Biology subjects

Spence, J. R.

Publications and source records attributed to Spence, J. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Diverse Intestinal Injuries Drive Heterogeneous Transcriptional Responses and Limited Reactivation of Developmental Gene Programs in Human Enteroids

The murine intestine reactivates developmental gene programs following various forms of damage in vivo and in vitro; however, injury response mechanisms used by the human intestine remain unclear. Using adult human small intestinal epithelium-only organoids ("enteroids"), we characterized the early response to eight injury conditions and injury-associated signaling pathways (P53, PGE2, YAP, TGFB) to interrogate whether human developmental genes were activated. P53 activation and decreased proliferation were common features across treatments. Most (7/8) injuries did not activate human development genes. Butyrate is a notable exception given it inhibited P53 and promoted a human developmental transcriptional signature. We observe that P53 induces a human adult gene signature while TGFB and YAP promote a developmental signature. Together our data characterizes various transcriptional responses to injury, supports injury-associated signaling pathways as regulators of human adult and developmental genes, and highlights how our data can be mined to predict injury-specific interventions for epithelial protection.

cell biology↗

bioRxiv2026

Suspension-Based Human Esophagoids Recapitulate WNT2B-Dependent Regulation of Esophageal Basal Progenitors

Background & AimsThe human esophagus undergoes a tightly regulated developmental program, transitioning from a simple columnar epithelium in early development to a mature stratified squamous tissue essential for adult barrier function. Here, we constructed a developmental cell atlas spanning early development to adulthood and leveraged it to generate physiologically relevant in vitro models. MethodsWe utilized single-cell RNA sequencing and spatial multiplex proteomics of human esophageal tissue from early development through adulthood. We established a feeder-supported 2D culture system and a Matrigel-free, suspension-based 3D esophagoid model in a 96-well format. To interrogate WNT2B function, we analyzed patient tissue harboring WNT2B loss-of-function mutations and performed WNT inhibition in esophagoids. ResultsSequencing profiling identified stage-specific epithelial populations: multiciliated and GPC3 basal cells were unique to early development; KRT14 basal and CRNN luminal cells were adult-specific; and COL17A1, LY6D, and KRT4 populations were shared across stages. Spatially organized WNT2B, KIT, and VWC2 mesenchymal subtypes were identified. The 2D system preserved both epithelial and mesenchymal compartments with transcriptional fidelity. Esophagoids exhibited basal-to-luminal stratification, mesenchymal compartmentalization, and required stromal interactions for formation. WNT2B repressed self-renewal of TP63 basal progenitors and inhibited proliferation, confirmed by pharmacologic inhibition of WNT in the in vitro esophagoids. ConclusionsWe present a stage-resolved atlas of human esophageal development and a scalable esophagoid platform recapitulating esophageal architecture. WNT2B regulates progenitor dynamics by restraining basal cell self-renewal. Esophagoids provide a physiologically relevant system for modeling esophageal development and disease. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/733451v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1af5743org.highwire.dtl.DTLVardef@8a061dorg.highwire.dtl.DTLVardef@1975c3forg.highwire.dtl.DTLVardef@292ce9_HPS_FORMAT_FIGEXP M_FIG C_FIG Key Findings and ImplicationsO_LIDevelopmental Atlas: The study presents a comprehensive transcriptional and structural atlas of the human esophageal epithelium, identifying conserved and stage-specific epithelial populations from early development to adulthood. Notably, stage-specific gene expression of multiciliated and GPC3 basal cells were unique to early development, while KRT14 basal and CRNN luminal cells were adult specific, with COL17A1+ (basal), LY6D+ (epibasal), and KRT4+ (middle), shared at all stages. C_LIO_LIMesenchymal Diversity: Spatial and transcriptional profiling revealed distinct mesenchymal subtypes, including WNT2B, KIT, and VWC2 populations, which are spatially organized and contribute to epithelial-mesenchymal signaling. These findings reinforce the role of stromal-epithelial interactions in esophageal development. C_LIO_LI2D Esophagus Cell Culture System: A feeder-supported 2D cell culture system was developed that retains both epithelial and mesenchymal populations, preserving transcriptional fidelity and enabling long-term expansion for mechanistic studies. C_LIO_LI3D Esophagoid Model: A suspension-based 3D organoid system was optimized using a 96-well format, enabling high-throughput generation of esophagoids with robust epithelial stratification and mesenchymal compartmentalization. These organoids recapitulate key features of the human esophagus, including basal-to-luminal organization, and require stromal interactions for formation. C_LIO_LIFunctional Role of WNT2B in esophagus development: Both in vivo and in vitro analyses demonstrated that WNT2B regulates epithelial progenitor dynamics and tissue architecture by repressing self-renewal of basally localized TP63+ cells and inhibiting proliferation. Loss-of-function models and WNT pathway modulation confirmed its role in epithelial-mesenchymal crosstalk and organoid integrity. C_LI

developmental biology↗

bioRxiv2026

Analysing antimicrobial resistance mobility patterns using a diverse dataset of over 8000 bacterial species

2.Advances in genomics have enhanced surveillance of antimicrobial resistance (AMR), yet the factors governing resistance gene mobility, and therefore their risk of spread, remain poorly characterized. Here, we analyzed AMR gene distributions across thousands of bacterial genomes from NCBI RefSeq, encompassing clinical, agricultural and environmental isolates, to quantify associations with plasmids and predicted mobile genomic islands. AMR genes were identified using the Resistance Gene Identifier, and in addition to plasmid identification, mobile chromosomal elements were predicted using IslandViewer 4. Analysing both the full dataset and subsets of data with corrections for sampling bias, we show that known AMR genes are significantly enriched in mobile regions overall. However, stratification by resistance mechanism supported marked heterogeneity: certain drug classes and mechanisms are strongly associated with mobile elements, whereas others are predominantly chromosomal and non-mobile. Notably, mechanisms with specialized functions showed higher mobility, consistent with their role as "ecological public goods" that need not be present in all cells to confer community-level benefit. Differences were also observed across bacteria with distinct cell envelope structures. Together, these findings lay the groundwork for predictive models of AMR gene mobility and provide a framework for incorporating gene-level mobility into AMR risk assessment and antimicrobial stewardship policy. 3. Impact statementUnderstanding which antimicrobial resistance (AMR) genes are most likely to spread is central to managing resistance in clinical, agricultural, and environmental settings. This study reports a large-scale, systematic analysis of antimicrobial resistance (AMR) gene mobility across >8000 bacterial species, spanning diverse clinical and other ecological sources. By linking resistance mechanisms to their genomic location (plasmids, genomic islands, or the rest of chromosomes), our study demonstrates that AMR genes are overall enriched in mobile elements, but that mobility varies substantially by mechanism and bacterial context. In particular, functionally specialized resistance mechanisms are more frequently associated with mobile elements, consistent with an "ecological public goods" model for AMR dissemination. These findings extend beyond gene-specific case studies to identify generalizable patterns that help explain why some resistance determinants spread more readily than others. The breadth of relevance spans clinical microbiology, evolutionary biology, environmental microbiology, agri-foods, and public health. The work provides evidence supporting an improved framework for AMR mobility risk assessment, and antimicrobial stewardship strategies, with direct implications for forecasting the durability of existing and future antibiotics. 4. Data summaryThe complete dataset and analysis code used to generate the results can be accessed via the Open Science Framework (OSF) repository under DOI:10.17605/OSF.IO/WE3TX

genomics↗

bioRxiv2026

Baseline Inventory of the Bryophytes of Grand Staircase-Escalante National Monument, Utah

Abstract summaryThis report presents a preliminary bryoflora for Grand Staircase-Escalante National Monument (GSENM) in southern Utah. The inventory included over 1000 collections made across 40 localities (i.e. macrohabitat types) spanning two ecologically important gradients in bryophyte habitat: shade and moisture availability. At present, the growing checklist contains 117 taxa of liverworts and mosses including 27 families, 65 genera, 116 species, 9 varieties, and 1 subspecies. Noteworthy records include 49 putative taxa new for the state of Utah, and 2 undescribed species in the genera Grimmia and Schistidium. We propose 4 of these species be considered for addition to the recently revised bryoflora of North America. As expected for arid and semiarid environments, the bryophytes of GSENM are predominantly acrocarpous mosses (75%) followed by pleurocarpous mosses (16%), thalloid liverworts (7%), and leafy liverworts (2%). The most diverse families included xeric-soil acrocarpous mosses in the Pottiaceae (35%) and xeric-rock acrocarpous mosses in the Grimmiaceae (15%). Both xeric and mesic species were recovered in the Bryaceae (10% of species) while the pleurocarpous Amblystegiaceae included mesic and hydric species (7%). Most species in the bryoflora have broad global or disjunct distributions, but notably, the known distribution of 17 species appears limited in the United States, or globally, and warrant monitoring in GSENM. Using floristic habitat sampling across 19 macrohabitat types (combinations of 6 topography and 7 vegetation classes), mean site richness was 17.2 {+/-} 9 (SD) and ranged from 4 to 34 species. Six diversity hotspots supported [≥]30 species and were canyons with perennial or ephemeral streams dominated by mixed conifer, hardwood-riparian, riparian, or pinyon-juniper vegetation. High richness is likely supported by greater habitat diversity including xeric, mesic, and hydric conditions on variable substrates (e.g. rock, soil, biocrust, downed wood, seeps, and riparian aquatic/semi-aquatic habitat). Consequently, managing and monitoring diversity under future climate change and land-use alterations will necessitate a habitat-stratified approach that utilizes repeated floristic habitat sampling to document changes in site-level richness and to predict other candidate diversity hotspots on the basis of microhabitat-level diversity, which could be assessed by trained non-bryologists. Collection data are available to the public as georeferenced and photographed observations of half of the bryophyte collections on our iNaturalist.com project, Bryophytes of Grand Staircase Escalante, available for scientific, educational, or outreach activities. Observations are accessible to visitors (via the smartphone app) who wish to know what species have been found along popular trails in GSENM. Landscape-level richness may not reach that of the neighboring Grand Canyon National Park (>155 species), which supports a unique high-elevation bryophyte community sheltered in the mixed conifer and spruce-fir forests of the North Rims Kaibab Plateau. Future collecting by experts will inevitably uncover more species in this ecologically diverse monument important to conserving dryland bryophyte diversity and ecosystem function. This study will serve as a baseline for future research and long-term monitoring related to climate change impacts on dryland bryophytes including biocrust species. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/708354v1_ufig1.gif" ALT="Figure 1"> View larger version (106K): org.highwire.dtl.DTLVardef@c50925org.highwire.dtl.DTLVardef@1db65c3org.highwire.dtl.DTLVardef@f60909org.highwire.dtl.DTLVardef@cb5244_HPS_FORMAT_FIGEXP M_FIG C_FIG Cover photos (by T. A. Clark): View of sandstone canyon wall along the Escalante River Trail taken during a July collection trip in 2015 (top) during which riparian bryophytes were collected by authro, T. A. Clark, (shown in photo) at a sandstone seep (bottom). Bureau of Land Managements National Landscape Conservation System Grant Cooperative Agreement #L14AC00275 issued to P.I. Lloyd R. Stark, UNLV O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/708354v1_ufig2.gif" ALT="Figure 2"> View larger version (88K): org.highwire.dtl.DTLVardef@86109eorg.highwire.dtl.DTLVardef@1ad0efborg.highwire.dtl.DTLVardef@b2c239org.highwire.dtl.DTLVardef@1ed4a4a_HPS_FORMAT_FIGEXP M_FIG C_FIG Copyright 2020 by the authorsAll content contained herein is the property of the authors and all images the property of Theresa A. Clark and should not be used without permission except for education, in which case inclusion of the author/photographers name in citation or superimposed over any image(s) is requested.

plant 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

RUNX2 promotes epigenetic WNT signaling in inflamed intestinal epithelial cells

Ulcerative colitis (UC) is characterized by chronic mucosal inflammation, recurrent epithelial injury, and impaired colonic mucosal wound healing. While WNT/{beta}-catenin dysregulation has been reported in UC, the mechanisms of such abnormalities remain unclear. To investigate epithelial intrinsic alterations associated with UC, we performed single-nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) multiomics on human primary colonic epithelial cells (colonoids) from healthy donors and patients with inactive or active UC. Colonoids were cultured in a 3D matrix recapitulating crypt base cells or grown as 2D monolayers in differentiation medium to recapitulate luminal epithelial cells. Colonoids from active UC had a unique cell population with elevated CTNNB1 and reduced APC expression. Chromatin profiling identified enrichment of RUNX2 motifs in this UC-associated cell population. Active UC colonoids exhibited reduced OLFM4 expression in 3D and the differentiation marker VIL1 in 2D, suggesting impaired self-renewal and maturation. RUNX2 inhibition using CADD522 reduced {beta}-catenin levels in 3D colonoids and restored VIL1 expression and junctional {beta}-catenin localization in 2D cultures. These findings reveal an intrinsic defect in epithelial renewal in UC, driven in part by RUNX2-dependent WNT dysregulation. Our study identifies RUNX2 as a transcriptional regulator of epithelial stem cell function and WNT signaling in the inflamed human colon. Graphical Abstract summarySingle-nucleus RNA and ATAC sequencing of UC patient-derived colonoids reveals a RUNX2-associated WNT signature in active inflammation. Elevated {beta}-catenin and reduced OLFM4 and VIL1 expression indicate impaired self-renewal and differentiation. Pharmacologic inhibition of RUNX2 restores epithelial maturation, identifying RUNX2 as a key regulator of epithelial dysfunction in UC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/688458v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@14c0093org.highwire.dtl.DTLVardef@b9b6a3org.highwire.dtl.DTLVardef@858ee2org.highwire.dtl.DTLVardef@67b98c_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2025

Clinical Interventions and Inflammatory Signaling Shape the Transcriptional and Cellular Architecture of the Early Postnatal Lung

The early postnatal period in human development is characterized by extensive remodeling of the distal lung to support gas exchange, but this critical period remains poorly understood. Here, we constructed a comprehensive cellular atlas of the early postnatal human lung (0 to 2 years) using single-nucleus RNA sequencing of histologically normal specimens from 23 individuals. Our analysis identified two previously unknown and mutually exclusive transcriptional states of alveolar type 2 (AT2) cells, one defined by upregulation of genes involved in lipid metabolism and identified by unique expression of FMO5, and the other defined by upregulation of inflammatory response genes and identified by unique expression of CFTR. Using spatial transcriptomics, we discovered that AT2 cell states reside in specific niches and interact with distinct alveolar fibroblast subtypes. Clinical data and organoid experiments further suggest that the environment dictates which state prevails as the pro-inflammatory/pro-regenerative signals TNF- and IL-1{beta} promoted the CFTR+ state in vitro, while patients treated with the anti-inflammatory drug dexamethasone (Dex) had more abundant FMO5+ AT2 cells, and Dex induced the FMO5+ state in vitro. These observations link inflammatory signaling and anti-inflammatory clinical interventions to shifts in transcriptional state of the alveolar epithelium. We benchmarked two neonatal lung diseases, bronchopulmonary dysplasia and pulmonary interstitial glycogenosis, revealing a profound disruption in the balance of AT2 states, a broad arrest of postnatal cellular development, and impaired cellular maturation. Our work uncovers a fundamental new understanding of early postnatal human lung biology, linking pro- and anti-inflammatory signaling to AT2 transcriptional phenotypes and providing a new framework for understanding lung disease.

developmental biology↗

bioRxiv2025

A scalable long-term in vitro model of human colonic epithelium with continuous barrier function

Background & AimsHuman in vitro models of differentiated intestinal epithelium are valuable research tools for discovery and translational medicine. Primary adult colonic epithelium barrier function is difficult to assess in a 2-dimensional (2D) in vitro format for more than a few days due to rapid cellular turnover. We developed a transwell model of the colonic epithelium that maintains proliferative and differentiated cell types, allowing long-term culture. MethodsUsing 3-dimensional (3D) primary human colonoids, we seeded dissociated epithelium onto 96-well transwell membranes and added different media that was growth factor specific to apical or basolateral transwell chambers. Barrier integrity was assessed using a variety of stimuli and TEER was continuously monitored in real time for up to 2 weeks, followed by quantitative single-cell image analysis using high-content imaging and deep-learning-based cell segmentation. ResultsEpithelium cultured up to 30 days sustained a time-dependent differentiation, leading to the establishment of a monolayer with proliferative stem/progenitor and differentiated cell types. While monitoring continuous TEER across multiple donor organoids, barrier challenge assays recorded donor-specific responses to IFN-{gamma}, TNF-, Clostridium difficile toxin B (TcdB), and ethylenediaminetetraacetic acid (EDTA). Endpoint immunofluorescence analysis of tight junctions and whole cell staining identified unique cellular features among treatment groups which significantly correlated with TEER, including cell-cell orientation (e.g., adjacent cell mean angle). ConclusionsWe demonstrate a high-throughput in vitro model of the homeostatic human colonic epithelium that maintains a long-term functional barrier. This model can be used to investigate complex epithelial barrier responses over time.

cell biology↗

bioRxiv2025

Human-mouse cross-species comparison identifies common and unique aspects of intestinal mesenchyme development

Using single-cell RNA sequencing (scRNA-seq) and histological approaches, we examine cross-species cellular identity, diversity and organization of mesenchymal (fibroblast) populations in the developing mouse and human intestines. In both species, we defined 7 fibroblast populations. Using cross-species integration and label transfer approaches we find that each mesenchymal cell subtype in the murine intestine is highly concordant to a cell type/state in the human intestine, and vice-versa, suggesting a strong conservation of mesenchymal cell types/states across species. Despite this conservation, we also observe that individual lineage-defining genes are not always shared and can be found in different mesenchymal populations. High resolution spatial analysis via fluorescent in situ hybridization (FISH) and immunofluorescence (IF) confirmed these findings and revealed that transcriptionally-defined sub-types of intestinal mesenchymal cells in mice and humans are organized within similar spatial domains.

developmental biology↗

bioRxiv2025

Dynamic Reprogramming of Stromal Pdgfra-expressing cells during WNT-Mediated Transformation of the Intestinal Epithelium

Stromal fibroblasts regulate critical signaling gradients along the intestinal crypt-villus axis1 and provide a niche that supports adjacent epithelial stem cells. Here we report that Pdgfra-expressing fibroblasts secrete ligands that promote a regenerative-like state in the intestinal mucosa during early WNT-mediated tumorigenesis. Using a mouse model of WNT-driven oncogenesis and single-cell RNA sequencing (RNA-seq) of mesenchyme cell populations, we revealed a dynamic reprogramming of Pdgfra+ fibroblasts that facilitates WNT-mediated tissue transformation. Functional assays of potential mediators of cell-to-cell communication between these fibroblasts and the oncogenic epithelium revealed that TGFB signaling is notably induced in Pdgfra+ fibroblasts in the presence of oncogenic epithelium, and TGFB was essential to sustain regenerative-like growth of organoids ex vivo. Genetic reduction of Cdx2 in the {beta}-catenin mutant epithelium elevated the fetal-like/regenerative transcriptome and accelerated WNT-dependent onset of oncogenic transformation of the tissue in vivo. These results demonstrate that Pdgfra+ fibroblasts are activated during WNT-driven oncogenesis to promote a regenerative state in the epithelium that precedes and facilitates formation of tumors.

cancer biology↗

bioRxiv2024

A spatially-resolved blueprint of the developing human lung reveals a basal cell population regulated by a WNT-driven niche

Organs are composed of diverse cell types that change across space and time during development. To interrogate this diversity, we micro-dissected developing human lungs along the proximal-distal axis during the late pseudoglandular stage and generated an integrated analysis of single-nucleus sequencing and spatial transcriptomics, creating a cellularly-resolved atlas of the lung. These rich datasets revealed positional niches and cellular heterogeneity along the proximal-distal axis, including the identification of a unique population of TP63+ basal cells, the primary stem cell of the airway, marked by expression of LGR5 and LGR6. Analysis of the LGR5+ basal cell niche and functional experiments with primary organoid models suggest a tonic level of WNT pathway activity in LGR5+ basal cells that is potentiated by mesenchyme-derived R-SPONDIN. We found that basal cell self-renewal is enhanced by WNT activity, suggesting that the WNT pathway plays a previously unappreciated but critical role in airway stem cell maintenance during human development. These results enhance our fundamental understanding of the positional and cellular heterogeneity in the developing human lungs and begin to reveal unique niches that maintain homeostasis throughout the lung.

developmental 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

Stably-Inverted Apical-Out Human Upper Airway Organoids for SARS-CoV-2 Infection and Therapeutic Testing

While breakthroughs with organoids have emerged as next-generation in vitro tools, standardization for drug discovery remains a challenge. This work introduces human airway organoids with reversed biopolarity (AORBs), cultured and analyzed in a high-throughput, single-organoid-per-well format, enabling milestones towards standardization. AORBs exhibit a spatio-temporally stable apical-out morphology, facilitating high-yield direct intact-organoid virus infection. Single-cell RNA sequencing and immunohistochemistry confirm the physiologically relevant recapitulation of differentiated human airway epithelia. The cellular tropism of five severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains along with host response differences between Delta, Washington, and Omicron variants, as observed in transcriptomic profiles, also suggest clinical relevance. Dose-response analysis of three well-studied SARS-CoV-2 antiviral compounds (remdesivir, bemnifosbuvir, and nirmatrelvir) demonstrates that AORBs efficiently predict human efficacy, comparable to gold-standard air-liquid interface cultures, but with higher throughput ([~]10-fold) and fewer cells ([~]100-fold). This combination of throughput and relevance allows AORBs to robustly detect false negative results in efficacy, preventing irretrievable loss of promising lead compounds. While this work leverages the SARS-CoV-2 study as a proof-of-concept application, the standardization capacity of AORB holds broader implications in line with regulatory efforts to push alternatives to animal studies.

bioengineering↗

bioRxiv2023

An integrated transcriptomic cell atlas of human endoderm-derived organoids

Human stem cells can generate complex, multicellular epithelial tissues of endodermal origin in vitro that recapitulate aspects of developing and adult human physiology. These tissues, also called organoids, can be derived from pluripotent stem cells or tissue-resident fetal and adult stem cells. However, it has remained difficult to understand the precision and accuracy of organoid cell states through comparison with primary counterparts, and to comprehensively assess the similarity and differences between organoid protocols. Advances in computational single-cell biology now allow the integration of datasets with high technical variability. Here, we integrate single-cell transcriptomes from 218 samples covering organoids of diverse endoderm-derived tissues including lung, pancreas, intestine, liver, biliary system, stomach, and prostate to establish an initial version of a human endoderm organoid cell atlas (HEOCA). The integration includes nearly one million cells across diverse conditions, data sources and protocols. We align and compare cell types and states between organoid models, and harmonize cell type annotations by mapping the atlas to primary tissue counterparts. To demonstrate utility of the atlas, we focus on intestine and lung, and clarify ontogenic cell states that can be modeled in vitro. We further provide examples of mapping novel data from new organoid protocols to expand the atlas, and showcase how integrating organoid models of disease into the HEOCA identifies altered cell proportions and states between healthy and disease conditions. The atlas makes diverse datasets centrally available, and will be valuable to assess organoid fidelity, characterize perturbed and diseased states, and streamline protocol development.

cell biology↗

bioRxiv2023

Coordinated differentiation of human intestinal organoids with functional enteric neurons and vasculature

Human intestinal organoids (HIOs) derived from human pluripotent stem cells co-differentiate both epithelial and mesenchymal lineages in vitro but lack important cell types such as neurons, endothelial cells, and smooth muscle. Here, we report an in vitro method to derive HIOs with epithelium, mesenchyme, enteric neuroglial populations, endothelial cells, and organized smooth muscle in a single differentiation, without the need for co-culture. When transplanted into a murine host, these populations expand and organize to support organoid maturation and function. Functional experiments demonstrate enteric nervous system function, with HIOs undergoing peristaltic-like contractions, suggesting the development of a functional neuromuscular unit. HIOs also form functional vasculature, demonstrated in vitro using microfluidic devices to introduce vascular-like flow, and in vivo following transplantation, where HIO endothelial cells anastomose with host vasculature. Collectively, we report an in vitro model of the human gut that simultaneously co-differentiates epithelial, stromal, endothelial, neural, and organized muscle populations.

bioengineering↗

bioRxiv2023

Identification of amnion progenitor-like cells at the amnion-epiblast boundary in the primate peri-gastrula

Amniogenesis is triggered in a collection of pluripotent epiblast cells as the human embryo implants. To gain insights into the critical but poorly understood transcriptional machinery governing amnion fate determination, we examined the evolving transcriptome of a developing human pluripotent stem cell-derived amnion model at the single cell level. This analysis revealed several continuous amniotic fate progressing states with state-specific markers, which include a previously unrecognized CLDN10+ amnion progenitor state. Strikingly, we found that expression of CLDN10 is restricted to the amnion-epiblast boundary region in the human post-implantation amniotic sac model as well as in a peri-gastrula cynomolgus macaque embryo, bolstering the growing notion that, at this stage, the amnion-epiblast boundary is a site of active amniogenesis. Bioinformatic analysis of published primate peri-gastrula single cell sequencing data further confirmed that CLDN10 is expressed in cells progressing to amnion. Additionally, our loss of function analysis shows that CLDN10 promotes amniotic but suppresses primordial germ cell-like fate. Overall, this study presents a comprehensive amniogenic single cell transcriptomic resource and identifies a previously unrecognized CLDN10+ amnion progenitor population at the amnion-epiblast boundary of the primate peri-gastrula.

developmental biology↗

bioRxiv2023

Opposing roles for TGFbeta and BMP-signaling during nascent alveolar differentiation in the developing human lung

Alveolar type 2 (AT2) cells function as stem cells in the adult lung and aid in repair after injury. The current study aimed to understand the signaling events that control differentiation of this therapeutically relevant cell type during human development. Using lung explant and organoid models, we identified opposing effects of TGF{beta}- and BMP-signaling, where inhibition of TGF{beta}- and activation of BMP-signaling in the context of high WNT- and FGF-signaling efficiently differentiated early lung progenitors into AT2-like cells in vitro. AT2-like cells differentiated in this manner exhibit surfactant processing and secretion capabilities, and long-term commitment to a mature AT2 phenotype when expanded in media optimized for primary AT2 culture. Comparing AT2-like cells differentiated with TGF{beta}-inhibition and BMP-activation to alternative differentiation approaches revealed improved specificity to the AT2 lineage and reduced off-target cell types. These findings reveal opposing roles for TGF{beta}- and BMP-signaling in AT2 differentiation and provide a new strategy to generate a therapeutically relevant cell type in vitro.

developmental biology↗

bioRxiv2023

TGFB1 Induces Fetal Reprogramming and Enhances Intestinal Regeneration

The adult gut epithelium has a remarkable ability to recover from damage. To achieve cellular therapies aimed at restoring and/or replacing defective gastrointestinal tissue, it is important to understand the natural mechanisms of tissue regeneration. We employed a combination of high throughput sequencing approaches, mouse genetic models, and murine and human organoid models, and identified a role for TGFB signaling during intestinal regeneration following injury. At 2 days following irradiation (IR)-induced damage of intestinal crypts, a surge in TGFB1 expression is mediated by monocyte/macrophage cells at the location of damage. Depletion of macrophages or genetic disruption of TGFB-signaling significantly impaired the regenerative response following irradiation. Murine intestinal regeneration is also characterized by a process where a fetal transcriptional signature is induced during repair. In organoid culture, TGFB1-treatment was necessary and sufficient to induce a transcriptomic shift to the fetal-like/regenerative state. The regenerative response was enhanced by the function of mesenchymal cells, which are also primed for regeneration by TGFB1. Mechanistically, integration of ATAC-seq, scRNA-seq, and ChIP-seq suggest that a regenerative YAP-SOX9 transcriptional circuit is activated in epithelium exposed to TGFB1. Finally, pre-treatment with TGFB1 enhanced the ability of primary epithelial cultures to engraft into damaged murine colon, suggesting promise for the application of the TGFB-induced regenerative circuit in cellular therapy. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/523825v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@147f3b7org.highwire.dtl.DTLVardef@d1a64dorg.highwire.dtl.DTLVardef@1fd0dc0org.highwire.dtl.DTLVardef@91f7d8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗