Single-cell RNA sequencing provides insights into the potential cellular origins and microenvironment of Extramammary Paget's disease.

Aberrant accumulation of basal cells in the distal lung is a hallmark of impaired epithelial regeneration and is closely associated with fibrotic remodeling; however, their cellular origins and the mechanisms governing their expansion remain unclear. Here, this study establishes human distal lung organoids (DLOs) as a physiologically relevant model to investigate epithelial repair. Single-cell transcriptomic and functional analyses identify a CD66c+ basal cell subset in DLOs that resembles basal cell states enriched in idiopathic pulmonary fibrosis (IPF) lungs and exhibits inflammatory and profibrotic transcriptional programs. The data further demonstrate that secretory cells, alveolar type 2 (AT2) cells, and resident basal cells can each generate CD66c+ basal cells, indicating substantial epithelial lineage plasticity. Mechanistically, the GSK3 inhibitor CHIR99021 stabilizes secretory and AT2 identities and prevents their conversion into CD66c+ basal cells, accompanied by activation of ERBB4-MAPK signaling. Moreover, exogenous NRG1 directly restricts this transdifferentiation through ERBB4-dependent signaling, reinforcing the role of ERBB4 in maintaining epithelial lineage stability. Notably, ERBB4 expression is reduced in IPF tissues, coinciding with expansion of the CD66c+ basal cell population. Together, these findings identify ERBB4 signaling as a critical regulator that constrains pathological epithelial remodeling during severe lung injury.

The human olfactory epithelium possesses remarkable neurogenic capacity, sustained by a resident population of basal stem cells; however, establishing a dynamic in vitro model that faithfully recapitulates this regenerative process has been proven challenging. Here, we present an established protocol for generating olfactory organoids from progenitor cells isolated from adult human superior turbinate tissue. These organoids maintain stemness, as evidenced by SOX2 expression, and display a spatially organized differentiation pattern, characterized by uniform expression of the immature neuronal marker GAP43 and peripheral localization of the mature olfactory sensory neuron marker OMP. Subsequent targeted differentiation with the Notch inhibitor LY411575, the Wnt activator CHIR-99021, and retinoic acid generated marker-positive olfactory sensory neuron-like cells that co-expressed OMP and the neuronal marker Tuj1 (class III β-tubulin). This human-derived model circumvents the limitations of murine systems and provides a robust in vitro platform for studying olfactory neurogenesis, COVID-19-associated anosmia, and other olfactory-related neurodegenerative disorders.

Breast cancer remains a leading cause of death among women, with the HER2+ subtype being particularly aggressive due to acquired resistance to HER2-targeted therapies. Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2, represses the expression of genetic programs crucial for differentiation, proliferation, and apoptosis. To investigate the role of EZH2 in HER2+ tumor progression, we crossed a genetically engineered mouse model of HER2-driven breast cancer with a conditional Ezh2 knockout strain and showed that Ezh2 is essential for accelerating tumor initiation and metastatic dissemination. Combined bulk and single cell RNA sequencing analyses revealed a significant downregulation of basal cell populations in the absence of Ezh2, and an upregulation of luminal progenitor cell populations, driven by crucial transcription factors such as Esr1. Further, inhibition of EZH2 in vitro resulted in increased expression of ER in HER2+ human breast cancer cell lines and conferred sensitivity to Tamoxifen. These findings demonstrate that EZH2 dictates cancer plasticity and provides rationale for combining EZH2 inhibitors with endocrine therapies to improve HER2+ breast cancer outcomes.

The cellular composition and disease susceptibilities of the distinct zones of the human prostate remain incompletely understood. Through extensive single-cell RNA sequencing (scRNA-seq) of benign regions from prostatectomy specimens, we identified a basal cell population expressing WIF1, VCAN , and NRG1 , among other genes, that was significantly enriched in the transition zone (TZ). Benign prostatic hyperplasia (BPH) is a common condition that causes widespread morbidity and is nearly exclusively localized to the TZ. Analysis of previously published scRNA-seq datasets further confirmed that WIF1 + basal cells were significantly enriched in BPH compared to normal prostate. Pathway and cell-cell communication analyses revealed that this basal subtype is associated with programs related to cell proliferation, epithelial-mesenchymal transition (EMT), angiogenesis, and hormone response. Together, the molecular signature, zonal distribution, and pathway enrichment suggest that TZ-enriched WIF1 + basal cells may contribute to BPH pathogenesis by promoting epithelial and stromal remodeling.

Although histologically normal, esophageal preneoplastic cells harbor early genetic alterations and likely exhibit lineage plasticity. However, their origins and trajectories remain unclear. To address this, we combined genetic barcoding with single-cell RNA sequencing to trace the lineage of esophageal preneoplastic cells. We identified a distinct progenitor-like cell population with high plasticity. Through a scoring system, these high-plasticity cells are mapped, revealing their contributions to proliferative and basal cell populations. This approach uncovers molecular markers, including Nfib and Qk, that define these precursor cells, validated by spatial transcriptomics and a Trp53 Cdkn2a Notch1 mouse model. These findings provide critical insights into early tumorigenesis, highlighting the potential of precursor cells as biomarkers for early detection and therapeutic targets of esophageal squamous cell cancer. By elucidating the cellular dynamics underlying esophageal preneoplasia, this research lays the foundation for strategies to prevent malignant progression, offering broader implications for improving cancer diagnostics and treatment approaches.

Sox9 is a transcription factor with multiple roles during development and in adult organ homeostasis. In the adult eye, Sox9 expression persists in several cell types, including the retinal pigmented epithelium cells and the Müller glial (MG) cells, as well as in the limbal and corneal basal epithelia. To uncover the role of Sox9 in these cell types, we induced the deletion of the gene in adult mice. We found that, after Sox9 ablation, mutant mice undergo a severe process of retinal degeneration characterized by the loss of MG cells and complete depletion of the photoreceptors layer. Moreover, by combining single-cell RNA sequencing and Sox9 lineage tracing, we found that Sox9 is expressed in a basal limbal stem cell population with the ability to form two types of long-lived cell clones involved in stem cell maintenance and homeostasis. Mosaic analysis of Sox9 positive and negative cells confirmed that the gene is essential for limbal stem cell differentiation. Our results show that Sox9 is required for the maintenance of retinal integrity and for limbal stem cell differentiation in the adult mouse eye.

Sox9 is a transcription factor with multiple roles during development and in adult organ homeostasis. In the adult eye, Sox9 expression persists in several cell types, including the retinal pigmented epithelium cells and the Müller glial (MG) cells, as well as in the limbal and corneal basal epithelia. To uncover the role of Sox9 in these cell types, we induced the deletion of the gene in adult mice. We found that, after Sox9 ablation, mutant mice undergo a severe process of retinal degeneration characterized by the loss of MG cells and complete depletion of the photoreceptors layer. Moreover, by combining single-cell RNA sequencing and Sox9 lineage tracing, we found that Sox9 is expressed in a basal limbal stem cell population with the ability to form two types of long-lived cell clones involved in stem cell maintenance and homeostasis. Mosaic analysis of Sox9 positive and negative cells confirmed that the gene is essential for limbal stem cell differentiation. Our results show that Sox9 is required for the maintenance of retinal integrity and for limbal stem cell differentiation in the adult mouse eye.

Abstract Description Airway epithelial cells (AECs) are the primary interface between the respiratory system and environmental exposures such as pathogens, allergens, and particulate matter. While AECs are not typically known as professional antigen-presenting cells, recent studies revealed AEC populations capable of antigen processing and presentation during viral and bacterial infections. However, MHCII expression by these AECs did not influence disease outcomes. Therefore, we hypothesized that MHCII expression on AECs may instead play a critical role in response to inhaled allergens, another significant environmental exposure. Our study demonstrates constitutive MHCII expression across several distinct AEC populations in the mouse lung via flow cytometry. In response to house dust mite (HDM), we observed increased MHCII expression and upregulation of costimulatory molecules in AECs. In addition, using fluorescently labeled HDM, we identified a unique MHCII+ basal AEC subpopulation that doubles in number upon allergen exposure and phagocytoses allergen in vivo. This observation also translates to humans, since bronchial epithelial cells from asthmatic patients also show dysregulated MHCII expression compared to controls. Our findings suggest the existence of a specific population of airway epithelial basal cells may act as an antigen-presenting cell in the context of allergy. The dysregulation of this process may modify the outcomes of allergic disease. Funding Sources Supported by NSF GRFP (Grant No. DGE1746891) Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Although histologically normal, esophageal preneoplastic cells harbor early genetic alterations and likely exhibit lineage plasticity. However, their origins and trajectories remain unclear. To address this, we combined genetic barcoding with single-cell RNA sequencing to trace the lineage of esophageal preneoplastic cells. We identified a distinct progenitor-like cell population with high plasticity. Through a newly developed scoring system, these high-plasticity cells are mapped, revealing their contributions to proliferative and basal cell populations. This approach uncovers molecular markers, including Nfib and Qk, that define these precursor cells, validated by spatial transcriptomics and a Trp53 Cdkn2a Notch1 mouse model. These findings provide critical insights into early tumorigenesis, highlighting the potential of precursor cells as biomarkers for early detection and therapeutic targets of esophageal squamous cell cancer. By elucidating the cellular dynamics underlying esophageal preneoplasia, this research lays the foundation for strategies to prevent malignant progression, offering broader implications for improving cancer diagnostics and treatment approaches.

Estrogen receptor β (ERβ) plays an important role in both the mouse and human prostate. The endogenous ligand for ERβ is the dihydrotestosterone metabolite, 5β-androstane-3β, 17β-diol (3β-Adiol). Thus, treatment with 5-α reductase inhibitor (5-ARI) should produce a phenotype similar to that seen in ERβ-/- mice. By comparing RNA-Seq of the ventral prostates (VP) of ERβ knockout mice (ERβcrispr-/-) and wild-type (WT) mice, we confirmed that ERβ modulates androgen receptor (AR) signaling indirectly by suppressing AR coactivators. Compared to WT mice, basal cell genes from ERβcrispr-/- mouse VP were significantly upregulated. A population of abnormal basal cells coexpressing P63 and AR was identified in the ERβcrispr-/- mouse VP by immunohistochemistry. In men treated with 5-ARI for treatment of benign prostatic hyperplasia (BPH), there was induction of a P63-positive intermediate cell population characterized by down regulation of Krt14 without significant change in the expression of Krt15, upregulation of AR and NKX3.1, and increased proliferation. In both VP of aging ERβcrispr-/- mice and in human prostates after 5-ARI treatment, there was substantial immune infiltration. Testosterone treatment inhibited immune infiltration in the VP of ERβcrispr-/- mice. We conclude that ERβ is a gene critical in maintaining normal basal cells and modulating immune environment in the prostate. Its loss leads to histological changes suggesting prostatitis and increases the number of intermediate cells, which are considered to be the cells of origin of prostate cancers. We suggest that an ERβ agonist could protect against 5-ARI-induced inflammatory cell infiltration and defects in the basal cell layer in BPH.

JAK/STAT signaling sustains an intermediate basal cell population in prostate homeostasis and disease.

Transplantation of airway basal stem cells could achieve a durable cure for genetic diseases of the airway, such as cystic fibrosis and primary ciliary dyskinesia. Recent work demonstrated the potential of primary and pluripotent stem cell-derived basal cells to efficiently engraft into the mouse trachea after injury. However, there are many hurdles to overcome in translating these approaches to humans, including developing safe and efficient methods for delivery in larger animal models. We propose a model that targets preconditioning and cell delivery to intrapulmonary airways using a microbronchoscope for delivery. The detergent polidocanol was adapted for distal lung preconditioning, inducing intrapulmonary airway epithelial denudation by 5 and 24 hours after delivery. Although initial reepithelialization of airways occurred later than that of tracheas, complete repair was observed within 7 days. Both pluripotent stem cell-derived and primary basal cells delivered via microbronchoscope after polidocanol injury engrafted in tracheas and intrapulmonary airways, respectively. Transplanted cells differentiated into ciliated and secretory lineages while maintaining a population of basal cells. These findings demonstrate the utility of bronchoscopically targeted preconditioning and cell delivery to the conducting intrapulmonary airways, thus providing an important framework for preclinical translation of approaches for engineered airway epithelial regeneration.

Single-cell transcriptomics reveals extracellular matrix remodeling and collagen dynamics during lactation in sheep mammary gland.

Abstract RATIONALE: The prevalence of SARS-CoV2 (SCov2) infection and severity of COVID-19 illness differs markedly based upon age. Whether children and infants are resistant to, or resilient from, infection is not entirely clear. METHODS: We studied the acute response to SCoV2 infection in a physiological model of human airways using single cell RNAseq (scRNAseq). Confluent, early passage primary cultures of human airway epithelial cells, derived from adult or infant lungs (obtained from LungMAP biorepository), were differentiated at air-liquid interface (ALI) and infected apically with SCoV2 under BSL3 conditions (or sham infected). Cells were recovered 48 hours later by enzymatic dissociation, fixed using a novel method for viral inactivation (Osborne et al., PLoS One 2023), and captured on a 10X Chromium system. Gene expression analysis of the dataset (Osborne et al, Clin Exp Immunol, In Press) was performed in Seurat, and ontologies were identified using ToppFun. RESULTS: As expected, scRNAseq identified multiple distinct epithelial cell sub-types (basal, ciliated, secretory, etc.) in our culture system. Both adult and infant cells were reliably infected by SCoV2 in vitro, showing a preferential, but non-specific tropism for ciliated cells. Gene expression responses were markedly different between adult and infant cells. Adult, but not infant cells demonstrated a robust induction of interferon signaling genes, and a switch from eukaryotic to viral translation. This was evident particularly in Club and basal cell populations. Conversely, infant cells demonstrated a marked reduction in secretory protein and mitochondrial gene expression, and evidence for widespread shutdown of all RNA translation in all cell types. SUMMARY: Significant differences in the prevalence and severity of COVID-19 in adults and children is associated with divergent transcriptional responses to SCoV2 infection. These studies provide new insight into factors contributing to resilience to infection in children.

Abstract RATIONALE: Obesity and asthma frequently co-occur, with 60% of asthma patients being obese. This obese asthma (OA) subgroup has worse clinical outcomes, including poor asthma control and reduced corticosteroid responsiveness, likely driven by poorly understood non-T2 inflammatory mechanisms. Here, we examine airway biospecimens from obese asthma patients to explore airway inflammation and epithelial remodeling phenotypes. METHODS: Bulk RNA-sequencing was performed on nasal airway brushings from the Asthma Characterization Protocol (ACP). Bronchial brushings and biopsies from the Obesity and Metabolic Dysfunction in Asthma study were analyzed using bulk/scRNA-sequencing and immunofluorescent histology. Co-expression networks were obtained from the bulk data and then used to identify disease endotypes and investigate responses driven by obese asthma. Airway specimen single-cell data were analyzed using Seurat analysis pipeline. RESULTS: Network analysis of nasal RNA-seq data from obese (n=135) and lean (n=55) asthma patients identified T1 (interferon), T2 (IL-4, IL-13, IL-5), T17 (IL-17), and IL36G-driven inflammatory networks and endotyping based on network expression showed 26%, 25%, 21%, and 30% of obese asthmatics highly expressed these networks, respectively. Obese asthmatics were more likely to exhibit at least one of these endotypes (64%) than lean patients (47%, p=0.032). These inflammatory changes coincided with downregulation in the expression of genes involved in epithelial integrity, including CLD17, JAM3, and several cadherin/protocadherin and integrin genes. Examining lower airway effects, gene networks from bronchial brushings revealed a higher proportion of obese asthmatics expressing the T1 endotype compared to lean asthmatics and healthy controls (p=0.026). Pathway analysis of dysregulated genes in obsese versus lean asthma (FDR<0.05) indicated upregulation of interferon-driven gene expression, including ISG, IFIT, and OAS gene families, and dysregulation of metabolic pathways related to obesity (arginine/proline, glucose, sphingolipid metabolism). Bronchial biopsy scRNA-seq demonstrated elevated expression of squamous metaplasia markers, KRT13 and KRT6A, in club and basal cell populations among obese asthmatics (FDR<0.05). This matched epithelial remodeling and squamous metaplasia signatures indicated by upregulation of keratinization and mucin biosynthesis pathways (p<0.05) in the obese asthma bronchial brush RNA-seq data. Squamous epithelial remodeling was also visualized by SPRR3+ labeling and H&E staining observed in 29% of obese asthmatics, which was entirely absent in biopsies from lean asthmatics. CONCLUSIONS: Obese asthmatics are more prone to airway inflammation of various forms, but most commonly involves interferon-driven, T1 inflammation. Moreover, systemic metabolic dysfunction in these patients extends to the airway epithelium. This pronounced inflammation in patients with obese asthma likely drives observed epithelial metaplastic and remodeling phenotypes.

Abstract Introduction: Mitochondria play an important role in progenitor cell function. Although mitochondrial dysfunction has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD), it is unclear whether mitochondrial dysfunction contributes to abnormal airway progenitor (basal) cell function in COPD. Methods: We compared gene expression between 1) basal cells isolated from the distal lungs of COPD patients and non-diseased controls using our published single-cell RNA sequencing (scRNA-seq) atlas; and 2) individually cultured COPD and control basal cell clones. Additionally, we examined whether media supplementation with pyruvate, uridine, and glucose (which are known to support the growth of mitochondria-deficient cells) normalizes differences in gene and protein expression between heterogenous populations of COPD and control basal cells. We also examined functional abnormalities in COPD and control basal cells using the Seahorse Mito Stress test and examined their mitochondrial content by flow cytometry using MitoTracker Green. Finally, we induced mitochondrial dysfunction in healthy basal cells by inhibiting mitochondrial DNA polymerase γ (POLG) with 10uM zidovudine while they were differentiating in air-liquid interface and examined goblet cell numbers by Alcian Blue Periodic Acid-Schiff (AB-PAS) staining and mucin gene expression by RT-qPCR. Results: We observed marked differences in the expression of genes related to mitochondria and metabolism between COPD and control basal cells in our scRNA-seq atlas. Moreover, we identified a COPD-specific subset of basal cell clones with high expression of the mitophagy markers PINK1 and PRKN and high expression of goblet cell genes including MUC5AC and MUC5B. Overall, transcriptional and proteomic differences between COPD and control basal cells were reduced by media supplementation with pyruvate, uridine, and glucose, suggesting mitochondrial dysfunction contributes to altered gene and protein expression in these cells. The Seahorse assay indicated COPD basal cells have reduced basal and peak oxygen consumption rates and reduced glycolytic function. They also had reduced mitochondrial content by flow cytometry. Zidovudine-treated healthy basal cells produced increased numbers of AB-PAS positive goblet cells relative to vehicle-treated controls and increased expression of MUC5AC and MUC5B. Conclusions: These data support the concept that mitochondrial dysfunction influences differentiation outcomes in COPD basal cells and that restoration of normal mitochondrial function may represent a novel therapeutic strategy in COPD.

Epidermal Loss of PRMT5 Leads to the Emergence of an Atypical Basal Keratinocyte-Like Cell Population and Defective Epidermal Stratification.

Abstract RATIONALE: Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia (DIPNECH) is a rare lung disease defined by hyperplasia of pulmonary neuroendocrine cells (PNECs) in the bronchiolar epithelium with risk of subsequent development of carcinoid tumors. The mechanism by which this PNEC hyperplasia occurs has not been established. We hypothesized that single-cell RNA sequencing (scRNA-seq) could be used to infer transcriptional programs and cell-cell communications in the DIPNECH epithelium, illuminating potential avenues for more targeted treatments informed by the mechanisms of the disease. METHODS: Single cell suspensions were generated from a surgical lung biopsy of one DIPNECH lung and lung explants from 25 declined donor (control) lungs underwent scRNA-seq library preparation using the 10X Genomics Chromium system, and libraries were sequenced on an Illumina Novaseq6000. Following quality control filtering, data integration, and cell type annotation, normalized gene expression counts were analyzed using a variety of methods to infer up-regulated genes, gene co-expression modules (using CEMiTool) and cell-cell communication (using CellChat) in DIPNECH and unaffected lungs. RESULTS: There were significant differences in the cell type compositions of the DIPNECH epithelium, including a larger proportion of PNECs and respiratory airway secretory cells (RASCs) and a smaller proportion of multiciliated cells, compared to non-DIPNECH “unaffected” epithelia. Additionally, we found a large increase in the fraction of NK cells in the immune population of the DIPNECH sample compared to unaffected samples. Gene co-expression module analysis on PNECs revealed an enriched module in DIPNECH neuroendocrine cells including hub genes HGF (hepatocyte growth factor), EGFR, EGR1, and NR4A1. Further investigation into the DIPNECH epithelium showed that several epithelial cell types including basal and secretory cell populations were enriched for MET, the tyrosine kinase receptor for HGF. Further, HGF was one of the most enriched ligands expressed by PNECs in the DIPNECH sample compared to controls. Using CellChat to infer cell-cell communication, we orthogonally identified HGF-MET signaling as one of the most significantly enriched pathways in the DIPNECH lung epithelium compared to the unaffected epithelium. CONCLUSIONS: These data indicate that HGF signaling may contribute to widespread epithelial changes in DIPNECH lungs beyond neuroendocrine cell hyperplasia. Further investigation into the HGF-MET signaling pathway in DIPNECH patients is necessary to determine its significance in disease progression.

Collagen VII is an essential anchoring protein in the basement membrane zone, maintaining the attachment of stratified and pseudostratified epithelia to the underlying interstitial matrix. However, collagen VII is largely unexplored in normal lungs and idiopathic pulmonary fibrosis (IPF), a disease characterized by excessive accumulation of extracellular matrix and aberrant re-epithelialization of fibrotic lung parenchyma. Analysis of collagen VII protein and mRNA encoded by COL7A1 gene in IPF distal lungs demonstrated elevated levels compared with those in normal lungs. To investigate its cellular source and spatial distribution in lung tissue, immunohistochemistry, RNAscope in situ hybridization, and cell culture experiments, in combination with analysis of public transcriptomic data sets were performed. In the IPF lungs, collagen VII was abundant in pathologically remodeled airways and honeycomb cysts, associated with increased basal cell populations. In contrast, in the control lungs, collagen VII was mainly localized in larger airways. RNA sequencing data revealed that epithelial basal cells and KRT5–/KRT17+ aberrant basaloid cells are the primary sources of COL7A1 mRNA expression. Furthermore, COL7A1 mRNA was observed in mesenchymal subsets, and both COL7A1 mRNA and the protein were observed in fibroblast foci, another histopathologic feature of IPF. In vitro, COL7A1 mRNA expression was increased in normal human lung fibroblasts treated with transforming growth factor-β1. These findings suggest that collagen VII could be involved in the process of abnormal re-epithelialization in lung fibrosis.