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The versatility of somatosensation arises from heterogenous human dorsal root ganglion (DRG) neurons. The critical information to decipher their functions, i.e., the soma transcriptome, is lacking due to technical difficulties. Here we developed a novel approach to isolate individual human DRG neuron somas for deep RNA sequencing (RNA-seq). On average, >9000 unique genes per neuron were detected, and 16 neuronal types were identified. Cross-species analyses revealed that touch-, cold-, and itch-sensing neuronal types were relatively conserved, while the pain-sensing neurons displayed marked divergence. Soma transcriptomes of human DRG neurons predicted novel functional features, which were confirmed using single-cellin vivoelectrophysiological recordings. These results support a close relationship the between physiological properties of human sensory afferents and molecular profiles uncovered by the single-soma RNA-seq dataset. In summary, by conducting single-soma RNA-seq of human DRG neurons, we generated an unprecedented neural atlas for human somatosensation.

Skin is composed of diverse cell populations that cooperatively maintain homeostasis. Upregulation of the Nuclear factor-kappa B pathway may lead to the development of chronic inflammatory disorders of the skin, but its role during the early events remains unclear. Through analysis of single cell RNA sequencing data via iterative Random Forest Leave One Out Prediction, an explainable artificial intelligence method, we identified an immunoregulatory role for a unique Prx1(+) fibroblast subpopulation. Disruption of Ikkb-NF-kB under homeostatic conditions in these fibroblasts paradoxically induced skin inflammation due to the overexpression of C-C Motif Chemokine Ligand 11 (CCL11, or eotaxin-1) characterized by eosinophil infiltration and a subsequent Th2 immune response. Because the inflammatory phenotype resembled that seen in human atopic dermatitis (AD), we examined validated human AD skin samples and found that human AD fibroblasts also over-expressed CCL11 and that perturbation of Ikkb-NFkB in primary human dermal fibroblasts upregulates CCL11. We also demonstrated that monoclonal antibody treatment against CCL11 was effective in reducing the eosinophilia and Th2 inflammation in a mouse model. Taken together, the murine model and human AD specimens point to dysregulated Prx1(+) fibroblasts as a previously unrecognized etiologic factor that may contribute to the pathogenesis of AD and suggest targeting CCL11 as a way to treat AD-like skin lesions.

RATIONALE AND OBJECTIVES: Three-dimensional (3D) visualization has been shown to benefit new generations of medical students and physicians-in-training in a variety of contexts. However, there is limited research directly comparing student performance after using 3Dtools to those using two-dimensional (2D) screens. MATERIALS AND METHODS: A CT was performed on a donated cadaver and a 3D CT hologram was created. A total of 30 first-year medical students were randomly assigned into two groups to review head and neck anatomy in a teaching session that incorporated CT. The first group used an augmented reality headset, while the second group used a laptop screen. The students were administered a five-question anatomy test before and after the session. Two-tailed t-tests were used for statistical comparison of pretest and posttest performance within and between groups. A feedback survey was distributed for qualitative data. RESULTS: Pretest vs. posttest comparison of average percentage of questions answered correctly demonstrated both groups showing significant in-group improvement (p < 0.05), from 59% to 95% in the augmented reality group, and from 57% to 80% in the screen group. Between-group analysis indicated that posttest performance was significantly better in the augmented reality group (p = 0.022, effect size = 0.73). CONCLUSION: Immersive 3D visualization has the potential to improve short-term anatomic recall in the head and neck compared to traditional 2D screen-based review, as well as engage millennial learners to learn better in anatomy laboratory. Our findings may reflect additional benefit gained from the stereoscopic depth cues present in augmented reality-based visualization.

Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer in humans, with an incidence of approximately 700,000 cases per year in the United States (Rogers et al., 2010). It is known that cSCC is strongly associated with sun exposure, specifically UVB and UVA, as well as other risk factors, such as human papillomavirus infection, immunodeficiency, and specific medications (Ratushny et al., 2012). However, the precise sequence of biological events leading to tumor development remains unknown. With projected higher incidence of patients with cSCCs in the future, there is a strong need to elucidate the molecular pathways that regulate formation of cSCCs.

Stem cells support lifelong maintenance of adult organs, but their specific roles during injury are poorly understood. Here we demonstrate that Lgr6 marks a regionally restricted population of epidermal stem cells that interact with nerves and specialize in wound re-epithelialization. Diphtheria toxin-mediated ablation of Lgr6 stem cells delays wound healing, and skin denervation phenocopies this effect. Using intravital imaging to capture stem cell dynamics after injury, we show that wound re-epithelialization by Lgr6 stem cells is diminished following loss of nerves. This induces recruitment of other stem cell populations, including hair follicle stem cells, which partially compensate to mediate wound closure. Single-cell lineage tracing and gene expression analysis reveal that the fate of Lgr6 stem cells is shifted toward differentiation following loss of their niche. We conclude that Lgr6 epidermal stem cells are primed for injury response and interact with nerves to regulate their fate.

Squamous cell carcinoma in situ (SCCIS) is a prevalent precancerous lesion that can progress to cutaneous squamous cell carcinoma. Although SCCIS is common, its pathogenesis remains poorly understood. To better understand SCCIS development, we performed laser captured microdissection of human SCCIS and the adjacent epidermis to isolate genomic DNA and RNA for next-generation sequencing. Whole-exome sequencing identified UV-signature mutations in multiple genes, including NOTCH1–3 in the epidermis and SCCIS and oncogenic TP53 mutations in SCCIS. Gene families, including SLFN genes, contained UV/oxidative-signature disruptive epidermal mutations that manifested positive selection in SCCIS. The frequency and distribution of NOTCH and TP53 mutations indicate that NOTCH mutations may precede TP53 mutations. RNA sequencing identified 1,166 differentially expressed genes; the top five enriched gene ontology biological processes included (i) immune response, (ii) epidermal development, (iii) protein phosphorylation, (iv) regulation of catalytic activity, and (v) cytoskeletal regulation. The NEURL1 ubiquitin ligase, which targets Notch ligands for degradation, was upregulated in SCCIS. NEURL1 protein was found to be elevated in SCCIS suggesting that increased levels could represent a mechanism for downregulating Notch during UV-induced carcinogenesis. The data from DNA and RNA sequencing of epidermis and SCCIS provide insights regarding SCCIS formation.

Graft versus host disease (GVHD) is a common, morbid, and potentially fatal complication of allogeneic hematopoietic stem cell transplantation (aHSCT).1-3 The exact timing of the arrival of donor-derived immune cells and their profile in the skin have not been well described in humans.4-6 Fluorescent in situ hybridization (FISH) is a technique that has been used to distinguish host versus donor-derived immune cells in patients who have undergone sex-mismatched aHSCT.7 This article is protected by copyright. All rights reserved.

Fibroblast growth factor receptor 3 (FGFR3) activating mutations are drivers of malignancy in several human tissues, including bladder, lung, cervix, and blood. However, in skin, these mutations are associated predominantly with benign, common epidermal growths called seborrheic keratoses (SKs). How epidermis resists FGFR3 mediated transformation is unclear, but previous studies have suggested that FGFR3 activation in skin keratinocytes may serve a tumor-suppressive role by driving differentiation and antagonizing Ras signaling. To define the role of FGFR3 in human normal and neoplastic epidermis, and to directly test the hypothesis that FGFR3 antagonizes Ras, we engineered human skin grafts in vivo with mutant active FGFR3 or shRNA FGFR3 knockdown. We show that FGFR3 active mutants drive mild hyperproliferation, but are insufficient to support benign or malignant tumorigenesis, either alone, or in combination with G 1-S checkpoint release. This suggests that additional cell-intrinsic or stromal cues are required for formation of benign SKs with FGFR3 mutations. Further, FGFR3 activation does not alter the growth kinetics or differentiation status of engineered human epidermal SCCs driven by Ras, and FGFR3 protein itself is dispensable for Ras-driven SCC. To extend these findings to patients, we examined a uniquely informative human tumor in which SCC developed in continuity with a SK, raising the hypothesis that one of the tumors evolved from the other. However, mutational analysis from each tumor indicates that the overlapping SK and SCC evolved independently and supports our conclusion that FGFR3 activation is insufficient to drive SCC.

Background: In vivo confocal scanning laser microscopy (CSLM) is a recently developed non-invasive technique for visualizing microscopic structures with the skin. CSLM has been used to characterize proliferative and inflammatory skin diseases, neoplastic skin lesions and pigmented lesions. Objective: Here, we assessed the ability of CSLM to evaluate the formation of neogenic hair follicles after a full-thickness wound in mice. Methods: Full-thickness wounds were made on the dorsal skin of 3-week-old mice. After scab detachment (SD), the number, width, length, space and volume of neogenic hair follicles were analyzed using CSLM. The results were compared with those from conventional methods, including staining for alkaline phosphatase (AP) and keratin 17 (K17) as well as histology. Results: Quantification of neogenic hair follicles using CSLM compared favorably with the results from direct measurements on isolated epidermal tissue after immunostaining for K17, a marker for the epithelial portion of new hair follicles. CSLM detected 89% of K17-stained follicles. CSLM more accurately quantified the number of new follicles compared with AP staining, which detects the dermal portion of the new follicle. The width and length measurement from CSLM and histology were very close and correlated with each other. The minimum length of a neogenic hair follicle that could be detected by CSLM was 21 μm. The space between neogenic hair follicles was decreased in histological sections compared with CSLM. Conclusion: CSLM is an accurate and valuable method for counting and measuring neogenic hair follicles non-invasively. CSLM produces images similar to histology in mice. Measurements of microstructures using CSLM more accurately reflect actual sizes as this technique avoids fixation artifacts. In vivo visualization of developing follicles with CSLM allows the detection of serial changes in hair follicle formation, thus conserving the numbers of mice required for studies and improving the detection of temporal changes in developing hair follicles.