, and Hes1 in ESCs after the treatment of pre-miR-203 or anti-miR-203. Finally, we examined the effects local anti-miR-203 treatment on would closure and scar formation using a mouse skin wound model. RESULTS: Pre-miR-203 treatment increased ESCs differentiation to MFB cells, as indicated by decreased CK15 ratio and increased MFB biomarkers. This phenomenon was reversed by overexpression of Hes1 in ESCs. In addition, skin incision increased expression of miR-203 in wound tissue. Local treatment of anti-miR-203 could accelerate wound closure and reduce scar formation in vivo, which was associated with increased re-epithelialization, skin attachment regeneration, and collagen reassignment. Finally, we confirmed that anti-miR-203 treatment could inhibit ESCs differentiation in vivo via increasing Hesl expression. CONCLUSION: Taken together, our results suggested that overexpression of miR-203 in ESCs after skin wound may be a critical mechanism underlying the scar formation.

Regulation of keratin 5/14 intermediate filaments by CDK1, Aurora-B, and Rho-kinase.

We previously reported that vimentin, GFAP, and desmin (type III intermediate filament [IF] proteins) are mitotically phosphorylated by CDK1, Aurora-B, and Rho-kinase. This phosphorylation is critical for efficient separation of these IFs and completion of cytokinesis. Keratin 5 (K5) and K14 form a heterodimer, which constitutes IF network in basal layer cells of stratified squamous epithelia. Here, we report that the solubility of K5/K14 increased in mitosis. The in vitro assays revealed that three mitotic kinases phosphorylate K5 more than K14. We then identified Thr23/Thr144, Ser30, and Thr159 on murine K5 as major phosphorylation sites for CDK1, Aurora-B, and Rho-kinase, respectively. Using site- and phosphorylation-state-specific antibodies, we demonstrated that K5-Thr23 was phosphorylated in entire cytoplasm from prometaphase to metaphase, whereas K5-Ser30 phosphorylation occurred specifically at the cleavage furrow from anaphase to telophase. Efficient K5/K14-IF separation was impaired by K5 mutations at the sites phosphorylated by these mitotic kinases. K5-Thr23 phosphorylation was widely detected in dividing K5-positive cells of murine individuals. These results suggested that mitotic reorganization of K5/K14-IF network is governed largely through K5 phosphorylation by CDK1, Aurora-B, and Rho-kinase.

Canine corneal epithelial cells possess a sustained proliferative capacity and generate a spontaneously derived cell line.

We have previously reported characteristics of canine corneal epithelial cells in vitro and found that canine corneal epithelial cells could maintain their proliferative capacity even after continuous culture without the use of feeder cells and growth promoting additives. The objective of this study was to elucidate proliferative characteristics of canine corneal epithelial cells independent of feeder cells and growth promoting additives, with the aim of developing a spontaneously derived corneal epithelial cell line. Canine and rabbit corneal epithelial cells were harvested from the limbus and cultured with, or without, feeder cells and growth promoting additives, and both were passaged continuously until growth arrest. Canine corneal epithelial cells could proliferate independently, and could be passaged more times than rabbit cells. A canine corneal epithelial cell line, cCEpi, which could be passaged more than 100 times without using feeder cells and growth promoting additives, was established. cCEpi cells maintained a cell morphology close to the primary culture and expressed p63, cytokeratin 15 (K15), and K3. Although changes in colony morphology, shortening of the population doubling time and a heteroploid karyotype were observed, cCEpi was not tumorigenic. Stratified cell sheets cultured from cCEpi were morphologically and immunohistologically similar to sheets cultivated from early passage cells. In conclusion, canine corneal epithelial cells can proliferate independent of feeder cells and growth promoting additives. cCEpi maintains properties similar to normal corneal epithelial cells and could be a useful source for studies in cellular biology and for developing novel therapies.

Identification of gene pairs through penalized regression subject to constraints.

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.

Urothelial proliferation and regeneration after spinal cord injury.

The basal, intermediate, and superficial cell layers of the urothelium undergo rapid and complete recovery following acute injury; however, the effects of chronic injury on urothelial regeneration have not been well defined. To address this discrepancy, we employed a mouse model to explore urothelial changes in response to spinal cord injury (SCI), a condition characterized by life-long bladder dysfunction. One day post SCI there was a focal loss of umbrella cells, which are large cells that populate the superficial cell layer and normally express uroplakins (UPKs) and KRT20, but not KRT5, KRT14, or TP63. In response to SCI, regions of urothelium devoid of umbrella cells were replaced with small superficial cells that lacked KRT20 expression and appeared to be derived in part from the underlying intermediate cell layer, including cells positive for KRT5 and TP63. We also observed KRT14-positive basal cells that extended thin cytoplasmic extensions, which terminated in the bladder lumen. Both KRT14-positive and KRT14-negative urothelial cells proliferated 1 day post SCI, and by 7 days, cells in the underlying lamina propria, detrusor, and adventitia were also dividing. At 28 days post SCI, the urothelium appeared morphologically patent, and the number of proliferative cells decreased to baseline levels; however, patches of small superficial cells were detected that coexpressed UPKs, KRT5, KRT14, and TP63, but failed to express KRT20. Thus, unlike the rapid and complete restoration of the urothelium that occurs in response to acute injuries, regions of incompletely differentiated urothelium were observed even 28 days post SCI.