Biology subjects

Kotton, D. N.

Publications and source records attributed to Kotton, D. N..

At least 19 recordsLinked to original sources

bioRxiv2026

Immunomodulatory RIG-I Agonist SLR14 Confers Protection Against a Range of Highly Pathogenic Negative Sense RNA viruses

Emerging and re-emerging zoonotic RNA viruses, including filoviruses, henipaviruses and Rift Valley Fever virus (RVFV), pose a significant threat to human health. Due to their epidemic potential and high pathogenicity, these viruses are categorized as WHO priority pathogens, yet we still lack effective medical countermeasures (MCMs) against most of these dangerous pathogens, supporting that host-directed antivirals will be advantageous for broadly acting MCMs. Here we assessed the antiviral efficacy of SLR14, an immunomodulatory stem-loop RNA that activates RIG-I, for its activity against a panel of highly pathogenic viruses. SLR14 treatment provided broad-spectrum protection, markedly reducing infection with Ebola virus (EBOV), Marburg virus, and Sudan virus in human primary monocyte-derived macrophages, and Nipah virus replication in iPSC-derived endothelial cells, whether administered prophylactically or therapeutically. In vivo, a single dose of SLR14 protected mice against lethal RVFV infection when administered prophylactically, and, notably, retained significant efficacy when administered up to two days post-infection. Therapeutic SLR14 administration also improved survival rates of EBOV-infected mice. Although EBOV infection and SLR14 treatment activated overlapping innate immune pathways in human macrophages, viral infection induced a nonprotective response, whereas SLR14 elicited a well-regulated, markedly stronger and protective interferon-stimulated and inflammatory gene signature while avoiding prolonged IFN gene upregulation. Our data identify SLR14 as a potent host-directed therapeutic with broad antiviral efficacy against some of the most lethal viral pathogens.

microbiology↗

bioRxiv2026

Vessel Organoids Reveal FOXF1 Variant-Specific Regulation of Mesoderm and Capillary Development

How allelic variants in lineage-regulating transcription factors drive diverging human developmental outcomes remains poorly understood. This is partly due to the lack of human model systems. Here, we used vessel organoids from human induced pluripotent stem cells (hiPSCs) to resolve variant-specific functions of Forkhead Box F1 (FOXF1), a critical regulator of mesoderm and vascular development. Using three patient-derived hiPSC lines harboring unique FOXF1 variants, we show that heterozygous variants cause capillary maldevelopment of varying severity. Single-nucleus multiomic analysis revealed variant-specific mechanisms - a severe variant impairs differentiation of nascent mesoderm to lateral plate mesoderm and disrupts vascular progenitor specification, while moderate variants permit mesoderm differentiation but rewire vascular progenitor states and function. Restoration of wild-type FOXF1 via lipid nanoparticle-mediated mRNA delivery rescued capillary formation in a variant- and developmental-stage-dependent manner. Together, these findings demonstrate that different variants disrupt stage-specific FOXF1 functions in human mesoderm-to-vascular development, underscoring the importance of variant-specific therapeutic strategies. HIGHLIGHTS O_LIHuman vessel organoids reveal variant-specific roles of FOXF1 in mesoderm patterning and capillary development. C_LIO_LISevere FOXF1 variant c.253T>A (p.F85I) impairs nascent mesoderm-to-lateral plate mesoderm differentiation and disrupts vascular progenitor specification. C_LIO_LI Moderate FOXF1 variants differentially rewire endothelial and mural progenitor cell states and function. C_LIO_LILipid nanoparticle-mediated FOXF1 mRNA delivery rescues capillary formation in a variant- and developmental-stage-dependent manner. C_LI

developmental biology↗

bioRxiv2026

A chimeric human-mouse lung vascular model using induced pluripotent stem cells reveals insights into the pathogenesis of BMPR2-related pulmonary hypertension

Advances in tissue biology have revealed remarkable transcriptomic heterogeneity of endothelial cells between and within organ systems. This necessitates more precise models of organ-specific endothelium to understand the pathogenesis of genetic vascular disorders, such as pulmonary hypertension (PH), where gene-disease associations have implicated endothelial cell dysfunction as a key driver of disease pathogenesis. Towards this end, human induced pluripotent stem cells (hiPSCs) hold immense promise for PH disease modeling where hiPSCs are generated from an affected individual and undergo gene correction to generate syngeneic controls that can be differentiated to endothelial cells (hiEndos), providing a limitless source of material for downstream studies; however, the ability to generate lung-specific hiEndos to model pulmonary vascular disease has been limited. To overcome this challenge, we developed a chimeric human-mouse lung vascular model wherein hiEndos are first patterned via BMP9-induced signaling towards a lung-like molecular phenotype in vitro and are then intravenously transplanted into the mouse lung vasculature in vivo to generate orthotopic lung-specific endothelium for downstream studies. Transplanted pre-patterned hiEndos form functional connections to the native mouse lung vasculature and upregulate differentiated lung-specific molecular cell subtype profiles that include capillary- and arterial-like cell populations. To apply this approach for disease modeling, we generated new hiPSC lines by reprogramming fibroblasts from individuals of the 2001 landmark cohort of BMPR2 gene variant-associated PH and developed a novel in vivo competitive lung endothelial reconstitution assay to quantify functional and molecular differences between human BMPR2-variant vs syngeneic gene-corrected/edited hiEndos. Our approach revealed novel insights into PH disease pathogenesis, not previously evident with prior models, including BMPR2 variant-induced in vivo defects in human lung capillary gene expression, elevated lncRNA H19 expression, increased AHR signaling, and diminished functional capacity to repopulate the pulmonary vascular endothelium.

genetics↗

bioRxiv2026

The chromatin remodeling complex PRC2 safeguards cell fate in alveolar epithelial type 2 cells

Maintenance of the gas exchange surface throughout life and regeneration of the lung after injury requires tight regulation of epithelial cell fate and function. Alveolar epithelial type 2 (AT2) cells serve as the progenitors of the distal epithelium, differentiating into alveolar epithelial type 1 (AT1) cells or proliferating to maintain the quorum of AT2 cells. Here we describe the role of the chromatin regulator polycomb repressive complex 2 (PRC2) in the maintenance of AT2 cell fate in the adult alveolus. Cross-species single-cell transcriptomic analyses identified PRC2 activation in proliferative AT2 populations. PRC2 loss of function in human iPSC-derived AT2 (iAT2) cells and primary murine AT2 cells in vitro resulted in loss of AT2 cell state and emergence of programs reminiscent of alveolar-basal intermediate (ABI) cell states, while overexpression of the PRC2 enzymatic component EZH2 in human iAT2 cells augmented the AT2 cell program. Genetic loss of PRC2 function in the AT2 lineage in adult mice in vivo led to emphysematous remodeling of the lung and induced a time-dependent series of transitions of AT2 cells through an alveolar-basal intermediate (ABI) state into Krt5+ basal-like cells. Comparison of murine ABI cells to human disease-associated ABI cells demonstrates de-repression of canonical PRC2 targets during transition to ABI and basal-like states in human fibrosis, implicating PRC2 is a conserved regulator of AT2 cell fate. Together, these findings define PRC2 complex function during AT2 cell self-renewal as a critical guardrail for maintaining epithelial cell fate in the adult lung.

genetics↗

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

Divergent Pathways of Surfactant Protein C Maturation for Disease-Associated Isoforms

Surfactant Protein C (SP-C), a hydrophobic protein exclusively synthesized and secreted by alveolar type II (AT2) cells, is important for reducing alveolar surface tension in the distal lung. Chronic interstitial pulmonary diseases have been associated with SFTPC mutations. However, a detailed understanding of SP-C maturation in the secretory pathway and disruptions caused by mutations has remained incomplete. The goal of this study was to comprehensively ascertain differences in trafficking and post-translational processing between wild-type and disease-associated SP-C mutants using doxycycline-inducible mouse lung epithelial (MLE-12) cell lines expressing either wildtype SP-C or the common clinical variant SP-CI73T, validated using primary AT2 cells isolated from a murine SP-CI73T pulmonary fibrosis model and induced pluripotent stem cell (iPSC)-derived human alveolar type 2 cells (iAT2s) expressing the same mutant. In all 3 models SP-CWT was highly concentrated in acidic LROs while SP-CI73T accumulated on the plasma membrane, which was corroborated by inhibition of clathrin-mediated endocytosis, surface biotinylation, immunogold EM, immunofluorescent staining in non-permeabilized cells, and proteinase K protection assays supporting divergence of SP-CI73T trafficking from SP-CWT. The exclusion of SP-CI73T from normal routing occurred early in the biosynthetic pathway as Brefeldin A blocked processing of both SP-C proproteins, while a 20{degrees}C temperature shift caused selective accumulation of a processed proSP-CWT intermediate, suggesting initial C-terminal cleavage of proSP-CWT occurs in late-Golgi/ trans-Golgi network (TGN). This cleavage event was sensitive to DC1, an inhibitor of furin-related subtilisin-like proprotein convertase (PPC) family members. Site-directed mutagenesis of canonical residues K160, R167 within a predicted PPC recognition site in the proSP-C BRICHOS domain blocked its processing. Expression constructs encoding inhibitory pre-proprotein (pp) peptide fragments of Furin and ppPC7 each inhibited cleavage of proSP-CWT by MLE-12 cells. Collectively, our data demonstrate that trafficking pathways for maturation of WT and mutant I73T SP-C diverge prior to the TGN where initial cleavage of the COOH-terminal SP-C propeptide occurs via a Furin-like proprotein convertase.

cell biology↗

bioRxiv2025

Lentiviral-mediated gene complementation rescues pathogenic ABCA3 variants

The ATP-binding cassette subfamily A member 3 (ABCA3) protein on the limiting membrane of lamellar bodies in alveolar type 2 (AT2) cells transports phospholipids required for pulmonary surfactant assembly. ABCA3 deficiency results from biallelic pathogenic variants in ABCA3 and causes progressive neonatal respiratory failure or childhood interstitial lung disease (chILD). Supportive/compassionate care or lung transplantation are the only current definitive treatments for ABCA3 deficiency and progressive respiratory failure. Complementing dysfunctional ABCA3 by gene addition has therapeutic potential. Previous studies show that repairing or complementing ABCA3 in induced pluripotent stem cell (iPSC)-derived AT2 cells rescues lamellar body morphology and surfactant phospholipid composition. Pathogenic variants disrupt ABCA3 function through altered protein trafficking (type 1) or by impaired phospholipid transport (type 2) into lamellar bodies. Here we tested ABCA3 gene complementation using a human pulmonary epithelial cell line (A549) with a genomically silenced ABCA3 locus (ABCA3KO). From this line, additional cell lines that stably express individual ABCA3 variant cDNA constructs from a single genomic locus were tested: L101P (type 1), E292V (type 2), E690K (type 2), or wild-type ABCA3. Lentiviral-mediated ABCA3 delivery to each cell line partially rescued localization to LAMP3+ vesicles, lamellar body-like structure morphology, and cell proliferation. A functional assay measuring NF-{kappa}B signaling suggested that ABCA3 complementation ameliorated aberrant inflammatory signaling in E292V or E690K (type 2) mutant lines, but not in L101P (type 1) or knockout lines. These studies highlight the therapeutic potential of gene addition as well as differences between ABCA3 pathogenic variants that may influence genetic therapy outcomes.

molecular biology↗

bioRxiv2024

Targeted pre-conditioning and cell transplantation in the murine lower respiratory tract

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

cell biology↗

bioRxiv2024

Complex optogenetic spatial patterning with split recombinase

Light is a powerful and flexible input into engineered biological systems and is particularly well-suited for spatially controlling genetic circuits. While many light-responsive molecular effectors have been developed, there remains a gap in the feasibility of using them to spatially define cell fate. We addressed this problem by employing recombinase as a sensitive light-switchable circuit element which can permanently program cell fate in response to transient illumination. We show that by combining recombinase switches with hardware for precise spatial illumination, large scale heterogeneous populations of cells can be generated in situ with high resolution. We envision that this approach will enable new types of multicellular synthetic circuit engineering where the role of initial cell patterning can be directly studied with both high throughput and tight control.

synthetic biology↗

bioRxiv2024

Nuclear size-regulated emergence of topological packing order on growing human lung alveolospheres

Within multicellular living systems, cells coordinate their positions with spatiotemporal accuracy to form various structures, setting the clock to control developmental processes and trigger maturation. These arrangements can be regulated by tissue topology, biochemical cues, as well as mechanical perturbations. However, the fundamental rules of how local cell packing order is regulated in forming three-dimensional (3D) multicellular architectures remain unclear. Furthermore, how cellular coordination evolves during developmental processes, and whether this cell patterning behavior is indicative of more complex biological functions, is largely unknown. Here, using human lung alveolospheres as a model system, by combining experiments and numerical simulations, we find that, surprisingly, cell packing behavior on alveolospheres resembles hard-disk packing but with increased randomness; the stiffer cell nuclei act as the hard disks surrounded by deformable cell bodies. Interestingly, we observe the emergence of topological packing order during alveolosphere growth, as a result of increasing nucleus-to-cell size ratio. Specifically, we find more hexagon-concentrated cellular packing with increasing bond orientational order, indicating a topological gas-to-liquid transition. Additionally, by osmotically changing the compactness of cells on alveolospheres, we observe that the variations in packing order align with the change of nucleus-to-cell size ratio. Together, our findings reveal the underlying rules of cell coordination and topological phases during human lung alveolosphere growth. These static packing characteristics are consistent with cell dynamics, together suggesting that better cellular packing stabilizes local cell neighborhoods and may regulate more complex biological functions such as organ development and cellular maturation.

biophysics↗

bioRxiv2024

Impaired AMPK Control of Alveolar Epithelial Cell Metabolism Promotes Pulmonary Fibrosis

Alveolar epithelial type II (AT2) cell dysfunction is implicated in the pathogenesis of familial and sporadic idiopathic pulmonary fibrosis (IPF). We previously described that expression of an AT2 cell exclusive disease-associated protein isoform (SP-CI73T) in murine and patient-specific induced pluripotent stem cell (iPSC)-derived AT2 cells leads to a block in late macroautophagy and promotes time-dependent mitochondrial impairments; however, how a metabolically dysfunctional AT2 cell results in fibrosis remains elusive. Here using murine and human iPSC-derived AT2 cell models expressing SP-CI73T, we characterize the molecular mechanisms governing alterations in AT2 cell metabolism that lead to increased glycolysis, decreased mitochondrial biogenesis, disrupted fatty acid oxidation, accumulation of impaired mitochondria, and diminished AT2 cell progenitor capacity manifesting as reduced AT2 self-renewal and accumulation of transitional epithelial cells. We identify deficient AMP-kinase signaling as a key upstream signaling hub driving disease in these dysfunctional AT2 cells and augment this pathway to restore alveolar epithelial metabolic function, thus successfully alleviating lung fibrosis in vivo.

molecular biology↗

bioRxiv2024

Nascent matrix deposition supports alveolar organoid formation from aggregates in synthetic hydrogels

HighlightsO_LIAlveolar organoids are formed with a two-step, Matrigel-free method in a semi-synthetic hyaluronic acid (HA) hydrogel C_LIO_LIThe two-step method offers control over alveolar size, density, and growth C_LIO_LIAlveolar organoids maintain their AT2 identity in HA hydrogels C_LIO_LIAlveolar organoids secrete nascent extracellular matrix supporting organoid growth without Matrigel C_LI SummaryHuman induced pluripotent stem cell (iPSC) derived alveolar organoids have emerged as a system to model the alveolar epithelium in homeostasis and disease. However, alveolar organoids are typically grown in Matrigel, a mouse-sarcoma derived basement membrane matrix that offers poor control over matrix properties, prompting the development of synthetic hydrogels as a Matrigel alternative. Here, we develop a two-step culture method that involves pre-aggregation of organoids in hydrogel-based microwells followed by embedding in a synthetic hydrogel that supports alveolar organoid growth, while also offering considerable control over organoid and hydrogel properties. We find that the aggregated organoids secrete their own nascent extracellular matrix (ECM) both in the microwells and upon embedding in the synthetic hydrogels. Thus, the synthetic gels described here allow us to de-couple exogenous and nascent ECM in order to interrogate the role of ECM in organoid formation.

cell biology↗

bioRxiv2024

Deciphering Endothelial and Mesenchymal Organ Specification in Vascularized Lung and Intestinal Organoids

To investigate the co-development of vasculature, mesenchyme, and epithelium crucial for organogenesis and the acquisition of organ-specific characteristics, we constructed a human pluripotent stem cell-derived organoid system comprising lung or intestinal epithelium surrounded by organotypic mesenchyme and vasculature. We demonstrated the pivotal role of co-differentiating mesoderm and endoderm via precise BMP regulation in generating multilineage organoids and gut tube patterning. Single-cell RNA-seq analysis revealed organ specificity in endothelium and mesenchyme, and uncovered key ligands driving endothelial specification in the lung (e.g., WNT2B and Semaphorins) or intestine (e.g., GDF15). Upon transplantation under the kidney capsule in mice, these organoids further matured and developed perfusable human-specific sub-epithelial capillaries. Additionally, our model recapitulated the abnormal endothelial-epithelial crosstalk in patients with FOXF1 deletion or mutations. Multilineage organoids provide a unique platform to study developmental cues guiding endothelial and mesenchymal cell fate determination, and investigate intricate cell-cell communications in human organogenesis and disease. HighlightsO_LIBMP signaling fine-tunes the co-differentiation of mesoderm and endoderm. C_LIO_LIThe cellular composition in multilineage organoids resembles that of human fetal organs. C_LIO_LIMesenchyme and endothelium co-developed within the organoids adopt organ-specific characteristics. C_LIO_LIMultilineage organoids recapitulate abnormal endothelial-epithelial crosstalk in FOXF1-associated disorders. C_LI

developmental biology↗

bioRxiv2023

Generation of human alveolar epithelial type I cells from pluripotent stem cells

In the distal lung, alveolar epithelial type I cells (AT1s) comprise the vast majority of alveolar surface area and are uniquely flattened to allow the diffusion of oxygen into the capillaries. This structure along with a quiescent, terminally differentiated phenotype has made AT1s particularly challenging to isolate or maintain in cell culture. As a result, there is a lack of established models for the study of human AT1 biology, and in contrast to alveolar epithelial type II cells (AT2s), little is known about the mechanisms regulating their differentiation. Here we engineer a human in vitro AT1 model system through the directed differentiation of induced pluripotent stem cells (iPSC). We first define the global transcriptomes of primary adult human AT1s, suggesting gene-set benchmarks and pathways, such as Hippo-LATS-YAP/TAZ signaling, that are enriched in these cells. Next, we generate iPSC-derived AT2s (iAT2s) and find that activating nuclear YAP signaling is sufficient to promote a broad transcriptomic shift from AT2 to AT1 gene programs. The resulting cells express a molecular, morphologic, and functional phenotype reminiscent of human AT1 cells, including the capacity to form a flat epithelial barrier which produces characteristic extracellular matrix molecules and secreted ligands. Our results indicate a role for Hippo-LATS-YAP signaling in the differentiation of human AT1s and demonstrate the generation of viable AT1-like cells from iAT2s, providing an in vitro model of human alveolar epithelial differentiation and a potential source of human AT1s that until now have been challenging to viably obtain from patients.

cell biology↗

bioRxiv2022

Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1Omicron

The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date1-7. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes3,8. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor-binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.

microbiology↗

bioRxiv2022

Directed differentiation of mouse pluripotent stem cells into functional lung-specific mesenchyme

The successful generation of endodermal, ectodermal, and most mesodermal lineages from pluripotent stem cells has resulted in basic discoveries and regenerative medicine clinical trials of cell-based therapies. In contrast, the derivation of tissue-specific mesenchyme via directed differentiation in vitro has markedly lagged, due in part to a limited understanding of the signaling pathways regulating in vivo mesenchymal development and a lack of specific markers or reporters able to purify such lineages. The derivation of lung-specific mesenchyme is a particularly important goal since this tissue plays important roles in lung development and respiratory disease pathogenesis. Here we generate a mouse induced pluripotent stem cell (iPSC) line carrying a lung-specific mesenchymal reporter/lineage tracer facilitating the tracking and purification of engineered lung-specific mesenchyme. We identify the key signaling pathways (RA and Shh) necessary to specify lung mesenchyme from lateral plate mesodermal precursors and find that mouse iPSC-derived lung mesenchyme (iLM) expresses the molecular and functional phenotypes of primary developing lung mesenchyme. Purified iLM can be recombined with separately engineered lung epithelial progenitors, self-organizing into 3-dimensional organoids featuring significantly augmented structural complexity and lineage purity, including interacting juxtaposed layers of epithelium and mesenchyme. Co-culture with iLM increases the yield of lung epithelial progenitors and impacts epithelial and mesenchymal differentiation programs, suggesting functional epithelial-mesenchymal crosstalk. Our iPSC-derived population thus expresses key features of developing lung mesenchyme, providing an inexhaustible source of cells for studying lung development, modeling diseases, and developing therapeutics.

developmental biology↗

bioRxiv2022

Durable alveolar engraftment of PSC-derived lung epithelial cells into immunocompetent mice

Durable reconstitution of the injured distal lung epithelium with pluripotent stem cell (PSC) derivatives, if realized, would represent a promising potential therapy for diseases that result from alveolar damage. Here we differentiate murine PSCs in vitro into self-renewing lung epithelial progenitors able to engraft into the injured distal lung epithelium of immunocompetent, syngeneic mouse recipients. Emulating the roadmap of the developing embryo, we generate transplantable PSC-derived Nkx2-1+/Sox9+ lung epithelial progenitors that are highly similar to cultured primary embryonic distal lung bud tip progenitors. These cells display a stable phenotype after frozen archiving or extensive expansion in culture, providing a nearly inexhaustible source of cells that can be engrafted into syngeneic injured mouse lungs without the need for immunosuppression. After transplantation PSC-derived tip-like progenitors downregulate Sox9 and mature in the distal lung, upregulating alveolar type 2 cell markers or assuming the flat morphology and molecular phenotype of terminally differentiated alveolar type 1 cells. After months in vivo, donor-derived cells retain their alveolar epithelial type 2-like and type 1-like phenotypes, as characterized by single cell RNA sequencing, ultrastructural analyses, in vivo histologic profiling, and ex vivo organoid assays that demonstrate continued capacity of the engrafted cells to proliferate and differentiate. These results indicate durable reconstitution of the distal lungs facultative progenitor and differentiated epithelial cell compartments in vivo with PSC-derived cells, thus establishing a novel model for pulmonary cell therapy which can be utilized to better understand the mechanisms and utility of engraftment prior to future clinical studies.

cell biology↗