Epithelial Organoids Research Articles

Organoid co-culture model of the human endometrium in a fully synthetic extracellular matrix enables the study of epithelial-stromal crosstalk

The human endometrium undergoes recurring cycles of growth, differentiation, and breakdown in response to sex hormones. Dysregulation of epithelial-stromal communication during hormone-mediated signaling may be linked to myriad gynecological disorders for which treatments remain inadequate. Here, we describe a completely defined, synthetic extracellular matrix that enables co-culture of human endometrial epithelial and stromal cells in a manner that captures healthy and disease states across a simulated menstrual cycle. We parsed cycle-dependent endometrial integrin expression and matrix composition to define candidate cell-matrix interaction cues for inclusion in a polyethylene glycol (PEG)-based hydrogel crosslinked with matrix metalloproteinase-labile peptides. We semi-empirically screened a parameter space of biophysical and molecular features representative of the endometrium to define compositions suitable for hormone-driven expansion and differentiation of epithelial organoids, stromal cells, and co-cultures of the two cell types. Each cell type exhibited characteristic morphological and molecular responses to hormone changes when co-encapsulated in hydrogels tuned to a stiffness regime similar to the native tissue and functionalized with a collagen-derived adhesion peptide (GFOGER) and a fibronectin-derived peptide (PHSRN-K-RGD). Analysis of cell-cell crosstalk during interleukin 1B (IL1B)-induced inflammation revealed dysregulation of epithelial proliferation mediated by stromal cells. Altogether, we demonstrate the development of a fully synthetic matrix to sustain the dynamic changes of the endometrial microenvironment and support its applications to understand menstrual health and endometriotic diseases. This work was supported by The John and Karine Begg Foundation, the Manton Foundation, and NIH U01 (EB029132).

The Importance of Effective Ligand Concentration to Direct Epithelial Cell Polarity in Dynamic Hydrogels.

Epithelial cysts and organoids are multicellular hollow structures formed by correctly polarized epithelial cells. Important in steering these cysts from single cells is the dynamic regulation of extracellular matrix presented ligands, and matrix dynamics. Here, control over the effective ligand concentration is introduced, decoupled from bulk and local mechanical properties, in synthetic dynamic supramolecular hydrogels formed through noncovalent crosslinking of supramolecular fibers. Control over the effective ligand concentration is realized by 1) keeping the ligand concentration constant, but changing the concentration of nonfunctionalized molecules or by 2) varying the ligand concentration, while keeping the concentration of non-functionalized molecules constant. Theresults show that in 2D, the effective ligand concentration within thesupramolecular fibers rather than gel stiffness (from 0.1 to 8kPa) regulates epithelial polarity. In 3D, increasing the effective ligand concentration from 0.5 × 10-3 to 2 × 10-3 m strengthens the effect of increased gel stiffness from 0.1 to 2kPa, to synergistically yield more correctly polarized cysts. Through integrin manipulation, it isshown that epithelial polarity is regulated by tension-based homeostasis between cells and matrix. Theresults reveal the effective ligand concentration as influential factor in regulating epithelial polarity and provide insights on engineering of synthetic biomaterials for cell and organoid culture.

Effect of aging on the formation and growth of colonic epithelial organoids by changes in cell cycle arrest through TGF-β-Smad3 signaling

BackgroundThis study aimed to investigate how aging alters the homeostasis of the colonic intestinal epithelium and regeneration after tissue injury using organoid models and to identify its underlying molecular mechanism.MethodsTo investigate aging-related changes in the colonic intestinal epithelium, we conducted organoid cultures from old (older than 80 weeks) and young (6–10 weeks) mice and compared the number and size of organoids at day 5 of passage 0 and the growth rate of organoids between the two groups.ResultsThe number and size of organoids from old mice was significantly lower than that from young mice (p < 0.0001) at day 5 of passage 0. The growth rate of old-mouse organoids from day 4 to 5 of passage 0 was significantly slower than that of young-mouse organoids (2.21 times vs. 1.16 times, p < 0.001). RNA sequencing showed that TGF-β- and cell cycle-associated genes were associated with the aging effect. With regard to mRNA and protein levels, Smad3 and p-Smad3 in the old-mouse organoids were markedly increased compared with those in the young-mouse organoids. Decreased expression of ID1, increased expression of p16INK4a, and increased cell cycle arrest were observed in the old mouse-organoids. Treatment with SB431542, a type I TGF-β receptor inhibitor, significantly increased the formation and growth of old-mouse organoids, and TGF-β1 treatment markedly suppressed the formation of young-mouse organoids. In the acute dextran sulfate sodium-colitis model and its organoid experiments, the colonic epithelial regeneration after tissue injury in old mice was significantly decreased compared with young mice.ConclusionsAging reduced the formation ability and growth rate of colonic epithelial organoids by increasing cell cycle arrest through TGF-β-Smad3-p16INK4a signaling.

Vitamin D promotes epithelial tissue repair and host defense responses against influenza H1N1 virus and Staphylococcus aureus infections

BackgroundEarly studies indicated that vitamin D (VD) exerted pleiotropic extra-skeletal effects in the airway, but the definite linkage between VD deficiency and airway host responses remains unclear.Methods142 cases of clinical data from Department of Otolaryngology, the Seventh Affiliated Hospital of Sun Yat-sen University, were collected to characterize the relationship between VD deficiency and chronic rhinosinusitis (CRS). Based on the clinical observations, 2.5-D airway epithelial organoids cultured at the air–liquid interface (ALI) were used to simulate the effects of VD treatment in the development of airway epithelium and the modulation of the host responses against influenza H1N1 virus (representing viral infections) and Staphylococcus aureus (representing bacterial infections) infections in the airway. The intrinsic mechanisms of VD deficiency underlying epithelial remodeling were mapped by transcriptomic as well as proteomic analyses.ResultsIn this study we observed prevailing VD deficiency among inpatients suffering from CRS, a common disease predominantly characterized by epithelial impairment and remodeling. Relative to control organoids cultured without VD, long-term incubation with VD accelerated basal cell proliferation during nasal epithelial development. Under infectious conditions, VD treatment protected the organoids against influenza H1N1 virus and Staphylococcus aureus invasions by reinforcing the respiratory host defenses, including upregulation of LL37, suppression (or inhibition) of proinflammatory cytokines, strengthening of epithelial integrity, and mucociliary clearance. In silico analysis of transcriptomics and proteomics suggested that VD modulated the epithelial development and remodeling, involving epithelial cell proliferation/differentiation, epithelial–mesenchymal transition (EMT), and cytokine signaling in the immune system, as well as responses to microbe, cell junction organization, and extracellular matrix organization via PTEN signaling, independent of TGF-β signaling.ConclusionsOur findings emphasize the importance of managing VD deficiency in clinical settings for the sake of alleviating pathological epithelial remodeling. Vitamin D promotes epithelial tissue repair and host defense responses against influenza H1N1 and Staphylococcus aureus infections.

The Oral Transglutaminase 2 Inhibitor ZED1227 Accumulates in the Villous Enterocytes in Celiac Disease Patients during Gluten Challenge and Drug Treatment.

The enzyme transglutaminase 2 (TG2) plays a key role in celiac disease (CeD) pathogenesis. Active TG2 is located mainly extracellularly in the lamina propria but also in the villous enterocytes of the duodenum. The TG2 inhibitor ZED1227 is a promising drug candidate for treating CeD and is designed to block the TG2-catalyzed deamidation and crosslinking of gliadin peptides. Our aim was to study the accumulation of ZED1227 after oral administration of the drug. We studied duodenal biopsies derived from a phase 2a clinical drug trial using an antibody that detects ZED1227 when bound to the catalytic center of TG2. Human epithelial organoids were studied in vitro for the effect of ZED1227 on the activity of TG2 using the 5-biotin-pentylamine assay. The ZED1227-TG2 complex was found mainly in the villous enterocytes in post-treatment biopsies. The signal of ZED1227-TG2 was strongest in the luminal epithelial brush border, while the intensity of the signal in the lamina propria was only ~20% of that in the villous enterocytes. No signal specific to ZED1227 could be detected in pretreatment biopsies or in biopsies from patients randomized to the placebo treatment arm. ZED1227-TG2 staining co-localized with total TG2 and native and deamidated gliadin peptides on the enterocyte luminal surface. Inhibition of TG2 activity by ZED1227 was demonstrated in epithelial organoids. Our findings suggest that active TG2 is present at the luminal side of the villous epithelium and that inhibition of TG2 activity by ZED1227 occurs already there before gliadin peptides enter the lamina propria.

Suspended hydrogel culture as a method to scale up intestinal organoids

Primary tissue-derived epithelial organoids are a physiologically relevant in vitro intestinal model that have been implemented for both basic research and drug development applications. The existing method of culturing intestinal organoids in surface-attached native extracellular matrix (ECM) hydrogel domes is not readily amenable to large-scale culture and contributes to culture heterogeneity. We have developed a method of culturing intestinal organoids within suspended basement membrane extract (BME) hydrogels of various geometries, which streamlines the protocol, increases the scalability, enables kinetic sampling, and improves culture uniformity without specialized equipment or additional expertise. We demonstrate the compatibility of this method with multiple culture formats, and provide examples of suspended BME hydrogel organoids in downstream applications: implementation in a medium-throughput drug screen and generation of Transwell monolayers for barrier evaluation. The suspended BME hydrogel culture method will allow intestinal organoids, and potentially other organoid types, to be used more widely and at higher throughputs than previously possible.

Particulate matter 10 exposure affects intestinal functionality in both inflamed 2D intestinal epithelial cell and 3D intestinal organoid models.

A growing body of evidence suggests that particulate matter (PM10) enters the gastrointestinal (GI) tract directly, causing the GI epithelial cells to function less efficiently, leading to inflammation and an imbalance in the gut microbiome. PM10 may, however, act as an exacerbation factor in patients with inflamed intestinal epithelium, which is associated with inflammatory bowel disease. The purpose of this study was to dissect the pathology mechanism of PM10 exposure in inflamed intestines. In this study, we established chronically inflamed intestinal epithelium models utilizing two-dimensional (2D) human intestinal epithelial cells (hIECs) and 3D human intestinal organoids (hIOs), which mimic in vivo cellular diversity and function, in order to examine the deleterious effects of PM10 in human intestine-like in vitro models. Inflamed 2D hIECs and 3D hIOs exhibited pathological features, such as inflammation, decreased intestinal markers, and defective epithelial barrier function. In addition, we found that PM10 exposure induced a more severe disturbance of peptide uptake in inflamed 2D hIECs and 3D hIOs than in control cells. This was due to the fact that it interferes with calcium signaling, protein digestion, and absorption pathways. The findings demonstrate that PM10-induced epithelial alterations contribute to the exacerbation of inflammatory disorders caused by the intestine. According to our findings, 2D hIEC and 3D hIO models could be powerful in vitro platforms for the evaluation of the causal relationship between PM exposure and abnormal human intestinal functions.

P-539 Modelling embryo implantation in vitro using 3D ‘instant’ assembloids and human blastocysts

Abstract Study question Can embryo implantation be recapitulated in vitro using human blastocysts and a new 3D model of the human endometrium representing the architecture of the tissue? Summary answer We present an innovative implantation model consisting of blastocysts attaching on endometrial assembloids containing stromal cells, gland-like epithelial organoids, and overlaid with an epithelial monolayer. What is known already A successful pregnancy denotes the implantation of an embryo to the receptive, luminal lining of the uterus, the endometrium. The tissue transforms into the semi-permanent decidua which provides an optimal microenvironment and nourishes a healthy embryo. The transformation of the tissue is driven by a differentiation process, known as decidualisation, which accounts for the change of tissue-resident stromal cells to specialised decidual cells or senescent decidual cells. Simultaneously, endometrial glands of epithelial origin become secretory and provide histotrophic nutrition to the implanting embryo. Perturbations in this process of decidualisation may also result in an implantation failure or a pregnancy loss. Study design, size, duration Endometrial 'instant' assembloids, consisting of gland organoids and primary stromal cells in collagen hydrogels, were established from freshly isolated cells from endometrial biopsies. Additional cell types were incorporated including immune and endothelial cells creating a layer of epithelial cells recapitulating the luminal epithelium. Hormonal stimulated assembloids were further co-cultured with human blastocysts at 37 °C, 5% O2 and 6% CO2. Attachment of the blastocysts to the luminal epithelium was assessed upon overnight co-culture. Participants/materials, setting, methods Endometrial biopsies (n = 3) were obtained from consenting hormonally stimulated, healthy oocyte donors. After informed consent, human embryos (n = 14) were donated to research after 5 years of cryopreservation. Decidualisation was monitored by RT-qPCR analysis using epithelial and stromal cells from undifferentiated or decidualised assembloids. Attachment of the embryos (n = 11) to the model was assessed by disturbance of the liquid and this was further confirmed by immunofluorescence antibody labelling. Main results and the role of chance 'Instant' assembloids mimic the endometrium morphologically and functionally since hormonal stimulation results in the induction of decidual stromal (PRL, SCARA5 and DIO2) and epithelial (PAEP and SPP1) marker genes. The presence of immune and endothelial cells was confirmed with the induction of IL2RB and VWF respectively. Immunofluorescent antibody labelling confirmed the similarity in morphology of the model to the human endometrium. Aiming to mimic implantation, assembloids underwent a 3-day long differentiation in a chemically defined medium, 8-bromo-cAMP, estradiol, and a progestin, that induced in vitro decidualisation. Following the 3-day treatment, instant assembloids were overlaid with single epithelial cells, isolated from instant assembloids which had undergone prolonged decidualisation treatment to imitate the physiology of the endometrium. Following a 24-hour long incubation period, human hatched day 6 blastocysts were positioned on the top epithelial layer of the instant assembloid. Immunofluorescent antibody labelling using makers for the epiblast (NANOG), primitive endoderm (GATA4) and epithelial cells (E-cadherin) were used to visualise the co-culture. Our findings signify the newly established model as the most advanced method to study embryo implantation in vitro. The uniqueness of the model is owned by the preservation of endometrial cell types and the potential to model disease through patient specificity. Limitations, reasons for caution The proposed system is an innovative method to recapitulate apposition and adhesion during implantation. Our results are preliminary and further experiments will be performed to increase our repeats and therefore reliability. Moreover, due to the complex nature of the co-culture system, imaging remains a challenge that requires further optimisation. Wider implications of the findings The embryo-assembloid co-culture system may provide a useful tool to aid our understanding of the mechanisms of implantation. Future work will include the labelling of the embryos with a nuclear dye to enable tracking using time-lapse microscopy. We further aim to expand our protocol to study the process of invasion. Trial registration number N/A

Functional genomics of the human epididymis: Further characterization of efferent ducts and model systems by single-cell RNA sequencing analysis.

The human epididymis is poorly studied due to the lack of availability of tissue samples. Our understanding of its structure and function depends upon anatomical and histological observations of archived material. Here we used single-cell RNA sequencing (scRNA-seq) technologies to elucidate the identity of cells within the human efferent ducts (EDs) and compared them to caput epididymis cells. We also compared the cellularity of primary tissues with those of 2D and 3D (organoid) culture models used for functional studies. Human epididymis tissue was dissected to separate different anatomical regions and digested to release single cells for processing on the 10X Genomics Chromium platform. Primary human epididymis epithelial (HEE) cells and HEE organoids were grown as described previously and subjected to scRNA-seq. scRNA-seq data were processed by standard bioinformatics pipelines and used for comparative analysis. We define the cell types in the EDs which include specialized epithelial cells, connective tissue stromal cells, vascular endothelial cells, smooth muscle cells, and immune cells, but lack basal cells that are seen in the caput epididymis. Furthermore, we identify a sub-population of epithelial cells which have marker genes found in the bladder and urothelium. Comparative genomics analysis of the 2D and 3D culture models shows cellular identities adapted to the culture environment while still maintaining similarity to the primary tissue. Our data suggest that EDs are lined with a transitional epithelium, which like the urothelium is able to stretch and contract depending on luminal volume. This is consistent with its primary role in seminal fluid resorption and sperm concentration. Moreover, we describe the cellularity of models to study the human epididymis epithelium in vitro. Single-cell RNA-seq data from the human epididymis make a valuable contribution to our understanding of this highly specialized organ.

Clinical and functional efficacy of elexacaftor/tezacaftor/ivacaftor in people with cystic fibrosis carrying the N1303K mutation

BackgroundElexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) significantly improves health outcomes in people with cystic fibrosis (pwCF) carrying one or two F508del mutations. According to in vitro assays performed in FRT cells, 178 additional mutations respond to ELX/TEZ/IVA. The N1303K mutation is not included in this list of mutations. Recent in vitro data suggested that ELX/TEZ/IVA increases N1303K-CFTR activity. Based on the in vitro response, eight patients commenced treatment with ELX/TEZ/IVA. MethodsTwo homozygotes; and six compound heterozygotes N1303K/nonsense or frameshift mutation pwCF were treated off label with ELX/TEZ/IVA. Clinical data before and 8 weeks after starting treatment were prospectively collected. The response to ELX/TEZ/IVA was assessed in intestinal organoids derived from 5 study patients and an additional patient carrying N1303K that is not receiving treatment. ResultsCompared to the values before commencing treatment, mean forced expiratory volume in 1 second increased by 18.4 percentage points and 26.5% relative to baseline, mean BMI increased by 0.79 Kg/m2, and mean lung clearance index decreased by 3.6 points and 22.2%. There was no significant change in sweat chloride. Nasal potential difference normalized in four patients and remained abnormal in three. Results in 3D intestinal organoids and 2D nasal epithelial cultures showed a response in CFTR channel activity. ConclusionsThis report supports the previously reported in vitro data, performed in human nasal and bronchial epithelial cells and intestinal organoids, that pwCF who carry the N1303K mutation have a significant clinical benefit by ELX/TEZ/IVA treatment.

Luteolin Enhances Transepithelial Sodium Transport in the Lung Alveolar Model: Integrating Network Pharmacology and Mechanism Study.

Luteolin (Lut), a natural flavonoid compound existing in Perilla frutescens (L.) Britton, has been proven to play a protective role in the following biological aspects: inflammatory, viral, oxidant, and tumor-related. Lut can alleviate acute lung injury (ALI), manifested mainly by preventing the accumulation of inflammation-rich edematous fluid, while the protective actions of Lut on transepithelial ion transport in ALI were seldom researched. We found that Lut could improve the lung appearance/pathological structure in lipopolysaccharide (LPS)-induced mouse ALI models and reduce the wet/dry weight ratio, bronchoalveolar protein, and inflammatory cytokines. Meanwhile, Lut upregulated the expression level of the epithelial sodium channel (ENaC) in both the primary alveolar epithelial type 2 (AT2) cells and three-dimensional (3D) alveolar epithelial organoid model that recapitulated essential structural and functional aspects of the lung. Finally, by analyzing the 84 interaction genes between Lut and ALI/acute respiratory distress syndrome using GO and KEGG enrichment of network pharmacology, we found that the JAK/STAT signaling pathway might be involved in the network. Experimental data by knocking down STAT3 proved that Lut could reduce the phosphorylation of JAK/STAT and enhance the level of SOCS3, which abrogated the inhibition of ENaC expression induced by LPS accordingly. The evidence supported that Lut could attenuate inflammation-related ALI by enhancing transepithelial sodium transport, at least partially, via the JAK/STAT pathway, which may offer a promising therapeutic strategy for edematous lung diseases.

Airway Epithelial Cultures of Children with Esophageal Atresia as a Model to Study Respiratory Tract Disorders.

Esophageal atresia (EA) is a rare birth defect in which respiratory tract disorders are a major cause of morbidity. It remains unclear whether respiratory tract disorders are in part caused by alterations in airway epithelial cell functions such as the activity of motile cilia. This can be studied using airway epithelial cell culture models of patients with EA. Therefore, the aim of this study was to evaluate the feasibility to culture and functionally characterize motile cilia function in the differentiated air-liquid interface cultured airway epithelial cells and 3D organoids derived from nasal brushings and bronchoalveolar lavage (BAL) fluid from children with EA. We demonstrate the feasibility of culturing differentiated airway epithelia and organoids of nasal brushings and BAL fluid of children with EA, which display normal motile cilia function. EA patient-derived airway epithelial cultures can be further used to examine whether alterations in epithelial functions contribute to respiratory disorders in EA.

Deletion of Endogenous Neuregulin-4 Limits Adaptive Immunity During Interleukin-10 Receptor-Neutralizing Colitis.

Growth factors are essential for maintenance of intestinal health. We previously showed that exogenous neuregulin-4 (NRG4) promotes colonocyte survival during cytokine challenge and is protective against acute models of intestinal inflammation. However, the function(s) of endogenous NRG4 are not well understood. Using NRG4-/- mice, we tested the role of endogenous NRG4 in models of colitis skewed toward either adaptive (interleukin-10 receptor [IL-10R] neutralization) or innate (dextran sulfate sodium [DSS]) immune responses. NRG4-/- and wild-type cage mate mice were subjected to chronic IL-10R neutralization colitis and acute DSS colitis. Disease was assessed by histological examination, inflammatory cytokine levels, fecal lipocalin-2 levels, and single cell mass cytometry immune cell profiling. Homeostatic gene alterations were evaluated by RNA sequencing analysis from colonic homogenates, with real-time quantitative polymerase chain reaction confirmation in both tissue and isolated epithelium. During IL-10R neutralization colitis, NRG4-/- mice had reduced colonic inflammatory cytokine expression, histological damage, and colonic CD8+ T cell numbers vs wild-type cage mates. Conversely, in DSS colitis, NRG4-/- mice had elevated cytokine expression, fecal lipocalin-2 levels, and impaired weight recovery. RNA sequencing showed a loss of St3gal4, a sialyltransferase involved in immune cell trafficking, in NRG4-null colons, which was verified in both tissue and isolated epithelium. The regulation of St3gal4 by NRG4 was confirmed with ex vivo epithelial colon organoid cultures from NRG4-/- mice and by induction of St3gal4 in vivo following NRG4 treatment. NRG4 regulates colonic epithelial ST3GAL4 and thus may allow for robust recruitment of CD8+ T cells during adaptive immune responses in colitis. On the other hand, NRG4 loss exacerbates injury driven by innate immune responses.

Deterministic evolution and stringent selection during preneoplasia

The earliest events during human tumour initiation, although poorly characterized, may hold clues to malignancy detection and prevention1. Here we model occult preneoplasia by biallelic inactivation of TP53, a common early event in gastric cancer, in human gastric organoids. Causal relationships between this initiating genetic lesion and resulting phenotypes were established using experimental evolution in multiple clonally derived cultures over 2 years. TP53 loss elicited progressive aneuploidy, including copy number alterations and structural variants prevalent in gastric cancers, with evident preferred orders. Longitudinal single-cell sequencing of TP53-deficient gastric organoids similarly indicates progression towards malignant transcriptional programmes. Moreover, high-throughput lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programmes repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a marked degree of phenotypic convergence in premalignant epithelial organoids. These data imply predictability in the earliest stages of tumorigenesis and show evolutionary constraints and barriers to malignant transformation, with implications for earlier detection and interception of aggressive, genome-instable tumours.

AIM2 Promotes Epithelial Tuft Cell Development During Intestinal Type 2 Immune Responses

Abstract The epithelium performs a growing list of immunologically relevant functions at mucosal sites. Specialized epithelial cells of the intestine interact with the microbiota and lamina propria immune cells to regulate host defense, yet we know little about the molecular regulators linking these interactions during different immunological insults. Here, we found that the DNA sensor Absent in Melanoma 2 (AIM2) promotes development of epithelial tuft cells, which initiate intestinal type 2 immunity. Aim2-deficient intestinal epithelial organoids displayed reduced tuft cell gene signatures and numbers during treatment with the tuft cell-promoting cytokines IL-4 and IL-13. Tuft cells respond to the microbial metabolite succinate, leading to IL-13 production by type 2 innate lymphoid cells (ILC2s) and tuft cell hyperplasia in vivo. Succinate-fed Aim2 −/−mice displayed reduced tuft cells and IL13 expression compared to similarly treated wild type mice. Inflammasome-deficient Asc −/−mice and wild type mice displayed similar succinate-induced tuft cell profiles, suggesting AIM2 promotes tuft cell-ILC2 activation independently of its inflammasome function. RNA sequencing revealed Aim2 −/−intestinal organoids displayed diminished expression of O-linked N-acetylglucosamine transferase (OGT), which is required for IL13/STAT6-induced tuft cell expansion. In line with these findings, Aim2 −/−intestinal organoids and succinate-treated mice presented reduced OGT protein expression and STAT6 phosphorylation. These combined results reveal an novel role for AIM2 during type 2 intestinal inflammation in the intestine via promotion of tuft cell expansion, potentially through maintaining OGT-STAT6 signaling. Supported by grants from NIH (K22 CA212030) and the AZ Dept. of Health Services (AZBRC New Investigator Award).

Establishment of a vaginal organoid system to study local resident memory T cell differentiation

Abstract Over a million sexually transmitted diseases (STDs) are acquired every day worldwide, many of which preferentially target the female reproductive tract (FRT). Resident memory T cells (TRM) in the vaginal mucosa have proven protective roles against these STDs. Accordingly, a crucial objective of STD vaccinations is to establish a higher quantity and quality of TRM in the FRT. The vaginal epithelium has been implicated in regulation of local TRM differentiation and function, but the biological nature of this interaction has not been investigated. Here, we established a murine three dimensional (3D) vaginal organoid-CD8 coculture system to examine epithelial-T cell interactions at the molecular level. Differentiation of single epithelial stem cells led to the formation of 3D epithelial organoids. These organoids can be maintained long-term through serial passage and structurally resembled vaginal epithelium with a multilayer specialized epithelium. After culturing activated CD8 T cells with the organoids, we assessed the transcriptional and phenotypic composition of the T cells. We found that cocultured CD8 T cells were integrated into the extracellular matrix and acquired phenotypic and functional traits of in vivo TRM. This TRM-biased differentiation could be suppressed by a small molecule inhibitor of the cytokine TGF-β. Overall, the vaginal organoid coculture model recapitulates the in vivo environment far better than traditional 2D cell culture, while retaining high-throughput abilities. Importantly, it will enable highly reductionist experiments to examine the complex epithelial cell-T cell crosstalk to devise targeted approaches for improving CD8 T cell mediated immunosurveillance in the FRT. Supported by grants from NIH (2P20GM109035 &amp; P20GM121298) and by the Searle Scholars award.

Validating organoid-derived human intestinal monolayers for personalized therapy in cystic fibrosis.

Highly effective drugs modulating the defective protein encoded by the CFTR gene have revolutionized cystic fibrosis (CF) therapy. Preclinical drug-testing on human nasal epithelial (HNE) cell cultures and 3-dimensional human intestinal organoids (3D HIO) are used to address patient-specific variation in drug response and to optimize individual treatment for people with CF. This study is the first to report comparable CFTR functional responses to CFTR modulator treatment among patients with different classes of CFTR gene variants using the three methods of 2D HIO, 3D HIO, and HNE. Furthermore, 2D HIO showed good correlation to clinical outcome markers. A larger measurable CFTR functional range and access to the apical membrane were identified as advantages of 2D HIO over HNE and 3D HIO, respectively. Our study thus expands the utility of 2D intestinal monolayers as a preclinical drug testing tool for CF.

Fibroblast-derived EGF ligand neuregulin 1 induces fetal-like reprogramming of the intestinal epithelium without supporting tumorigenic growth.

Growth factors secreted by stromal fibroblasts regulate the intestinal epithelium. Stroma-derived epidermal growth factor (EGF) family ligands are implicated in epithelial regeneration and tumorigenesis, but their specific contributions and associated mechanisms remain unclear. Here, we use primary intestinal organoids modeling homeostatic, injured and tumorigenic epithelia to assess how the fibroblast-derived EGF family ligands neuregulin 1 (NRG1) and epiregulin (EREG) regulate the intestinal epithelium. NRG1 was expressed exclusively in the stroma, robustly increased crypt budding and protected intestinal epithelial organoids from radiation-induced damage. NRG1 also induced regenerative features in the epithelium, including a fetal-like transcriptome, suppression of the Lgr5+ stem cell pool and remodeling of the epithelial actin cytoskeleton. Intriguingly, unlike EGF and EREG, NRG1 failed to support the growth of pre-tumorigenic intestinal organoids lacking the tumor suppressor Apc, commonly mutated in human colorectal cancer (CRC). Interestingly, high expression of stromal NRG1 was associated with improved survival in CRC cohorts, suggesting a tumor-suppressive function. Our results highlight the power of stromal NRG1 in transcriptional reprogramming and protection of the intestinal epithelium from radiation injury without promoting tumorigenesis.

Modeling identifies variability in SARS-CoV-2 uptake and eclipse phase by infected cells as principal drivers of extreme variability in nasal viral load in the 48 h post infection

The SARS-CoV-2 coronavirus continues to evolve with scores of mutations of the spike, membrane, envelope, and nucleocapsid structural proteins that impact pathogenesis. Infection data from nasal swabs, nasal PCR assays, upper respiratory samples, ex vivo cell cultures and nasal epithelial organoids reveal extreme variabilities in SARS-CoV-2 RNA titers within and between the variants. Some variabilities are naturally prone to clinical testing protocols and experimental controls. Here we focus on nasal viral load sensitivity arising from the timing of sample collection relative to onset of infection and from heterogeneity in the kinetics of cellular infection, uptake, replication, and shedding of viral RNA copies. The sources of between-variant variability are likely due to SARS-CoV-2 structural protein mutations, whereas within-variant population variability is likely due to heterogeneity in cellular response to that particular variant. With the physiologically faithful, agent-based mechanistic model of inhaled exposure and infection from (Chen et al., 2022), we perform statistical sensitivity analyses of the progression of nasal viral titers in the first 0–48 h post infection, focusing on three kinetic mechanisms. Model simulations reveal shorter latency times of infected cells (including cellular uptake, viral RNA replication, until the onset of viral RNA shedding) exponentially accelerate nasal viral load. Further, the rate of infectious RNA copies shed per day has a proportional influence on nasal viral load. Finally, there is a very weak, negative correlation of viral load with the probability of infection per virus-cell encounter, the model proxy for spike-receptor binding affinity.

EPIREGULIN creates a developmental niche for spatially organized human intestinal enteroids.

Epithelial organoids derived from intestinal tissue, called enteroids, recapitulate many aspects of the organ in vitro and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identified an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells and feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown enteroids, and EREG-grown enteroids showed that EGF enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.