The airway epithelium interfaces with the external environment through its apical surface and with the extracellular matrix (ECM) through its basolateral surface. To model this organization in vitro, we developed a decellularized ECM-incorporated apical-out airway organoid (dECM-AoAO) platform in which human bronchial epithelial cells (HBECs) self-assemble around human lung-derived decellularized ECM microparticles (dECM-MPs). This configuration preserves apical-out polarity while enabling direct epithelial-ECM interactions. Here, we describe a protocol for the vacuum filtration and quantification of dECM-MPs, the generation of dECM-AoAOs, and ultimately, whole-mount immunofluorescence staining for organoid characterization. Key features • This protocol incorporates dECM as size-refined microparticles, enabling a consistent and reproducible ECM input. • The workflow uses suspension culture to generate apicalout airway organoids, eliminating the need for matrix embedding while preserving ECM-cell interactions. • dECM-MPs are directly wrapped by epithelial cells, simplifying handling and supporting native-like epithelial-ECM integration. • The system is compatible with wholemount immunofluorescence staining for comprehensive epithelial lineage analysis.

The World Health Organization and the U.S. Centre for Disease Control and Prevention have reported that antibiotic-resistant infections with Pseudomonas aeruginosa present a significant health risk worldwide. In the genetic disease cystic fibrosis (CF), chronic antibiotic-resistant Pseudomonas lung infections and persistent inflammation remain the leading causes of mortality. While highly effective modulator therapy (HEMT) dramatically improves lung function in CF, they fail to eradicate chronic infections or eliminate the associated hyperinflammatory state. Thus, there is an urgent need for innovative therapies that can simultaneously eliminate antibiotic-resistant P. aeruginosa lung infection and the attendant hyperinflammatory lung environment. Mesenchymal stromal cell-derived extracellular particles (MSC EPs) represent a promising solution, offering potent anti-inflammatory and antimicrobial properties while being safe and non-toxic. This study demonstrates, using a CF mouse model of infection, that MSC EPs reduce acute P. aeruginosa lung infection and inflammation. As the first investigation of MSC EPs in CF mice, this research underscores the dual effects of MSC EPs; reducing inflammation and bacterial burden. These findings mark an important advancement in antimicrobial therapy, addressing the unmet need for reducing antibiotic-resistant infections and hyperinflammation in CF as well as other diseases with chronic, antibiotic-resistant P. aeruginosa infections.

Ex vivo airway engineering approaches such as 3D bioprinting offer a promising strategy for generating functional airway replacements, but the fabrication of hollow, patient-specific proximal airway constructs using translationally relevant bioinks remains challenging. This study describes the development of biocompatible, polymer-blended human airway-derived decellularized extracellular matrix (AW-dECM) bioinks for engineering structurally and mechanically relevant airway tissues. An optimal formulation consisting of 30 mg/mL AW-dECM and nanofibrillar cellulose alginate conjugated to RGD supported the bioprinting of simple and complex hollow airway structures with mechanical properties comparable to native airways (∼8-10 kPa). The bioinks also promoted primary human airway epithelial cell viability, adhesion, and differentiation into mucociliary and secretory phenotypes during 28 days of air-liquid interface culture. Furthermore, subcutaneous implantation in immunocompetent rats demonstrated excellent biodegradative stability and overall biocompatibility over 30 days. Collectively, these findings establish a foundation for improved physiological airway models and future tissue-engineered airway replacements.

The airway epithelium is a dynamic barrier that interfaces with the external environment and internal matrix along its apicobasal axis. To recapitulate this spatial organization and associated cell-ECM interactions in an organoid format, we present the decellularized extracellular matrix-incorporated Apical-out Airway Organoid (dECM-AoAO), which integrates basolateral matrix cues through the incorporation of human lung dECM microparticles (dECM-MPs), while maintaining direct apical exposure to the exterior. Compared to ECM-free AoAOs, dECM incorporation diversifies lineage distribution, more closely recapitulating native epithelial composition and responsiveness to pathogenic stimulation. Harnessing dECM-AoAO locomotion driven by outward-facing ciliary beating, we developed an experimental and computational pipeline for batch analysis of organoid motility as a functional readout of ciliary activity. Furthermore, dECM-AoAOs are compatible with cryopreservation, retaining viability, lineage composition, and ciliary function upon revival. Together, this work establishes the dECM-AoAO as a physiologically relevant model system for investigating epithelial-ECM crosstalk during airway homeostasis, pathogenesis, and injury responses.

Three-dimensional human airway models are useful tools to study respiratory development, disease and regeneration. However, commonly used substrates such as Matrigel™ differ substantially from native airway ECM, and effects on cell behavior remain incompletely characterized. Human airway-derived decellularized ECM (AW-dECM) hydrogels represent a potentially more physiological alternative, but their properties relative to conventional matrices are undefined.We performed a comprehensive characterization of AW-dECM and Matrigel™ hydrogels by assessing ECM composition, stiffness and viscoelasticity and evaluated effects on primary human bronchial epithelial cell behavior at air-liquid interface. AW-dECM (15 mg/mL; 30 mg/mL) exhibited lower stiffness and greater similarity to native airway tissue than standard concentration (SC; 8 mg/mL) or high concentration (HC; 19 mg/mL) Matrigel™. All hydrogels displayed typical viscoelastic behavior. Proteomic analysis revealed collagen I and VI enrichment in AW-dECM that was absent in Matrigel™. These differences lead to distinct cellular organization: SC Matrigel™ promoted organoid formation, HC Matrigel™ supported tubule-like structures/monolayers, and AW-dECM favored confluent monolayers. Gene expression varied by substrate; stiffer matrices promoted greater secretory differentiation and AW-dECM increased expression of MMP9, MMP7, and TJP1. Inhibition of actomyosin contractility enhanced cell adhesion/spreading on softer matrices, further supporting the influence of matrix properties on airway epithelial cell behavior.AW-dECM and Matrigel™ exhibit distinct mechanical and biochemical properties that differentially influence airway epithelial cell growth, organization, and differentiation. This work establishes a detailed characterization of human airway-derived ECM hydrogels and provides a framework linking matrix properties with airway epithelial responses, thereby informing the development of more physiological airway models.

Over the past two decades there have been remarkable advances in stem cell biology, bioengineering, and lung regenerative research, transforming our understanding of pulmonary biology from development to repair, and disease. Strategies using endogenous lung progenitor cells, pluripotent stem cell technologies, and engineered tissue platforms have become central tools for interrogating lung biology. Major breakthroughs have included the identification of diverse cell populations that coordinate lung homeostasis and repair, facilitated by the extensive adoption of single cell, multiomic and spatialomics approaches. Simultaneous progress in biomaterials, organoid systems, decellularized lung scaffolds, and lung-on-chip platforms has uncovered how extracellular matrix composition, mechanical forces, and tissue architecture contribute to the regulation of cell fate and function. These advances have enabled increasingly physiologically relevant in vitro, and ex vivo models while informing tissue engineering strategies aimed ultimately at functional lung replacement. Translation toward the clinic has advanced through both cell-based and cell-free therapeutic strategies. Early efforts focused largely on mesenchymal stromal cell-based approaches and extracellular vesicles, which have demonstrated safety and context-dependent efficacy in inflammatory lung diseases, alongside emerging preclinical evidence of functional engraftment of induced pluripotent stem cell-derived lung lineages. The past twenty years of progress, captured at the 20th Anniversary Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, highlights the power of interdisciplinary collaboration in advancing lung regeneration from foundational discovery toward therapeutic reality.

Abstract Background The variable clinical outcomes of mesenchymal stromal cell (MSC)-based therapy in acute respiratory distress syndrome (ARDS) are attributed to a variety of factors, including host microenvironmental factors. Interleukin-1β (IL-1β) has been linked to the development and progression of ARDS, and we have previously found that IL-1β could be used to predict MSC activation in vitro. However, the exact mechanisms through which IL-1β alters the MSC function and its interaction with the host immune cells remains unknown. Therefore, the aim of this study was to assess how IL-1β alters MSC function, with a specific focus on MSC-neutrophil interaction. Methods Human bone marrow-derived MSCs were exposed to 20 ng/ml IL-1β for 1 or 24 h. Following exposure, MSCs were analyzed using bulk RNA sequencing and key secretome proteins were measured in their conditioned medium. A transwell culture system was used to evaluate the neutrophil recruitment capacity of IL-1β-exposed MSCs, with or without NF-kB inhibition. MSCs exposed to serum free medium were used as controls in all experiments. Results The sequencing data revealed that genes involved in response to biotic stimuli and immune response were altered in MSCs exposed to IL-1β compared to control cells. In particular, genes essential for neutrophil recruitment were significantly upregulated after IL-1β exposure. The functional in vitro studies further validated these results, demonstrating that MSCs exposed to IL-1β had a significantly higher neutrophil recruitment capacity compared to unstimulated MSCs. Finally, inhibition of the NF-kB pathway resulted in a significant decrease of the MSC’s capacity to recruit neutrophils to levels similar as to the unstimulated control MSCs. Conclusion These data provide mechanistic insight into how inflammatory factors present in the host microenvironment might affect the interaction between MSCs and immune cells. This further highlights the need to understand the MSC mode of action, and to map out how the MSC fate might change in different host environments after administration.

Three-dimensional bioprinting offers a promising strategy for the fabrication of physiologically relevant airway constructs; however, generating hollow, anatomically accurate proximal airway structures using tissue-specific bioinks with sufficient printability and mechanical stability remains challenging. This study describes the development and characterization of biocompatible, polymer-blended human airway-derived decellularized extracellular matrix (AW-dECM) bioinks for extrusion bioprinting of structurally and mechanically relevant proximal airway-like hollow constructs. A formulation consisting of 30 mg/mL AW-dECM and nanofibrillar cellulose-alginate conjugated to RGD supported the fabrication of simple and complex hollow airway structures with elastic moduli of approximately 8-10 kPa, within the range measured for bulk proximal airway tissue. The bioinks also supported preliminary primary human airway epithelial cell viability and adhesion, as well as gene expression patterns consistent with differentiation toward mucociliary and secretory phenotypes during 28 days of air-liquid interface culture. Following subcutaneous implantation in immunocompetent rats, acellular AW-dECM polymer-blended bioinks maintained structural integrity over 30 days and were tolerated without evidence of infection or tissue necrosis, although a localized foreign body response was observed. Collectively, these findings demonstrate proof of concept for combining tissue-specific AW-dECM with commercially available printable polymers to fabricate mechanically relevant hollow airway-like constructs. This approach provides a basis for further development of advanced ex vivo airway models and regenerative airway engineering strategies.

Background and aims Human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) and their extracellular vesicles (EVs) reduce lung inflammation and fibrosis in a variety of model systems, including in a Cystic Fibrosis (CF) mouse model. Many components of MSC-derived EVs, including cytokines, antimicrobial peptides, and miRNAs have been implicated in their anti-inflammatory effects. However, a major gap in our knowledge of using MSC as a therapeutic intervention for people with CF (pwCF) is whether the CF airway environment compromises miRNA cargo in hBM-MSC-derived EVs. Methods To assess this, hBM-MSCs were exposed to cell culture media (control) or to bronchoalveolar lavage fluid (BALF) obtained from pwCF or healthy controls (HC) and compositional analysis of EV miRNA content was conducted. Results Thirteen miRNAs (each ≥1% of the total miRNA content) were identified that collectively account for ∼70% of the miRNA content of EVs. These miRNAs were remarkably stable across treatments. To infer potential therapeutic effects, we identified predicted gene targets of these miRNAs and performed pathway enrichment analysis. Gene pathway analysis revealed that many of the 13 miRNAs are predicted to inhibit TLR signaling, NF-κB activation, TGF-β-mediated fibrosis, and cytokine production. Conclusions These results indicate that miRNAs secreted by hBM-MSCs in EVs may contribute to the observed anti-inflammatory and anti-fibrotic effects in experimental models and that exposure to CF BALF does not significantly diminish the abundance of the 13 miRNAs.

Cystic Fibrosis (CF) is a multiorgan disease caused by mutations in the CFTR gene, leading to chronic pulmonary infections and hyperinflammation. Among pathogens colonizing the CF lung, Pseudomonas aeruginosa is predominant, infecting over 50% of adults with CF, and becoming antibiotic-resistant over time. Current therapies for CF, while providing tremendous benefits, fail to eliminate persistent bacterial infections, chronic inflammation, and irreversible lung damage, necessitating novel therapeutic strategies. Our group engineered mesenchymal stromal cell derived extracellular vesicles (MSC EVs) to carry the microRNA let-7b-5p as a dual anti-infective and anti-inflammatory treatment. MSC EVs are low-immunogenicity platforms with innate antimicrobial and immunomodulatory properties, while let-7b-5p reduces biofilm formation and inflammation. In a preclinical CF mice model, we reported that let-7b-5p-loaded MSC EVs reduced P. aeruginosa burden, immune cells, and proinflammatory cytokines in the lungs. We hypothesize four complementary mechanisms for the observed in-vivo effects of the let-7b-5p loaded MSC EVs: antimicrobial activity, anti-inflammatory properties, inhibition of antibiotic-resistant P. aeruginosa biofilm formation in CF airways, and stimulation of anti-inflammatory macrophage behaviors. This study focused on the second and third mechanisms and demonstrates that MSC EVs engineered to contain let-7b-5p effectively blocked the formation of antibiotic-resistant P. aeruginosa biofilms on primary human bronchial epithelial cells (pHBECs) while also reducing P. aeruginosa-induced inflammation. This approach holds promise for improving outcomes for people with CF. Future work will focus on optimizing delivery strategies and expanding the clinical applicability of MSC EVs to target other CF-associated pathogens.

Cystic fibrosis (CF) is a multiorgan disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, leading to chronic pulmonary infections and hyperinflammation. Among pathogens colonizing the CF lung, Pseudomonas aeruginosa is predominant, infecting over 50% of adults with CF, and becoming antibiotic-resistant over time. Current therapies for CF, while providing tremendous benefits, fail to eliminate persistent bacterial infections, chronic inflammation, and irreversible lung damage, necessitating novel therapeutic strategies. Our group engineered mesenchymal stromal cell-derived extracellular vesicles (MSC EVs) to carry the microRNA let-7b-5p as a dual anti-infective and anti-inflammatory treatment. MSC EVs are low-immunogenicity platforms with innate antimicrobial and immunomodulatory properties, whereas let-7b-5p reduces inflammation. This study demonstrates that MSC EVs effectively blocked the formation of antibiotic-resistant P. aeruginosa biofilms on primary human bronchial epithelial cells (pHBECs), and let-7b-5p loading into MSC EVs conferred additional anti-inflammatory effects by reducing P. aeruginosa-induced IL-8 secretion by pHBECs. This approach holds promise for improving outcomes for people with CF, and future work will focus on optimizing delivery strategies and expanding the clinical applicability of MSC EVs to target other CF-associated pathogens.NEW & NOTEWORTHY This is the first study demonstrating that mesenchymal stromal cell extracellular vesicles (MSC EVs) block antibiotic-resistant P. aeruginosa biofilm formation and that let-7b-5p-loaded MSC EVs reduce inflammation in CF primary human bronchial epithelial cells.

The acute respiratory distress syndrome (ARDS) inflammatory environment alters mesenchymal stromal cell (MSC) gene and protein expression but effects on microRNA (miRNA) content of MSC-extracellular vesicle (EVs) remain unknown. To assess this, sequencing analysis of EV-miRNAs prepared from human bone marrow-derived MSCs (hMSCs) exposed ex vivo to bronchoalveolar lavage fluid (BALF) from ARDS patients or healthy volunteers (HV) identified a number of differentially expressed miRNAs. Discriminant, differential expression, and functional enrichment analyses identified 14 miRNAs significantly changed following ARDS versus HV BALF exposure. Network analysis showed 4 (miR-760, miR-3175, miR-885-3p, and miR-766-3p) of the 14 EV-miRNAs formed a regulatory “hub”, suggesting co-targeting of specific gene pathways. In silico prediction identified a number of pathways important in lung injury. Two miRNAs involved in regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), miRNA-145-5p and miRNA-138-5p, were also significantly increased in ARDS BALF-exposed hMSCs EVs. Functionally, EVs from hMSCs exposed to either ARDS or HV BALF had differential effects on CFTR Cl- secretion by cultured primary human bronchial epithelial cells, an effect predicted to reduce mucociliary clearance. The potential clinical impact of these finding highlights the need for further studies assessing the role of hMSC-EV miRNAs in regulating lung inflammation and mucociliary clearance.

The implications of climate change for malaria eradication in the 21st century remain poorly resolved. Many studies have focussed on parasite and vector ecology in isolation, neglecting the interactions between climate, malaria control, and the socioeconomic environment, including the disruptive impact of extreme weather. Here we integrate 25 years of data on climate, malaria burden, control interventions, socioeconomic factors, and extreme weather events in Africa. Using a geotemporal model linked to an ensemble of climate projections under the Shared Socioeconomic Pathway 2-4.5 (SSP 2-4.5) scenario, we estimate the future impact of climate change on malaria burden in Africa, accounting for both ecological and disruptive effects. Our findings suggest climate change could lead to 123 million (projection range 49.5 million - 203 million) additional malaria cases and 532,000 (195,000 - 912,000) additional deaths in Africa between 2024 and 2050 under current control levels. Contrary to the prevailing focus on ecological mechanisms, extreme weather events emerge as the primary driver of increased risk, accounting for 79% (50-94%) of additional cases and 93% (70%-100%) of additional deaths. Most increases are due to intensification in existing endemic areas rather than range expansion, with significant regional variation in impact. These results highlight the urgent need for climate-resilient malaria control strategies and robust emergency response systems to safeguard progress toward malaria eradication in Africa. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Funding for this work was primarily from the Bill and Melinda Gates Foundation (INV-055192, INV-075583). PWG is also supported by an NHMRC Investigator Grant (2025280). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The downscaled and bias-corrected CMIP6 climate projections used in this analysis are available from: https://registry.opendata.aws/nex-gddp-cmip6/. Citations to other supporting datasets (on historical climate variables, flood and hydrological modelling, cyclone modelling, topology, population density, remotely sensed landcover classifications, socioeconomic indicators, malaria infection prevalence and control coverage) are provided in the manuscript or in full in Supplementary Annex Table 1.

The December 2024 US Food and Drug Administration (FDA) approval of Mesoblast's Ryoncil (remestemcel-L-rknd)-allogeneic bone marrow mesenchymal stromal cell (MSC(M)) therapy-in pediatric acute steroid-refractory graft-versus-host-disease finally ended a long-lasting drought on approved MSC clinical products in the United States. While other jurisdictions-including Europe, Japan, India, and South Korea-have marketed autologous or allogeneic MSC products, the United States has lagged in its approval. The sponsor's significant efforts and investments, working closely with the FDA addressing concerns regarding clinical efficacy and consistent MSC potency through an iterative process that spanned several years, was rewarded with this landmark approval. This approval will revive investment and enthusiasm in MSC products, further approvals in major markets, and will continue to foreshadow the long-predicted success of MSCs as a pharmaceutical.

Urban population growth in Nigeria may exceed the availability of affordable housing and basic services, resulting in living conditions conducive to vector breeding and heterogeneous malaria transmission. Understanding the link between community-level factors and urban malaria transmission informs targeted interventions. We analyzed Demographic and Health Survey Program cluster-level data, alongside geospatial covariates, to describe variations in malaria prevalence in children under 5 years of age. Univariate and multivariable models explored the relationship between malaria test positivity rates at the cluster level and community-level factors. Generally, malaria test positivity rates in urban areas are low and declining. The factors that best predicted malaria test positivity rates within a multivariable model were post-primary education, wealth quintiles, population density, access to improved housing, child fever treatment-seeking, precipitation, and enhanced vegetation index. Malaria transmission in urban areas will likely be reduced by addressing socioeconomic and environmental factors that promote exposure to disease vectors. Enhanced regional surveillance systems in Nigeria can provide detailed data to further refine our understanding of these factors in relation to malaria transmission.

Languages vary in the mapping of relational terms onto events. For instance, English motion descriptions favor manner (how something moves) verbs over path (where something move) verbs, whereas those of other languages, like Spanish, show the opposite pattern. While these lexicalization biases are malleable, adopting a novel lexicalization pattern can be slow for second language learners. One potential mechanism for learning non-native verb mappings is cross-situational statistical learning (CSSL). However, the application of CSSL to verbs is limited and does not explicitly examine how lexicalization biases may complicate adults’ ability to resolve the referential uncertainty of multiple referents. We ask English-speaking monolingual adults to learn the mappings of ten verbs via CSSL. Verbs mapped onto either manner or path of motion, with the other event component held constant. Adults in both conditions demonstrated successful learning of novel verbs, with adults learning the manner verbs showing more consistent performance across accepting correct referents and rejecting incorrect ones. Our results are the first to demonstrate adults’ use of CSSL to acquire verb meanings that both align with and cut against native lexicalization biases and suggest a limited influence of lexicalization biases on adults’ learning in idealized CSSL conditions.

Multi-organ failure (MOF), particularly in the coexistence of acute kidney injury (AKI) and acute lung injury (ALI), presents a significant challenge in intensive care units (ICU) and is associated with exceedingly high mortality rates. Respiratory and renal failures are frequently managed by extracorporeal membrane oxygenation (ECMO) and continuous renal replacement therapy (CRRT), respectively. However, employing these therapies using separate devices requires specialized facilities, adds to complexity, and increases the risks of clotting due to the extensive artificial surface areas involved. Therefore, an integrated device capable of providing simultaneous respiratory and renal support is essential. This paper introduces the Pneuma-K ECLS system, which incorporates a multifunctional detoxifying filter (MDF) capable of performing gas exchange and renal replacement in a single cartridge. Ex-vivo blood tests confirmed the ability of the MDF to oxygenate blood, remove carbon dioxide, and eliminate uremic toxins. In addition, animal experiments demonstrated the considerable clinical potential of this novel integrated extracorporeal life support approach. Integrating respiratory and renal support into a singular device could mitigate risks, conserve resources, and enhance the survival rates of critically ill patients suffering from concurrent lung and kidney failure. ### Competing Interest Statement The authors have declared no competing interest.

Mesenchymal Stromal Cells (MSCs) have been used in multiple clinical trials for steroid-refractory moderate-severe (grade II-IV) acute Graft-versus-Host Disease (aGvHD) across the world over the last 2 decades. Despite very promising results in a variety of trials, it failed to get widespread approval by regulatory agencies such as the U.S. Food and Drug Administration and the European Medicines Agency. What lessons can we learn from this for future studies on MSCs and other cell therapy products? Broad heterogeneity among published trials using MSCs in aGVHD was likely the core problem. We propose a standardized approach in regards to donor-related factors, MSCs-related characteristics, as well as clinical trial design, to limit heterogeneity in trials for aGVHD and to fulfill the requirements of regulatory agencies. This approach may be expanded beyond MSCs to other Cell and Gene therapy products and trials in other diseases.