Children with congenital foregut malformations including tracheal pathology or long-gap esophageal atresia have limited treatment options. Severe malformations have suboptimal replacement options for both congenital and acquired anomalies despite maximal surgical intervention. Tissue engineering offers a potential solution to address this unmet need.For esophageal reconstruction, the combination of decellularized extracellular matrix scaffolds, myogenic progenitor cells, and bioreactor pre-conditioning has shown the most promising preclinical results. For tracheal replacement, there have been initial reports in pediatric patients, but no strategy has yet demonstrated consistent preclinical success, and the optimal approach remains to be defined. The two fields are at markedly different stages of translational readiness. Tracheal tissue engineering has not yet achieved consistent safety or durability in orthotopic large-animal models, and clinical use has been confined to a small number of compassionate-use cases with mixed outcomes partly related to inconsistent use of scaffold materials and cell replacement strategies. Esophageal reconstruction is closer to clinical translation, with a clinical trial of segmental replacement in adults underway and full-thickness circumferential replacement with evidence of secondary peristalsis demonstrated in a growing large-animal model.Pediatric patients impose unique constraints for tissue engineering but also offer advantages, including enhanced tissue remodeling capacity. Bridging the gap between current preclinical progress and safe clinical application will demand robust animal model validation, transparent documentation of setbacks, and sustained interdisciplinary engagement.This review summarizes current strategies for tracheal and esophageal reconstruction using tissue-engineered approaches, evaluating preclinical and clinical evidence across scaffold design, cell sourcing, and vascularization.

This mini-review, inspired by the 20th Anniversary Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases conference, summarizes two decades of advances in human-specific lung disease modeling. We discuss endogenous stem cell-based platforms for both distal alveolar and proximal airway epithelia, including primary alveolar type II cells, bronchial and nasal epithelial cultures established as organoids, air-liquid interface cultures, and ex vivo preparations. Advances in iPSC-derived basal and alveolar epithelial cells, humanized graft models, and high-throughput therapeutic screening are highlighted. Collectively, these in vitro and in vivo models are transforming the study of lung development, injury, and repair, while accelerating drug discovery and regenerative medicine strategies. Finally, we emphasize the critical need for integrating immune components into organotypic cultures and for establishing rigorous validation and benchmarking standards to maximize translational relevance.

Over the past two decades, progress in stem cell biology, bioengineering, and systems biology has improved our understanding of lung regeneration and repair. Building on work presented at the 20th Anniversary Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, this review examines the evolving trajectory of the field and outlines remaining challenges and opportunities for future research. We focus on three main areas: improving ex vivo lung models to better capture cellular heterogeneity and biomechanics; using single-cell, spatial, and computational approaches to support translation into clinical practice; and innovative therapeutic strategies, including gene therapies, epithelial cell therapies, immune cell engineering, extracellular matrix reconditioning, senescence targeting, and whole-organ bioengineering and xenotransplantation. Together, these approaches are shifting lung regeneration from descriptive studies toward precision, mechanism-driven therapies. Future progress in lung regenerative medicine will require integration of omics-driven insights with functional validation and biomaterial innovation to achieve meaningful clinical impact.

Fibrosis, marked by excess extracellular matrix (ECM) deposition, is the end stage of many diseases. Single-cell studies have highlighted the emergence of disease-specific fibroblast populations, including a high collagen-synthesizing CTHRC1+ subpopulation. The profibrotic cytokine TGF-β1 promotes fibrogenesis via cooperation between Smad and mTORC1/4E-BP1 signaling axes. Using CRISPR-Cas9 gene editing, we report that more than one-third of TGF-β1-regulated matrisome genes are under mTORC1 control. Mapping the transcriptome of TGF-β1-stimulated fibroblasts revealed similarity to CTHRC1+ fibroblasts identified in idiopathic pulmonary fibrosis (IPF). This overlap is lost when mTORC1 is disabled. Using the selective mTORC1 inhibitor RMC-5552, we confirm a causal role for mTORC1 in promoting the acquisition of the collagen-high, CTHRC1+ phenotype in response to TGF-β1 stimulation in fibroblasts derived from patients with either IPF or lung adenocarcinoma. We conclude that mTORC1 plays a key role in shaping the transcriptional identity of these fibroblasts, with implications for therapeutic inhibition of mTORC1 in fibrosis and cancer.

Cancers rarely respond completely to immunotherapy. While tumors consist of multiple genetically distinct clones, whether this affects the potential for immune escape remains unclear due to an inability to isolate and propagate individual subclones from human cancers. Here, we leverage the multi-region TRACERx lung cancer evolution study to generate a patient-derived organoid - T cell co-culture platform that allows the functional analysis of subclonal immune escape at single clone resolution. We establish organoid lines from 11 separate tumor regions from three patients, followed by isolation of 81 individual clonal sublines. Co-culture with tumor infiltrating lymphocytes (TIL) or natural killer (NK) cells reveals cancer-intrinsic and subclonal immune escape in all 3 patients. Immune evading subclones represent genetically distinct lineages with a unique evolutionary history. This indicates that immune evading and non-evading subclones can be isolated from the same tumor, suggesting that subclonal tumor evolution directly affects immune escape.

Background:Respiratory RNA viral infections significantly impact quality of life and productivity, in terms of both pandemics (e.g. severe acute respiratory syndrome coronavirus 2, influenza A viruses) and seasonal infections, such as respiratory syncytial virus (RSV). Globally, there are an estimated 33.8 million cases of RSV annually in children aged <5 years, leading to 2.8-4.3 million hospital admissions and up to 199 000 deaths. Human-relevant drug screening models are key to addressing the urgent need for effective antiviral treatments for RSV, particularly in vulnerable patient groups. Methods:Here, we present a donor-derived differentiated primary human nasal epithelial cell, high-throughput screening (HTS) Transwell-96 air-liquid interface (ALI) model designed to investigate the effects of combination antiviral therapies on RSV infection in primary human ciliated airway epithelium. In addition, we describe novel analytical tools using R (ciliR) to screen drug combinations by concurrently measuring efficacy and ciliary beat frequency, which we used as a sensitive marker of cell toxicity. Results:Our results demonstrate that the higher-throughput HTS Transwell-96 ALI cultures retain epithelial composition and ciliary function consistent within donor replicates. These cultures are permissible to infection with an RSV-green fluorescent protein (GFP) reporter virus, enabling quantitative comparison and combination treatment across multiple epithelial cultures from the same donor. Conclusion:We anticipate that our disease-relevant system will serve as a foundation for larger-scale experiments aimed at optimising combination therapy for RSV and other respiratory viruses.

Cystic fibrosis (CF), caused by mutations in the CFTR gene, is characterised by progressive dysfunction in multiple organs. The lung and pancreas largely define patient morbidity and survival, even in the era of highly effective modulator therapies. In the pancreas, CFTR activity loss obstructs exocrine ducts, leading to acinar atrophy and secondary β-cell dysfunction, culminating in CF-related diabetes. In the lung, impaired CFTR-mediated ion transport disrupts mucociliary clearance, driving chronic infection, inflammation, and airway remodelling. Despite these organ-specific manifestations, both tissues share a developmental origin and rely on resident stem and progenitor populations for postnatal maintenance and repair. Human pluripotent stem cells, including patient-derived induced pluripotent stem cells (hiPSCs), have enabled in vitro modelling of lung and pancreatic development. In CF, these models have demonstrated cell-type-specific CFTR defects and facilitated functional testing of modulators. Airway and pancreatic organoids support disease modelling and serve as preclinical platforms for gene correction and pharmacologic intervention. Recent studies highlight the potential of basal airway stem cells, alveolar type 2 cells, and pancreatic progenitors as targets for regenerative or cell-based therapies. Achieving durable organ repair, however, requires coordinated restoration of CFTR function, modulation of inflammation, and maintenance of tissue niches. These advances mark a shift in CF regenerative medicine, emphasising that therapeutic efficacy depends not only on replacing specific cell types, but on restoring a functional, CFTR-competent tissue microenvironment. Integrating these insights provides a roadmap for developing regenerative strategies capable of preserving both respiratory and metabolic functions, improving long-term outcomes for all people with CF.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants, yet therapeutics are lacking. The aim of this study was to develop a pre‑clinical model that recapitulates key clinical outcomes in infants with RSV bronchiolitis, such as neutrophil activation and migration into the airways. Peripheral blood neutrophils from infants with severe RSV disease admitted to the Paediatric Intensive Care Unit showed elevated myeloperoxidase (MPO) in children with RSV, compared to age-matched controls. To mechanistically model this response, we established an air-liquid interface (ALI) system incorporating paediatric airway epithelial cells, endothelial cells and neutrophils from adults, to recapitulate the blood-airway barrier. Following RSV infection, with and without treatment with antivirals remdesivir or RSV604, neutrophil migration and activation were assessed using flow cytometry. While both drugs reduced viral load, only RSV604 attenuated MPO expression. This model suggests that MPO could be useful as a readout of therapeutic efficacy. Targeting neutrophil-driven inflammatory pathways may be critical for reducing pathology in infant RSV infection.

Pulmonary homeostasis and regeneration require coordination between epithelial cells, immune populations, vascular networks, extracellular matrix, and stromal cells. This mini review examines recent advances in lung niche biology that were a focus of the 2025 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference. Here, we emphasize the impact of nonepithelial compartments essential for tissue function and repair. Reflecting the conference’s emphasis on comprehensive niche biology, we highlight how nonepithelial compartments, including stromal, vascular, and immune cells, serve as essential regulators of tissue function and repair, a perspective that unified many of the presentations and discussions. In diseases such as pulmonary fibrosis and chronic obstructive pulmonary disease, disruption of these niche interactions, rather than isolated cellular defects, drives regenerative failure. Spatial transcriptomic approaches reveal how pathological cell states colocalize within aberrant microniches. These insights suggest that effective therapeutics must target entire multicellular ecosystems, with advanced organoid platforms offering promising tools for developing niche-modulating interventions.

Age is a principal risk factor for chronic respiratory diseases. During aging, the airway epithelium undergoes structural and functional changes, including a reduced regenerative capacity. Basal cells act as stem/progenitor cells within the airway epithelium and are known to acquire age-dependent intrinsic defects, such as genomic mutations, transcriptomic changes and declining stem cell potential. However, the molecular mechanisms bridging transcriptomic change and stem cell function are unknown. Here we show that the transcription factor p63, a key regulator of epithelial cell identity, controls airway basal cell progenitor function in vitro and declines in expression with aging. We found impaired in vitro 2D colony and 3D organoid formation potential, as well as reduced culture longevity in primary human nasal basal cells isolated from older adults (>70 years) compared to those from pediatric (<10 years) donors. p63 protein expression was lower in basal cells from older adults and p63-regulated genes were enriched among genes whose expression changed with aging. Overexpression of p63 in older adult basal cells partially rescued progenitor cell functions, while gene knockdown in pediatric cells caused functional deficits. Integrative transcriptomic analyses revealed the network of p63-regulated genes in basal cells and demonstrated similarities in pathway engagement between TP63-knockdown cells and aged epithelium. Thus, while exogenous p63 expression alone could not rejuvenate older adult epithelial cells, p63 has an influential role in declining epithelial stem cell function during airway aging. Defining the upstream mechanisms controlling p63 decline during epithelial aging may reveal novel targets to promote healthy aging and reduce the burden of chronic lung disease.

Brain metastasis occurs in up to 40% of patients with non-small cell lung cancer (NSCLC). Considerable genomic heterogeneity exists between the primary lung tumor and respective brain metastasis; however, the identity of the genes capable of driving brain metastasis is incompletely understood. Here, we carried out an in vivo genome-wide CRISPR activation (CRISPRa) screen to identify molecular drivers of brain metastasis from an orthotopic NSCLC patient-derived xenograft model. We discovered activating expression of the Alzheimer’s disease associated β-site amyloid precursor protein cleaving enzyme 1 (BACE1) led to a significant increase in brain metastasis. Furthermore, genetic and pharmacological inhibition of BACE1 blocked NSCLC brain metastasis. Mechanistically, we identified BACE1 acts through its novel substrate EGFR to drive this metastatic phenotype. Together, our data highlights the power of in vivo CRISPR screening to unveil novel molecular drivers and potential therapeutic targets of NSCLC brain metastasis.

Animal-derived components in cell culture, such as foetal bovine serum (FBS) and extracellular matrix proteins (ECM), pose ethical concerns and contribute to variability in experimental outcomes. This study explores the use of animal-free cell culture media and substrates to support the growth and differentiation of primary human bronchial epithelial cells (BECs), as well as their infection by respiratory syncytial virus (RSV). We evaluated the performance of jellyfish collagen 0 and recombinant ECM proteins as alternatives to traditional mammalian substrates. Additionally, we assessed the use of animal-free media and human serum (HS) in viral propagation using HEp2 cells. Results demonstrate that the use of animal-free medium and matrix proteins and human serum can support primary epithelial cell growth and differentiation, with high-levels of ciliation and barrier integrity. RSV propagation in animal-free medium produced an increase in viral titres, indicating the potential of these systems for anti-viral research. Transitioning to include more animal-free medium and substrates for primary cell culture and viral propagation will help improve the ethical standing of research and offer more human-relevant models for studying viral diseases in the future.

Aberrant DNA methylation has been described in nearly all human cancers, yet its interplay with genomic alterations during tumor evolution is poorly understood. To explore this, we performed reduced representation bisulfite sequencing on 217 tumor and matched normal regions from 59 patients with non-small cell lung cancer from the TRACERx study to deconvolve tumor methylation. We developed two metrics for integrative evolutionary analysis with DNA and RNA sequencing data. Intratumoral methylation distance quantifies intratumor DNA methylation heterogeneity. MR/MN classifies genes based on the rate of hypermethylation at regulatory (MR) versus nonregulatory (MN) CpGs to identify driver genes exhibiting recurrent functional hypermethylation. We identified DNA methylation-linked dosage compensation of essential genes co-amplified with neighboring oncogenes. We propose two complementary mechanisms that converge for copy number alteration-affected chromatin to undergo the epigenetic equivalent of an allosteric activity transition. Hypermethylated driver genes under positive selection may open avenues for therapeutic stratification of patients.

Background:Animal-derived components in cell culture, such as fetal bovine serum and extracellular matrix proteins, pose ethical concerns and contribute to variability in experimental outcomes. This study explores the use of animal-free cell culture media and substrates to support the growth and differentiation of primary human bronchial epithelial cells, as well as their infection by respiratory syncytial virus (RSV). Methods:We evaluated the performance of jellyfish collagen 0 and recombinant extracellular matrix proteins as alternatives to traditional mammalian substrates. Additionally, we assessed the use of animal-free medium and human serum (HS) in viral propagation using HEp2 cells. Results:The use of animal-free medium, matrix proteins and HS can support primary epithelial cell growth and differentiation with high levels of ciliation and barrier integrity. RSV propagation in animal-free medium produced an increase in viral titres, indicating the potential of these systems for antiviral research. Conclusion:Transitioning to include more animal-free medium and substrates for primary cell culture and viral propagation will help improve the ethical standing of research and offer more human-relevant models for studying viral diseases in the future.

Neutrophils play a dual role in respiratory syncytial virus (RSV) infection, contributing to both viral clearance and lung damage. Analysis of peripheral blood-derived neutrophils from infants with RSV admitted to the Paediatric Intensive Care Unit (PICU) at Great Ormond Street Hospital (GOSH) highlighted myeloperoxidase (MPO), a hallmark of neutrophil degranulation, as a key indicator of disease severity when compared to control infants. The aim of this study is to investigate MPO as a functional readout of therapeutic efficacy. To mechanistically investigate these clinical observations, we developed a physiologically relevant in vitro model of the paediatric airway. Differentiated paediatric airway epithelial cells (AECs) were cultured at air-liquid interface (ALI), with or without an underlying layer of vascular endothelial cells. Following RSV infection, neutrophil migration and activation were assessed using flow cytometry. The inclusion of an endothelial layer enhanced physiological relevance and more accurately replicated in vivo MPO responses. To evaluate the therapeutic modulation of neutrophil activation, two antiviral compounds—remdesivir (RDV) and RSV604—were evaluated. While both compounds significantly reduced RSV viral load at 24 hours post-infection, only RSV604 attenuated MPO expression. RDV had no measurable effect on MPO levels, suggesting limited efficacy in mitigating neutrophil-driven inflammation. These findings highlight MPO as a biomarker of RSV severity and a functional readout of therapeutic impact. Targeting MPO-driven neutrophil activation may be key to reducing both viral load and inflammation. Take home message: Antiviral drug discovery should include neutrophil MPO reduction as a readout of therapeutic efficacy. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Animal Free Research UK, AFR19-20274 UK Research and Innovation, https://ror.org/001aqnf71, BB/V006738/1 [1]: pending:yes

Basal cells are key to maintaining and repairing a functioning airway epithelium. Understanding how basal cells maintain normal airways provides a foundation for interpreting their dysfunction in disease states and for the development of novel therapies. The airway epithelium exists in a dynamic state in which basal stem cells replace lost luminal mucosecretory and multiciliated cell types via an intermediate ‘suprabasal’ cell state. The ability to isolate basal cells with high progenitor cell potential would be beneficial in regenerative medicine applications, but the molecular identity of this population is unclear. Here, we evaluate candidate surface markers to isolate human basal cells. As an individual marker, we found that podoplanin (PDPN) had a favorable sensitivity and specificity compared with integrin alpha 6 (ITGA6) or nerve growth factor receptor (NGFR). We found that KRT5-expressing basal cells could be subdivided into those with high or low PDPN expression; KRT5-negative cells did not express PDPN. In vitro , PDPN-high basal cells had higher colony-forming capacity, increased population doubling potential and formed larger colonies than PDPN-low basal cells. PDPN-high basal cells expressed higher levels of TP63 , as well as other genes expressed by ‘quiescent’ or ‘resting’ basal cells identified in single cell RNA sequencing studies. PDPN-low basal cells expressed genes associated with a ‘differentiating’ basal cell state, including KRT4 , NOTCH3 and serpin B family genes. Our results demonstrate that PDPN expression can identify basal cells with high progenitor cell potential, enabling high efficiency sorting of airway stem cells.

Regeneration of the airway epithelium restores barrier function and mucociliary clearance following lung injury and infection. The mechanisms regulating the proliferation and differentiation of tissue-resident airway basal stem cells remain incompletely understood. To identify compounds that promote human airway basal cell proliferation, we performed phenotype-based compound screening of 1429 compounds (from the ENZO and Prestwick Chemical libraries) in 384-well format using primary cells transduced with lentiviral luciferase. A total of 17 pro-proliferative compounds were validated in independent donor cell cultures, including the antiretroviral therapy agent abacavir and several Wnt signalling pathway-activating compounds. The effects of compounds on proliferation were further explored in colony formation and 3D organoid assays. Structurally and functionally related compounds that more potently induced Wnt pathway activation were investigated. One such compound, 1-azakenpaullone, induced Wnt target gene activation and basal cell proliferation in mice. Our results demonstrate the pro-proliferative effect of small-molecule Wnt pathway activators on airway basal cells. These findings contribute to the rationale to develop novel approaches to modulate Wnt signalling during airway epithelial repair.

Modeling human epithelial diseases and developing cell-based therapies require robust methods to expand and manipulate epithelial stem and progenitor cells in vitro . Basal stem/progenitor cells from stratified epithelia can be expanded in 3T3-J2 fibroblast feeder cell co-culture systems, and the addition of the ROCK inhibitor Y-27632 enhances proliferation and culture longevity, a phenomenon described as ‘conditional reprogramming’. Here, we present a method incorporating the small molecule WS6 to further improve the proliferation and lifespan of cultured epithelial cells from multiple tissues, including airway, skin, and thymus. Cells maintained in this medium (‘EpMED’; FAD+Y+WS6) retain basal stem/progenitor cell identity and function, including the capacity to differentiate. We demonstrate their capacity to engraft in vivo in a tracheal transplantation model. In a second application, we generate clonal CRISPR-Cas9 genome edited nasal cultures, introducing targeted knockouts of DNAH5 or DNAI2 to create primary ciliary dyskinesia disease models. We anticipate that our method will have broad applications in epithelial cell biology, disease modeling, and regenerative medicine, while reducing reliance on immortalized or cancer cell lines and animal experimentation. ### Competing Interest Statement SMJ has received fees for advisory board membership from BARD1 Life Sciences. He has received grant income from GRAIL Inc. and is an unpaid member of a GRAIL advisory board. SMJ has received lecture fees for academic meetings from Chiesi and AstraZeneca and his wife works for AstraZeneca. REH has received speaker fees from AstraZeneca and REH and DRP have received royalties as inventors on licensed intellectual property licensed to AstraZeneca that is unrelated to the work in this manuscript. The remaining authors declare no conflicts of interest. Medical Research Council, https://ror.org/03×94j517, MR/R015635/1, MR/W025051/1 Debra, GR000070 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982 Rosetrees Trust NIHR Great Ormond Street Hospital Biomedical Research Centre Royal Society, RG\R1\241421 Great Ormond Street Hospital Children's Charity Cancer Research UK