Biology subjects

Ryan, A. L.

Publications and source records attributed to Ryan, A. L..

15 recordsLinked to original sources

bioRxiv2026

Defining Functional Correction Thresholds in Primary Ciliary Dyskinesia for Effective Gene Therapies

RationalePrimary ciliary dyskinesia (PCD) is an inherited disorder characterized by defective motile cilia and impaired mucociliary clearance. Mutations in CCDC40 disrupt axonemal organization, resulting in dyskinetic or immotile cilia. While emerging therapies may restore function in only a subset of cells, the functional consequences of mixed populations of mutant and healthy cilia are not well understood. ObjectivesTo determine how defined mixtures of CCDC40-deficient and wild-type ciliated cells influence mucociliary transport. MethodsHuman bronchial epithelial cells were combined at varying ratios of CCDC40-deficient and wild-type cells and differentiated to model heterogeneous epithelia. High-speed video microscopy and particle-tracking algorithms were used to assess ciliary motion and mucus transport and combined with electron microscopy to evaluate cilia ultrastructure. Measurements and Main ResultsAirway differentiation was largely preserved with marked ultrastructural defects observed in CCDC40 cells, including multiple central centrioles, absent inner dynein arms, and basal body misorientation. Ciliated surface coverage decreased, and goblet coverage increased with higher mutant representation. Mucociliary transport declined nonlinearly, with speeds dropping from [~]56 {micro}m/s (100% WT) to [~]9 {micro}m/s in 100% mutant cultures. Clearance-per-beat and flow coordination decreased sharply with rising mutant burden. Modeling revealed that transport efficiency was equivalent to that recorded in ex vivo human tissues and plateaued when [~]75% of the ciliated population was WT. ConclusionsTogether, these findings define a PCD-specific functional correction threshold and show that effective therapy must overcome the disruptive biomechanical and cellular influences of mutant epithelial cells, providing a quantitative benchmark to guide gene-therapy design and clinical translation.

cell biology↗

bioRxiv2025

Recapitulating apicobasal tissue polarity in extracellular matrix incorporated airway organoids

The airway epithelium is a dynamic barrier that interfaces with the external environment and internal matrix along its apicobasal axis. To recapitulate this tissue arrangement in an organoid format, we present the decellularized ExtraCellular Matrix-incorporated Apical-out Airway Organoid (dECM-AoAO) that integrates basolateral matrix cues through incorporation of human lung dECM microparticles, while maintaining direct apical exposure to the exterior. Compared to the ECM-free AoAO, dECM incorporation effectively diversifies lineage distribution that better recapitulates native epithelial composition. Harnessing dECM-AoAO locomotion powered by its outward-facing ciliary beating, we developed an experimental and computational pipeline for swarm analysis of organoid group motility as a functional readout of ciliary function. Lastly, dECM-AoAO withstood cryopreservation and revival with sustained viability, lineage composition, and ciliary function, enabling future scalability and broad distribution. Together, this work establishes dECM-AoAO as a more physiologically relevant model system for investigating epithelial-ECM crosstalk during airway homeostasis, pathogenesis, and injury responses.

cell biology↗

bioRxiv2024

Engineered hydrogel biomaterials facilitate lung progenitor cell differentiation from induced pluripotent stem cells

Lung progenitor (LP) cells identified by the expression of transcription factor NK2 homeobox 1 (NKX2.1) are essential for development of all lung epithelial cell types and hold tremendous potential for pulmonary research and translational regenerative medicine applications. Here we present engineered hydrogels as a promising alternative to the naturally derived materials that are often used to differentiate human induced pluripotent stem cells (iPSCs) into LP cells. Poly(ethylene glycol) norbornene (PEGNB) hydrogels with defined composition were used to systematically investigate the role of microenvironmental stiffness, cell origin, and splitting during the differentiation process. Results demonstrated each factor impacted LP differentiation efficiency. Soft hydrogels replicating healthy lung stiffness (Elastic modulus (E), E = 4.00 {+/-} 0.25 kPa) produced the highest proportion of LP cells (54% by flow cytometry), stiff hydrogels (E = 18.83 {+/-} 2.24 kPa) resulted in 48% differentiation efficiency, and a thin coating of Matrigel on tissue culture (TC) plastic (E[~]3 GPa) resulted in the lowest proportion of LP cells (32%) at the end of the non-split differentiation protocol. Collectively these results showed that engineered hydrogels enabled control over parameters that impacted differentiation and produced LP cells using well-defined microenvironments that may improve our ability to translate iPSC-derived LP cells into clinical applications. NEW & NOTEWORTHYStandard iPSC differentiation protocols rely on Matrigel, a basement membrane extract from mouse sarcoma cells that is poorly defined and exhibits significant batch-to-batch variation. Due to these limitations Matrigel-derived products have never been approved by the Food and Drug Administration. This study introduces a novel method for differentiating iPSCs into lung progenitor cells using well-defined hydrogel substrates. These biomaterials not only enhance differentiation efficiency, but also streamline the regulatory pathway, facilitating their potential therapeutic application.

bioengineering↗

bioRxiv2024

Culture Conditions Differentially Regulate the Inflammatory Niche and Cellular Phenotype of Tracheo-Bronchial Basal Stem Cells

Human bronchial epithelial cells (HBECs) derived from the tracheo-bronchial regions of human airways provide an excellent in vitro model for studying pathological mechanisms and evaluating therapeutics in human airway cells. This cell population comprises a mixed population of basal cells (BCs), the predominant stem cell in airways capable of both self-renewal and functional differentiation. Despite their potential for regenerative medicine, BCs exhibit significant phenotypic variability in culture. To investigate how culture conditions influence BC phenotype and function, we expanded three independent BC isolates in three media, airway epithelial cell growth medium (AECGM), dual-SMAD inhibitor (DSI)-enriched AECGM, and Pneumacult Ex plus (PEx+). Extensive RNA sequencing, immune assays and electrical measurements revealed that PEx+ media significantly drove cell proliferation and a broad pro-inflammatory phenotype in BCs. In contrast, BCs expanded in AECGM, displayed increased expression of structural and extracellular matrix components at high passage. Whereas culture in AECGM increased expression of some cytokines at high passage, DSI suppressed inflammation altogether thus implicating TGF-{beta} in BC inflammatory processes. Differentiation capacity declined with time in culture irrespective of expansion media except for PLUNC expressing secretory cells that were elevated at high passage in AECGM and PEx+ suggestive of an immune modulatory role of PLUNC in BCs. These findings underscore the profound impact of media conditions on inflammatory niche and function of in vitro expanded BCs. The broad pro-inflammatory phenotype driven by PEx+ media, in particular, should be considered in the development of cell-based models for airway diseases and therapeutic application. NEW & NOTEWORTHYAirway basal cells, vital for airway regeneration and potential therapies, show significant changes based on culture conditions. Our study reveals that media composition and culture duration greatly affect basal cell properties with profound changes in the pro-inflammatory phenotype and extracellular matrix deposition driven by changes in growth conditions. These results underscore the critical impact of culture conditions on BC phenotype, influencing cell-based models for airway disease research and therapy.

cell biology↗

bioRxiv2023

Structure-function relationships of mucociliary clearance in the human airways

Mucociliary clearance is a vital defense mechanism of the human airways, protecting against harmful particles and infections. When this process fails, it contributes to respiratory diseases like chronic obstructive pulmonary disease (COPD) and asthma. While advances in single-cell transcriptomics have revealed the complexity of airway composition, much of what we know about how airway structure impacts clearance relies on animal studies. This limits our ability to create accurate human-based models of airway diseases. Here we show that the airways in female rats and in humans exhibit species-specific differences in the distribution of ciliated and secretory cells as well as in ciliary beat, resulting in significantly higher clearance effectiveness in humans. We further reveal that standard lab-grown cultures exhibit lower clearance effectiveness compared to human airways, and we identify the underlying structural differences. By combining diverse experiments and physics-based modeling, we establish universal benchmarks to assess human airway function, interpret preclinical models, and better understand disease-specific impairments in mucociliary clearance.

biophysics↗

bioRxiv2023

MixOmics Integration of Biological Datasets Identifies Highly Correlated Key Variables of COVID-19 severity.

BackgroundDespite several years since the COVID-19 pandemic was declared, challenges remain in understanding the factors that can predict the severity of COVID-19 disease and complications of SARS-CoV-2 infection. While many large-scale Multiomic datasets have been published, integration of these datasets has the potential to substantially increase the biological insight gained allowing a more complex comprehension of the disease pathogenesis. Such insight may improve our ability to predict disease progression, detect severe cases more rapidly and develop effective therapeutics. MethodsIn this study we have applied an innovative machine learning algorithm to delineate COVID-severity based on integration of paired samples of proteomic and transcriptomic data from a small cohort of patients testing positive for SARS-CoV-2 infection with differential disease severity. Targeted plasma proteomics and an onco-immune targeted transcriptomic panel was performed on sequential samples from a cohort of 23 severe, 21 moderate and 10 mild COVID-19 patients. We applied DIABLO, a new integrative method, to identify multi-omics biomarker panels that can discriminate between multiple phenotypic groups, such as the varied severity of disease in COVID-19 patients. ResultsAs COVID-19 severity is known among our sample group, we can train models using this as the outcome variable and calculate features that are important predictors of severe disease. In this study, we detect highly correlated key variables of severe COVID-19 using transcriptomic discriminant analysis and multi-omics integration methods. ConclusionsThis approach highlights the power of data integration from a small cohort of patients offering a better biological understanding of the molecular mechanisms driving COVID-19 severity and an opportunity to improve prediction of disease trajectories and targeted therapeutics.

systems biology↗

bioRxiv2023

RBL2 represses the transcriptional activity of Multicilin to inhibit multiciliogenesis

A core pathophysiologic feature underlying many respiratory diseases is multiciliated cell dysfunction, leading to inadequate mucociliary clearance. Due to the prevalence and highly variable etiology of mucociliary dysfunction in respiratory diseases, it is critical to understand the mechanisms controlling multiciliogenesis that may be targeted to restore functional mucociliary clearance. Multicilin, in a complex with E2F4, is necessary and sufficient to drive multiciliogenesis in airway epithelia, however this does not apply to all cell types, nor does it occur evenly across all cells in the same cell population. In this study we further investigated how co-factors regulate the ability of Multicilin to drive multiciliogenesis. Combining data in mouse embryonic fibroblasts and human bronchial epithelial cells, we identify RBL2 as a repressor of the transcriptional activity of Multicilin. Knockdown of RBL2 in submerged cultures or phosphorylation of RBL2 in response to apical air exposure, in the presence of Multicilin, allows multiciliogenesis to progress. These data demonstrate a dynamic interaction between RBL2 and Multicilin that regulates the capacity of cells to differentiate and multiciliate. Identification of this mechanism has important implications for facilitating MCC differentiation in diseases with impaired mucociliary clearance.

cell biology↗

bioRxiv2023

Sorafenib inhibits invasion of multicellular organoids that mimic Lymphangioleiomyomatosis nodules.

Lymphangioleiomyomatosis (LAM) is a progressive lung disease with limited treatments, largely due to an incomplete understanding of its pathogenesis. Lymphatic endothelial cells (LECs) invade LAM cell clusters, which include HMB-45-positive epithelioid cells and smooth muscle -actin-expressing LAM-associated fibroblasts (LAMFs). Recent evidence shows that LAMFs resemble cancer-associated fibroblasts, with LAMF-LEC interactions contributing to disease progression. To explore these mechanisms, we used spatial transcriptomics on LAM lung tissues and identified a gene cluster enriched in kinase signaling pathways linked to myofibroblasts and co-expressed with LEC markers. Kinase arrays revealed elevated PDGFR and FGFR in LAMFs. Using a 3D co-culture spheroid model of primary LAMFs and LECs, we observed increased invasion in LAMF-LEC spheroids compared to non-LAM fibroblasts. Treatment with sorafenib, a multikinase inhibitor, significantly reduced invasion, outperforming Rapamycin. We also confirmed TSC2-null AML cells as key VEGF-A secretors, which was suppressed by sorafenib in both AML cells and LAMFs. These findings highlight VEGF-A and bFGF as potential therapeutic targets and suggest multikinase inhibition as a promising strategy for LAM. One Sentence SummaryUsing 3D spheroids and spatial transcriptomics, we identified LAMFs and LECs as key contributors to LAM, with bFGF and VEGF-A as potential therapeutic targets

cell biology↗

bioRxiv2022

Proteomic profiling identifies biomarkers of COVID-19 severity

Abstract/SummarySARS-CoV-2 infection remains a major public health concern, particularly for the aged and those individuals with co-morbidities at risk for developing severe COVID-19. Understanding the pathogenesis and biomarkers associated with responses to SARS-CoV-2 infection remain critical components in developing effective therapeutic approaches, especially in cases of severe and long-COVID-19. In this study blood plasma protein expression was compared in subjects with mild, moderate, and severe COVID-19 disease. Evaluation of an inflammatory protein panel confirms upregulation of proteins including TNF{beta}, IL-6, IL-8, IL-12, already associated with severe cytokine storm and progression to severe COVID-19. Importantly, we identify several proteins not yet associated with COVID-19 disease, including mesothelin (MSLN), that are expressed at significantly higher levels in severe COVID-19 subjects. In addition, we find a subset of markers associated with T-cell and dendritic cell responses to viral infection that are significantly higher in mild cases and decrease in expression as severity of COVID-19 increases, suggesting that an immediate and effective activation of T-cells is critical in modulating disease progression. Together, our findings identify new targets for further investigation as therapeutic approaches for the treatment of SARS-CoV-2 infection and prevention of complications of severe COVID-19.

bioinformatics↗

bioRxiv2022

Alveolar epithelial type 1 cells serve as a cell of origin for lung adenocarcinoma with distinct molecular and phenotypic presentation

Lung adenocarcinoma (LUAD) is the most common subtype of cancer arising in the distal lung. LUAD encompasses several pathologic subtypes, each with differing clinical outcomes and biological behaviors. However, the molecular and cellular underpinnings of the different subtypes are largely unknown. Understanding which cell populations in the distal lung contribute to LUAD could provide insights into the marked heterogeneity in pathologic features, clinical presentation and responses to therapy of LUAD. Differential expression analysis of lung adenocarcinoma transcriptomes from The Cancer Genome Atlas revealed distinct alveolar epithelial type 1 (AT1) and alveolar epithelial type 2 (AT2) cell signatures within human LUAD with significantly different survival outcomes between tumors expressing AT2 and AT1 gene signatures, suggesting AT1 cells might contribute to a subset of LUAD cases. To address this, we tested the ability of AT1 cells to give rise to LUAD following induction of KrasG12D, a known oncogenic driver of human LUAD. Activation of KrasG12D in Gram-domain containing 2 (Gramd2)+ AT1 cells gave rise to multiple LUAD lesions, primarily of papillary histology. In contrast, activation of KrasG12D in surfactant protein C (Sftpc+) AT2 cells resulted in LUAD lesions of lepidic histology. Immunohistochemistry established that Gramd2:KrasG12D lesions were of primary lung origin and not metastatic events. Spatial transcriptomic profiling revealed distinct pathway alterations within Gramd2- and Sftpc-derived LUAD. Immunofluorescence confirmed differences observed in the spatial transcriptomic analysis in expression patterns and distribution of cell-specific markers depending on cell of origin, while universal upregulation of the Krt8 intermediate cell state marker was observed. Our results are consistent with Gramd2+ AT1 cells serving as a putative cell of origin for LUAD and suggest that LUAD may be a collection of adenocarcinomas that share a common location within the distal lung but arise from different cells of origin.

cancer biology↗

bioRxiv2021

Neutrophil-epithelial interactions augment infectivity and pro-inflammatory responses to SARS-CoV-2 infection

In response to viral infection, neutrophils release inflammatory mediators as part of the innate immune response, contributing to pathogen clearance through virus internalization and killing. Pre-existing co- morbidities correlating to incidence of severe COVID-19 are associated with chronic airway neutrophilia. Furthermore, examination of COVID-19 explanted lung tissue revealed a series of epithelial pathologies associated with the infiltration and activation of neutrophils, indicating neutrophil activity in response to SARS- CoV-2 infection. To determine the impact of neutrophil-epithelial interactions on the infectivity and inflammatory responses to SARS-CoV-2 infection, we developed a co-culture model of airway neutrophilia. SARS-CoV-2 infection of the airway epithelium alone does not result in a notable pro-inflammatory response from the epithelium. The addition of neutrophils induces the release of proinflammatory cytokines and stimulates a significantly augmented pro-inflammatory response subsequent SARS-CoV-2 infection. The resulting inflammatory response is polarized with differential release from the apical and basolateral side of the epithelium. Additionally, the integrity of the epithelial barrier is impaired with notable epithelial damage and infection of basal stem cells. This study reveals a key role for neutrophil-epithelial interactions in determining inflammation and infectivity in response to SARS-CoV-2 infection.

cell biology↗

bioRxiv2021

Breathing on Chip: Biomechanical forces change airway epithelial cell biology ina human Airway Lung-Chip

Human lung function is intricately linked to blood flow and breathing cycles, but it remains unknown how these dynamic cues shape human airway epithelial biology. Here we report a state-of-the-art protocol for studying effects of physiological airflow and stretch on differentiation, cellular composition, and mucociliary clearance of human primary airway epithelial cells cultured on a perfused airway chip. Perfused epithelial tissue cultures developed a large airway-like cellular composition with accelerated maturation and polarization of mucociliary clearance when compared to traditional (static) culture methods. Additional application of airflow and stretch to the airway chip resulted in a cellular composition more comparable to the small(er) airways, reduced baseline secretion of interleukin-8 and other inflammatory proteins, and reduced gene expression of matrix metalloproteinase (MMP) 9, fibronectin, and other extracellular matrix factors. These results indicate that breathing-like mechanical stimuli are important modulators of airway epithelial cell differentiation and homeostasis and that their fine-tuned application could generate models of specific epithelial regions, pathologies, and mucociliary (dys)function.

cell biology↗

bioRxiv2021

Comprehensive epigenomic profiling of human alveolar epithelial differentiation identifies key epigenetic states and transcription factor co-regulatory networks for maintenance of distal lung identity

Disruption of alveolar epithelial cell (AEC) differentiation is implicated in peripheral lung diseases strongly impacting morbidity and mortality worldwide, such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma. Elucidating underlying disease pathogenesis requires a mechanistic molecular understanding of AEC differentiation. However, to date no study has comprehensively characterized the dynamic epigenomic alterations that facilitate this critical process in humans. We comprehensively profiled the epigenomic states of human AECs during type 2 to type 1-like cell differentiation, including the methylome and chromatin functional domains, and integrated this with transcriptome-wide RNA expression. Enhancer regions were drastically altered during AEC differentiation. Transcription factor binding analysis within enhancer regions revealed diverse interactive networks with enrichment for dozens of transcription factors, including NKX2-1 and FOXA family members, as well as transcription factors with previously uncharacterized roles in lung differentiation, such as members of the MEF2, TEAD, and AP1 families. Additionally, associations between transcription factors changed during differentiation, implicating a complex network of heterotrimeric complex switching may be involved in facilitating differentiation. Integration of AEC enhancer states with the catalog of enhancer elements in the Roadmap Epigenomics Mapping Consortium and Encyclopedia of DNA Elements (ENCODE) revealed that human mammary epithelial cells (HMEC) have a similar epigenomic structure to alveolar epithelium, with NKX2-1 serving as a distinguishing feature of distal lung differentiation. Taken together, our results suggest that enhancer regions with dynamic transcription factor interactions are hotspots of epigenomic alteration that help to facilitate AEC differentiation. Author SummaryHuman health and disease states are heavily influenced by the critical cellular processes that regulate and protect our genomes. One of these safeguards is the epigenome; the coordinated set of signals overlaid on top of our DNA that controls what can happen to a given stretch DNA. Hence, epigenomic signatures play a critical role in the development and maintenance of cellular fate and function. To determine the relationship between epigenomic alterations and cellular fates of distal lung cells in humans during the process that regenerates the human lung epithelial layer after injury, we performed comprehensive genome-wide profiling of many epigenetic modifications that have roles in regulating the function of the underlying DNA. We found that changes to enhancer regions, which act to turn on associated gene expression, were the major alterations to the epigenome during distal lung differentiation, and that within those regions dynamic changes in transcription factor associations were occurring to facilitate this process. We then characterize what was similar and distinct to the enhancers of distal lung from among other epithelial tissues and describe a novel role for specific transcription factors in this process that previously had no known role in normal lung repair.

genomics↗

bioRxiv2020

Development of novel in vitro human alveolar epithelial cell models to study distal lung biology and disease

Many acute and chronic lung diseases affect the distal lung alveoli. Although airway-derived human cell lines exist, alveolar epithelial cell (AEC)-derived lines are needed to better model these diseases. We have generated and characterized novel immortalized cell lines derived from human AECs. They grow as epithelial monolayers expressing lung progenitor markers SOX9 and SOX2, with little to no expression of mature AEC markers. Co-cultured in 3-dimensions (3D) with lung fibroblasts, the cells form NKX2-1+ organoids expressing mature AEC markers AQP5 and GPRC5A. Single-cell RNA sequencing of an AEC line in 2D versus 3D revealed increased cellular heterogeneity and induction of cytokine and lipoprotein signaling, consistent with organoid formation. Activating WNT and FGF pathways resulted in larger organoids. Our approach appears to yield lung progenitor lines that retain a genetic and structural memory of their alveolar cell lineage despite long-term expansion and whose differentiation may be modulated under various 3D conditions. These cell lines provide a valuable new system to model the distal lung in vitro.

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↗