Biology subjects

Kovbasnjuk, O.

Publications and source records attributed to Kovbasnjuk, O..

7 recordsLinked to original sources

bioRxiv2025

Mesenchymal WNT2B is Required for the Development and Function of the Human Intestine

Background and AimsWNT2B mutations result in Diarrhea-9 (DIAR9), a congenital diarrhea syndrome with an extreme phenotype and unique histological defects. Attempts to model DIAR9 in rodents and study patient epithelial tissue have not been able to fully reproduce the human phenotype, making understanding this condition challenging. Here, we aimed to interrogate the mechanisms and the specific cellular compartment contributing to DIAR9 using a human intestinal organoid model. MethodsHuman intestinal organoids (HIOs) generated from both a patient-derived WNT2BR69*iPSC line and a control line were transplanted into immunocompromised mice for 10 weeks. Grafts were harvested and histologically compared. Bulk RNA sequencing was performed on both organoid groups and on patient-biopsy derived enteroids. In vitro recombination experiments were performed to describe the causative cellular compartment. ResultsLive and histological imaging revealed partial epithelial delamination in WNT2BR69*HIOs, which was absent in controls. A significant number of crypts in WNT2BR69* HIOs lacked OLFM4, a surrogate marker of stem cell activity. Key transcriptomic pathways altered between groups included trafficking of apical digestion proteins, which was confirmed via immunofluorescence. Patient derived enteroid proteomic analysis revealed similar results. Recombination experiments in HIOs revealed that while both epithelial and mesenchymal WNT2B are important for stem cell function, lack of mesenchymal WNT2B was sufficient to elicit the phenotype. ConclusionWe demonstrated that mesenchymal WNT2B is critical for supporting human intestinal epithelial development and function.

developmental biology↗

bioRxiv2024

Intestinal epigenomic alterations are associated with a dysregulated nutrient absorption phenotype in obesity

Obesity is an epidemic with myriad health effects, but little is understood regarding individual obese phenotypes and how they may respond to therapy. Epigenetic changes associated with obesity have been detected in blood, liver, pancreas, and adipose tissues. Previous work using human organoids found that dietary glucose hyperabsorption is a steadfast trait in cultures derived from some obese subjects, but detailed transcriptional or epigenomic features of the intestinal epithelia associated with this persistent phenotype are unknown. This study evaluated differentially expressed genes and relative chromatin accessibility in intestinal organoids established from donors classified as non-obese, obese, or obese hyperabsorptive by body mass index and glucose transport assays. Transcriptomic analysis indicated that obese hyperabsorptive subject organoids have significantly upregulated dietary nutrient absorption transcripts and downregulated type I interferon targets. Chromatin accessibility and transcription factor footprinting predicted that enhanced HNF4G binding may promote the obese hyperabsorption phenotype. Quantitative RT-PCR assessment in organoids representing a larger subject cohort suggested that intestinal epithelial expression of CUBN, GIP, SLC5A11, and SLC2A5 were highly correlated with hyperabsorption. Thus, the obese hyperabsorption phenotype was characterized by transcriptional changes that support increased nutrient uptake by intestinal epithelia, potentially driven by differentially accessible chromatin. Recognizing unique intestinal phenotypes in obesity provides a new perspective in considering therapeutic targets and options to manage the disease.

genomics↗

bioRxiv2021

SARS-COV-2 induced Diarrhea is inflammatory, Ca2+ Dependent and involves activation of calcium activated Cl channels

Diarrhea occurs in 2-50% of cases of COVID-19 ([~]8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike-D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS-D614G exposure to enteroids that were pretreated for [~]24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by [~]8 min. The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco-2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca2+waves and an overall increase in intracellular Ca2+ with a delay of [~]10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).

physiology↗

bioRxiv2021

Human breast milk enhances intestinal mucosal barrier function and innate immunity in a pediatric human enteroid model

Breastfeeding has been associated with long lasting health benefits. Nutrients and bioactive components of human breast milk promote cell growth, immune development, and shield the infant gut from insults and microbial threats. The molecular and cellular events involved in these processes are ill defined. We have established human pediatric enteroids and interrogated maternal milks impact on epithelial cell maturation and function in comparison with commercial infant formula. Colostrum applied apically to pediatric enteroid monolayers reduced ion permeability, stimulated epithelial cell differentiation, and enhanced tight junction function by upregulating occludin expression. Breast milk heightened the production of antimicrobial peptide -defensin 5 by goblet and Paneth cells, and modulated cytokine production, which abolished apical release of pro-inflammatory GM-CSF. These attributes were not found in commercial infant formula. Epithelial cells exposed to breast milk elevated apical and intracellular pIgR expression and enabled maternal IgA translocation. Proteomic data revealed a breast milk-induced molecular pattern associated with tissue remodeling and homeostasis. Using a novel ex vivo pediatric enteroid model, we have identified cellular and molecular pathways involved in human milk-mediated improvement of human intestinal physiology and immunity.

immunology↗

bioRxiv2020

Obesity phenotypes are preserved in intestinal stem cell enteroids from morbidly obese patients

Obesity and obesity-related comorbidities are significant health care challenges. Bariatric surgery (BS) is the most effective therapy for treating obesity and type 2 diabetes. A barrier in the development of therapeutic alternatives is incomplete mechanistic understanding of the benefits of BS and the lack of human intestinal models that recapitulate the pathophysiology of obesity. Using adult intestinal stem cell-derived enteroid cultures established from healthy lean subjects and morbidly obese patients, including post-BS cases, four phenotypes correlating patient BMI and intestinal glucose absorption were identified suggesting that enteroids retain patient phenotype heterogeneity associated with healthy and diseased state. In a sub-population of obese patients, increased dietary glucose absorption and gluconeogenesis was due to significantly higher expression of intestinal carbohydrate transporters (SGLT1, GLUT2 and GLUT5) and gluconeogenic enzymes (PEPCK1 and G6Pase) compared to enteroids from lean subjects that demonstrated low glucose absorption and lacked gluconeogenesis. Enteroids established from successful BS cases exhibited low glucose absorption similar to that observed in lean subjects. These data show that human enteroids preserve the patient phenotype in long-term cultures and represent a reliable preclinical model to study the heterogeneity of the obesity mechanisms, which is necessary to determine the efficacy of therapeutic interventions.

physiology↗

bioRxiv2018

Epithelial WNT2B and Desert Hedgehog are necessary for human colonoid regeneration after bacterial cytotoxin injury

Intestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using the enterohemorrhagic E. coli secreted cytotoxin, EspP to induce injury to human colonoids, we evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. Here, we demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in regulating human colonic regeneration after injury.

physiology↗

bioRxiv2018

xMD-miRNA-seq to generate near in vivo miRNA expression estimates in colon epithelial cells

Accurate, RNA-seq based, microRNA (miRNA) expression estimates from primary cells have recently been described. However, this in vitro data is mainly obtained from cell culture, which is known to alter cell maturity/differentiation status, significantly changing miRNA levels. What is needed is a robust method to obtain in vivo miRNA expression values directly from cells. We introduce expression microdissection miRNA small RNA sequencing (xMD-miRNA-seq), a method to isolate cells directly from formalin fixed paraffin-embedded (FFPE) tissues. xMD-miRNA-seq is a low-cost, high-throughput, immunohistochemistry-based method to capture any cell type of interest. As a proof-of-concept, we isolated colon epithelial cells from two specimens and performed low-input small RNA-seq. We generated up to 600,000 miRNA reads from the samples. Isolated epithelial cells, had abundant epithelial-enriched miRNA expression (miR-192; miR-194; miR-200b; miR-200c; miR-215; miR-375) and overall similar miRNA expression patterns to other epithelial cell populations (colonic enteroids and flow-isolated colon epithelium). xMD-derived epithelial cells were generally not contaminated by other adjacent cells of the colon as noted by t-SNE analysis. xMD-miRNA-seq allows for simple, economical, and efficient identification of cell-specific miRNA expression estimates. Further development will enhance rapid identification of cell-specific miRNA expression estimates in health and disease for nearly any cell type using archival FFPE material.

molecular biology↗