Biology subjects

Gomperts, B. N.

Publications and source records attributed to Gomperts, B. N..

7 recordsLinked to original sources

bioRxiv2026

Transcriptional and spatial profiling of fibroblasts from human lungs highlights CTHRC1+ cells as fibrogenic signaling hubs in fibrosis

Lung fibroblasts are key regulators of tissue homeostasis and extracellular matrix (ECM) remodeling, and their aberrant activation drives the progressive parenchymal scarring characteristic of idiopathic pulmonary fibrosis (IPF), a fatal disease with limited therapeutic options. Despite their central pathogenic role, lung fibroblasts are difficult to isolate due to their embedded position within the ECM, and standard in vitro culture conditions may lead to the loss of their native functional and transcriptional characteristics, hampering the study of fibroblast behavior in disease. The transcriptional heterogeneity of lung fibroblast subtypes and the extent to which culture-induced alterations diverge from native tissue signatures remain poorly understood. Here, we integrated single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics of lung tissue from IPF patients and age-matched healthy donors with transcriptomic profiling of cultured fibroblasts collected at passages 1 and 6 after isolation using three optimized protocols: whole lung cell suspension (WLCS), negative fraction enrichment, and outgrowth. Tissue-based analysis identified six transcriptionally distinct mesenchymal subtypes: alveolar, adventitial, inflammatory, peribronchial, CTHRC1+ and smooth muscle cell (SMC). The fibroblast subtype CTHRC1+ represented the most transcriptionally activated pro-fibrotic subtype, showing the greatest upregulation of ECM biosynthesis genes, a prominent role in intercellular communication, and preferential enrichment within fibroblastic foci in IPF lung tissue. Pseudotime trajectory analysis supported a directional transcriptional continuum from alveolar and inflammatory fibroblasts toward the CTHRC1+ state, driven by coordinated activation of pro-fibrotic transcription factors, including RUNX2, CREB3L1, and SCX. In vitro culture progressively reshaped fibroblast transcriptional identity relative to native tissue, with increased collagen and matrix metalloproteinase (MMP) expression during passaging, loss of distinct CTHRC1+ fibroblasts, and gain of alveolar fibroblasts displaying pro-fibrotic activation across all isolation protocols. These findings provide a high-resolution transcriptional map of lung fibroblast heterogeneity in IPF and highlight critical limitations of standard in vitro culture systems for recapitulating native fibroblast diversity, with important implications for the development and evaluation of fibroblast-targeted therapeutic strategies in IPF.

molecular biology↗

bioRxiv2026

Modeling cell-cell interactions to advance drug discovery in Idiopathic Pulmonary Fibrosis

BackgroundIdiopathic Pulmonary Fibrosis (IPF) is characterized by scarring and remodeling of lung tissue, leading to progressive pulmonary dysfunction. Currently, very little is known about the steps involved in disease initiation and progression because models of IPF poorly replicate these processes. However, understanding the pathogenesis of IPF is essential for developing effective therapies. To address this, we have developed a scaffold-based co-culture IPF organoid that uses healthy and diseased human primary and iPSC-derived alveolar epithelial cells and fibroblasts to recapitulate the cellular communication and cell fate during progressive fibrosis. MethodsWe generated microbead scaffolds that mimic alveolar air-sacs and coated them in a rotating bioreactor with primary lung fibroblasts and induced pluripotent stem cell-derived type 2 alveolar cells (iAT2s). iAT2s with the surfactant protein C (SFTPC) I73T variant and the syngeneic corrected control iAT2s were cultured with primary healthy and IPF fibroblasts in different combinations. The epithelial-mesenchymal interactions during fibrosis initiation and progression were evaluated by single-cell RNA sequencing. ResultsWe found that the interaction between epithelial cells and fibroblasts plays a key role in inducing fibrotic responses in this model, with the secretion of chemokines, cytokines, TGF{beta}, and matrix metalloproteinases that mirror those observed in the serum of patients with pulmonary fibrosis. Single-cell RNA sequencing revealed the emergence of many cell subtypes observed in progressive lung fibrosis, along with key cellular interactions that correlated with the initial upregulation of fibrosis pathways, extracellular matrix (ECM) remodeling, inflammation, and changes in lipid metabolism. The anti-fibrotic compounds, Nintedanib and the TGF{beta} inhibitor, SB431542, demonstrated dose-dependent efficacy in the model, with IC50 values comparable to those observed in the clinic, and significantly reduced secretion of fibrosis-related factors. ConclusionOverall, this study shows that the three-dimensional, reductionist, cell co-culture organoid effectively models several components of progressive lung fibrosis, facilitating the investigation of epithelial-mesenchymal interactions and serving as a patient-relevant model to better predict the efficacy of therapeutics in the clinic.

cell biology↗

bioRxiv2026

Bidirectional fibrogenic cross-talk revealed in a human iPSC-derived epithelial-mesenchymal co-culture model of pulmonary fibrosis

Pulmonary fibrosis (PF) can arise from mutations in alveolar epithelial type 2 (AT2) cell-specific genes, but manifests in fibrotic activation of mesenchymal cells, thus involving fibrogenic epithelial-mesenchymal crosstalk. The ligand-receptor interactions underlying the onset and early progression of PF remain poorly understood. Induced pluripotent stem cell (iPSC)-derived models are powerful tools to study respiratory diseases, yet are currently limited to reductionist single lineage epithelial models or multi-lineage systems that lack purity and lung-specificity of the mesenchyme. Here we generate a human iPSC line carrying both a lung mesenchyme-specific reporter (TBX4-LERtdTomato) and a reporter for mesenchymal activation/differentiation (ACTA2GFP). Applying this line, we develop a directed differentiation protocol capable of generating cells that express key molecular and functional features of primary human developing lung mesenchyme across multiple iPSC genetic backgrounds. We then establish co-cultures of these iPSC-derived lung mesenchymal cells (iLM) with patient-specific iPSC-derived alveolar epithelial type 2 cells (iAT2s) carrying an SFTPCI73T mutation as a model for PF. We find increased expression of fibrotic markers in co-cultures with mutant iAT2s as compared to co-cultures with gene-corrected iAT2s. Moreover, mutant iAT2s express markers of alveolar-basal intermediate (ABI) cells only in the presence of iLM, suggesting that bidirectional crosstalk promotes this aberrant cell state. We identify ligand-receptor pairs enriched in co-cultures with mutant iAT2s, including TGF{beta}, multiple integrins, and additional genes that have not been previously linked to PF. Finally, we show that small molecule-mediated inhibition of TGF{beta} or integrins V{beta}1/V{beta}6 attenuates both fibrotic mesenchymal activation and the presence of ABI cells in iLM/iAT2 co-cultures. Thus, we have established a human iPSC-derived co-culture system that recapitulates key molecular hallmarks of bidirectional fibrogenic epithelial-mesenchymal crosstalk in pulmonary fibrosis, and enables the identification and study of potentially druggable pathways involved in disease initiation and progression.

molecular biology↗

bioRxiv2025

Investigation of Double-Stranded DNA Donors and CRISPR-Cas9 RNP for Universal Correction of Mutations Causing Cystic Fibrosis in Human Airway Cells

Cystic fibrosis (CF) is a devastating genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. As morbidity and mortality from CF results from a lack of mucus clearance that leads to chronic bacterial infections and progressive loss of lung function, site-specific insertion of a CFTR cDNA into the endogenous CFTR locus in airway basal stem cells (ABSCs) could prove curative for all disease-causing mutations. This study describes the development of nonviral genome editing reagents, designed to be packaged into nonviral delivery systems. An sgRNA targeting the 5 untranslated region of CFTR was characterized as directing high on-target cutting and displaying a safe off-target profile. Airway cell lines electroporated with chemically-modified (1-Aminohexane - AmC6), linear double-stranded DNA (ldsDNA) constructs were utilized as an Homology Directed Repair (HDR) donor, initially optimized with an mCitrine reporter. Expectedly, when the 780bp mCitrine cDNA was replaced with the 4.4kb CFTR cDNA, integration efficiency dropped significantly. However, 1-2% integration of codon optimized donors was sufficient to restore CFTR expression in the bulk edited population of human bronchial epithelial cell line, 16HBE14o- (16HBE), to levels reaching 50% of wildtype expression as measured by Western Blot. Electrophysiological validation of CFTR ion channel function measured via Ussing Chamber Assay revealed that these bulk edited populations exhibit greater than 40% restoration of the chloride ion currents of the measured wildtype controls. These results demonstrate that low levels of CFTR integration can be made therapeutically relevant by optimizing the designs of gene editing reagents. Importantly, this work utilizes nonviral editing reagents, an essential step towards in vivo gene therapy for CF.

genetics↗

bioRxiv2025

Lipid Nanoparticles for the Delivery of CRISPR/Cas9 Machinery to Enable Site-Specific Integration of CFTR and Mutation-Agnostic Disease Rescue

We report the engineering of lipid nanoparticles (LNPs) to transport CRISPR/Cas9 payloads, including double-stranded DNA (dsDNA) donor templates, designed for homology directed repair (HDR)-mediated site-specific insertion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to correct cystic fibrosis (CF) in diseased airway epithelium. We screened various nanoparticle formulations, adjusting ratios of Cas9-encoding mRNA, single guide RNAs (sgRNAs), and dsDNA donor templates to optimize gene editing using human bronchial epithelial cells (16HBE14o-) harboring a CF-causing mutation (G542X). Populations of G542X cells edited via LNP delivery of CFTR donors achieved 3 - 3.5% gene integration and yielded comparable CFTR protein expression compared to normal 16HBE14o- controls. These edited populations exhibit restoration of CFTR-dependent Cl- current to ca. 80% of values measured in normal 16HBE14o- cell monolayers. This LNP platform adds capabilities for transporting large gene editing machinery to airway epithelial cells for genomic integration of entire genes, enabling therapeutic solutions that achieve correction of any CF-causing mutation.

bioengineering↗

bioRxiv2020

Direct exposure to SARS-CoV-2 and cigarette smoke increases infection severity and alters the stemcell-derived airway repair response

Most demographic studies are now associating current smoking status with increased risk of severe COVID-19 and mortality from the disease but there remain many questions about how direct cigarette smoke exposure affects SARS-CoV-2 airway cell infection. We directly exposed mucociliary air-liquid interface (ALI) cultures derived from primary human nonsmoker airway basal stem cells (ABSCs) to short term cigarette smoke and infected them with live SARS-CoV-2. We found an increase in the number of infected airway cells after cigarette smoke exposure as well as an increased number of apoptotic cells. Cigarette smoke exposure alone caused airway injury that resulted in an increased number of ABSCs, which proliferate to repair the airway. But we found that acute SARS-CoV-2 infection or the combination of exposure to cigarette smoke and SARS-CoV-2 did not induce ABSC proliferation. We set out to examine the underlying mechanism governing the increased susceptibility of cigarette smoke exposed ALI to SARS-CoV-2 infection. Single cell profiling of the cultures showed that infected airway cells displayed a global reduction in gene expression across all airway cell types. Interestingly, interferon response genes were induced in SARS-CoV-2 infected airway epithelial cells in the ALI cultures but smoking exposure together with SARS-CoV-2 infection reduced the interferon response. Treatment of cigarette smoke-exposed ALI cultures with Interferon {beta}-1 abrogated the viral infection, suggesting that the lack of interferon response in the cigarette smoke-exposed ALI cultures allows for more severe viral infection and cell death. In summary, our data show that acute smoke exposure allows for more severe proximal airway epithelial disease from SARS-CoV-2 by reducing the mucosal innate immune response and ABSC proliferation and has implications for disease spread and severity in people exposed to cigarette smoke.

cell biology↗

bioRxiv2020

A molecular atlas of proximal airway identifies subsets of known airway cell types revealing details of the unique molecular pathogenesis of Cystic Fibrosis

Introduction/AbstractCystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts in excess of 70,000 people globally. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most significant determinant of morbidity and mortality. In this study we report results from a multi-institute consortium in which single cell transcriptomics were applied to define disease-related changes to the proximal airway of CF donors (n=19) undergoing transplantation for end-stage lung disease compared to the proximal airway of previously healthy lung donors (n=19). We found that all major airway epithelial cell types were conserved between control and CF donors. Disease-dependent differences were observed, including an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subtypes coupled with an unexpected decrease in cycling basal cells. This study developed a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease.

cell biology↗