Biology subjects

Burclaff, J.

Publications and source records attributed to Burclaff, J..

10 recordsLinked to original sources

bioRxiv2026

Transmural Single-Nucleus Transcriptomics Nominates Candidate Disease Programs in Human Necrotizing Enterocolitis

Background & Aims: Necrotizing enterocolitis (NEC) is a life-threatening disease of premature infants marked by inflammation, ischemia, mucosal injury, and, in advanced cases, necrosis extending into deeper layers of the intestinal wall. Because NEC affects specialized, interacting cells in a developing intestine, we used single-nucleus transcriptomics to build a transmural, cell-type-resolved map and nominate hypothesis-generating disease programs distinct from maturation. Methods: Full-thickness intestinal specimens from fetal/prenatal controls, acute NEC cases, and infant controls were analyzed by single-nucleus RNA sequencing (snRNA-seq). Fetal and infant samples served as developmental references. Analyses included cell annotation, NEC-versus-infant differential expression, gene ontology (GO), cross-lineage recurrence, cell death signatures, and developmental maturity. Results: We profiled 53,735 nuclei by snRNA-seq and resolved 34 epithelial, stromal, vascular, immune, neuronal, glial, and smooth muscle populations. We used a morphology-based nuclei scoring system for sample selection that predicted high-quality transcript recovery. NEC nuclei were represented across this cellular landscape, with recurrent gene-expression changes suggesting shared inflammatory recruitment, antimicrobial/redox defense, stress adaptation, complement/vascular activation, reduced homeostatic signaling, and cell death-associated regulation. Developmental maturity analysis identified fetal-like expression levels in subsets of NEC-regulated genes. Proliferative crypt populations retained DNA repair/cell-cycle programs with inflammatory, hypoxic, and ER-stress features, while absorptive enterocytes showed reduced digestive, transport, detoxification, and terminal villus-function programs. Secretory-lineage changes implicated goblet mucus-barrier responses with reduced glycosylation, trafficking, and hydration; enteroendocrine changes in epithelial defense, metabolic stress, and neural signaling; and Paneth cell retention of antimicrobial effector transcripts despite altered ER quality control, autophagy, and granule delivery in NEC. Non-epithelial programs implicated stromal Wnt/BMP niche loss, impaired lymphatic drainage/scavenging, macrophage cytokine activation with reduced clearance machinery, neuroglial circuit defects, and altered smooth muscle contraction-relaxation balance. Conclusions: This single nuclear intestinal cell atlas nominates multicellular programs that may govern NEC initiation, progression toward tissue destruction, and resolution toward functional recovery.

pathology↗

bioRxiv2026

SOX9-mediated G1 elongation confers reserve stem cell-associated injury resistance in human intestinal stem cells

Background & Aims Dynamic cell cycle control is critical for intestinal crypt maintenance and injury responses, yet genetic regulators driving these changes remain poorly defined. As reserve intestinal stem cells (rISCs) are often considered to be slowly-cycling and can resist replication-dependent injury, factors that restrain proliferation may confer cytoprotection. Here, we define SOX9 as a regulator of intestinal stem cell (ISC) cycling and injury resistance. Methods Primary human ISCs were engineered to tune SOX9 levels, visualize cell cycle state, and manipulate cell cycle regulators. Using this system, we tested how SOX9 dosage impacts stemness, differentiation, proliferative recovery after SOX9 washout, and survival after 5-FU-mediated injury. Transcriptional analyses identified candidate links between SOX9 levels and cell cycle control, which were functionally tested using inducible INK4A (CDKN2A) and Cyclin D2 (CCND2) ISC lines. Results SOX9 induction lengthens the cell cycle in a dose-dependent manner largely by elongating G1 phase through the INK4A-Rb pathway. The effects of high SOX9 levels repressing proliferation and stem cell activity are reversible. SOX9 induction protects against 5-FU toxicity. This protection is mimicked by INK4A overexpression or pharmacological G1 phase arrest and repressed by CCND2 induction. Conclusions These findings identify SOX9-mediated G1 elongation as a reversible cytoprotective program that confers key functional properties associated with rISCs: proliferative restraint, retained stem cell potential, and resistance to replication-dependent injury. This positions G1 length as a potential determinant of which crypt cells survive injury to act as reserve stem cells.

cell biology↗

bioRxiv2024

Sex differences in colonic inflammation are driven by epithelial-specific expression of estrogen receptor alpha

Background & AimsInflammatory bowel disease (IBD) patients exhibit altered expression of nuclear estrogen receptors alpha and beta (ER, ER{beta}) and G-protein coupled estrogen receptor 1 (GPER1). We previously showed that deletion of ER protects against intestinal damage selectively in female mice; however, the mechanisms conferring sex-specific protection are poorly understood. The goal of this study was to compare ER- and ER{beta}-specific mechanisms contributing to intestinal epithelial function in males and females. MethodsExpression of ER, ER{beta}, and GPER1 was evaluated in colonocytes from wild-type (WT) male and female mice. Intestinal epithelial cell (IEC)-specific ER and ER{beta} knockout mice were developed and challenged with dextran sulfate sodium (DSS). Colonic organoids were used to identify estrogen-dependent and -independent effects on cellular growth, differentiation, and transcriptional regulation in WT, ER-KO, and ER{beta}-KO IECs. ResultsColonic IECs showed significant expression of ER, ER{beta}, and GPER1 as well as Cyp19A1, which catalyzes production of 17{beta}-estradiol (estrogen). Female mice lacking ER specifically in colonic IECs showed protection from DSS-induced injury, whereas males showed increased pathology. Organoids derived from male ER-KO mice showed enhanced proliferation and decreased expression of key functional genes even without exogenous estrogen; however, colonoids derived from female ER-KO mice transcriptional analysis showed a protective gene signature. These findings reveal that deletion of ER differentially contributes to enhanced barrier function and resistance to inflammation in females, but to dysfunctional hyper-proliferation in males. ConclusionsER signaling within IECs drives opposing sex-dependent effects on the development, regenerative capacity, and inflammatory susceptibility of the intestinal epithelium.

cell biology↗

bioRxiv2024

Cathartocytosis: How Cells Jettison Unwanted Material as They Reprogram

Injury can cause differentiated cells to undergo massive reprogramming to become proliferative to repair tissue via a cellular program called paligenosis. Gastric digestive-enzyme-secreting chief cells use paligenosis to reprogram into progenitor-like Spasmolytic-Polypeptide Expressing Metaplasia (SPEM) cells. Stage 1 of paligenosis is the downscaling of mature cell architecture via a process involving lysosomes. Here, we noticed that sulfated glycoproteins were not only digested during paligenosis but also excreted into the gland lumen. Various genetic and pharmacological approaches showed that endoplasmic reticulum membranes and secretory granule cargo were also excreted and that the process proceeded in parallel with, but was mechanistically independent of autophagy. 3-dimensional light and electron-microscopy demonstrated that excretion occurred via unique, complex, multi-chambered invaginations of the apical plasma membrane. As this lysosome-independent cell cleansing process does not seem to have been priorly described, we termed it "cathartocytosis". Cathartocytosis allows a cell to rapidly eject excess material without waiting for autophagic and lysosomal digestion. We speculate the ejection of sulfated glycoproteins would aid in downscaling and might also help bind and flush pathogens away from tissue.

cell biology↗

bioRxiv2023

An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal stem cells

Background and AimsThe intestinal epithelium exhibits dynamic control of cell cycle phase lengths, yet no experimental platform exists for directly analyzing cell cycle phases in living human intestinal stem cells (ISCs). Here, we develop primary human ISC lines with two different reporter constructs to provide fluorescent readouts to analyze cell cycle phases in cycling ISCs. Methods3D printing was used to construct a collagen press for making chamber slides that support primary human ISC growth and maintenance within the working distance of a confocal microscope objective. The PIP-FUCCI fluorescent cell cycle reporter and a variant with H2A-mScarlet that allows for automated tracking of cell cycle phases (PIP-H2A) were used in human ISCs along with live imaging and EdU pulsing. An analysis pipeline combining free-to-use programs and publicly available code was compiled to analyze live imaging results. ResultsChamber slides with soft collagen pressed to a thickness of 0.3 mm concurrently support ISC cycling and confocal imaging. PIP-FUCCI ISCs were found to be optimal for snapshot analysis wherein all nuclei are assigned to a cell cycle phase from a single image. PIP-H2A ISCs were better suited for live imaging since constant nuclear signal allowed for more automated analysis. CellPose2 and TrackMate were used together to track cycling cells. ConclusionsWe present two complete platforms for analyzing cell cycle phases in living primary human ISCs. The PIP-FUCCI construct allows for cell cycle phase assignment from one image of living cells, the PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths in human ISCs using our computational pipeline. These platforms hold great promise for future studies on how pharmaceutical agents affect the intestinal epithelium, how cell cycle is regulated in human ISCs, and more.

cell biology↗

bioRxiv2023

A new microphysiological system shows hypoxia primes human ISCs for interleukin-dependent rescue of stem cell activity

Background & AimsHypoxia in the intestinal epithelium can be caused by acute ischemic events or conditions like Inflammatory Bowel Disease (IBD) where immune cell infiltration produces inflammatory hypoxia, a chronic condition that starves the mucosa of oxygen. Epithelial regeneration after ischemia and IBD suggests intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of acute and chronic hypoxia on human ISC (hISC) properties have not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs isolated from healthy human tissues. We then test the hypothesis that some inflammation-associated interleukins protect hISCs during prolonged hypoxia. MethodshISCs were exposed to <1.0% oxygen in the MPS for 6-, 24-, 48- & 72hrs. Viability, HIF1 response, transcriptomics, cell cycle dynamics, and hISC response to cytokines were evaluated. ResultsThe novel MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs remain viable until 72hrs and exhibit peak HIF1 at 24hrs. hISCs lose stem cell activity at 24hrs that recovers at 48hrs of hypoxia. Hypoxia increases the proportion of hISCs in G1 and regulates hISC capacity to respond to multiple inflammatory signals. Hypoxia induces hISCs to upregulate many interleukin receptors and hISCs demonstrate hypoxia-dependent cell cycle regulation and increased organoid forming efficiency when treated with specific interleukins ConclusionsHypoxia primes hISCs to respond differently to interleukins than hISCs in normoxia through a transcriptional response. hISCs slow cell cycle progression and increase hISC activity when treated with hypoxia and specific interleukins. These findings have important implications for epithelial regeneration in the gut during inflammatory events.

cell biology↗

bioRxiv2022

SOX9 elongates cell cycle phases and biases fate decisions in human intestinal stem cells

Background and AimsThe transcription factor SOX9 is expressed in many stem/progenitor cell populations and has biphasic correlations with proliferation rates across different biological systems. In murine intestinal crypts, distinct Sox9 levels mark three phenotypically different cell types, with lowest levels marking rapidly-dividing transit amplifying (TA) cells, intermediate levels marking intestinal stem cells (ISCs), and highest levels marking slowly-dividing label retaining secretory precursors. SOX9 expression levels and the impact of these levels on cell cycle and stem cell activity have not been characterized for humans. MethodsMonolayers of primary human ISCs isolated from healthy organ donors were engineered with stable SOX9-knockout (KO) and/or SOX9-overexpression (OE) genomic modifications to assess the impact of SOX9 levels on proliferative capacity by DNA content analysis, cell cycle phase length by live imaging for a PIP-FUCCI reporter, stem cell activity via organoid formation assays, and cell fate after ISC differentiation tracked via qPCR. ResultsSOX9 was expressed at diverse levels in human intestinal crypt lineages in vivo, repressed proliferation in human ISC monolayers, and predominantly lengthened G1 by >40% with lesser lengthening of S and G2/M phases. Overexpression of SOX9 caused slower proliferation yet increased organoid forming efficiency. Higher SOX9 levels biased ISC differentiation towards tuft cell and follicle-associated epithelium fates while loss of SOX9 biased cells toward absorptive enterocyte, goblet cell, BEST4+ cell, and enteroendocrine cell fates. ConclusionsSOX9 is a master regulator of stem cell activity in human ISCs, lengthening the cell cycle, promoting stemness, and altering differentiation fate. Interestingly, differences are noted between species, highlighting the importance of analyzing regulatory mechanisms in primary healthy human cells.

cell biology↗

bioRxiv2022

A Leaky Human Colon Model Reveals Uncoupled Apical/Basal Cytotoxicity in Early Clostridioides difficile Toxin Exposure

Background & AimsClostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis and increase morbidity and mortality. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop pre-clinical models for new therapies. Properties of cancer cell lines and 3D organoids inherently limit these efforts. Here, we develop adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigate the impact of toxin application to apical/basal aspects of monolayers, and evaluate whether a leaky epithelial barrier enhances toxicity. MethodsSingle-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to cell lineages across the human gut. Transcriptomics informed timing of stem cell differentiation to achieve in vitro colonocyte maturation like that observed in vivo. Transepithelial electrical resistance (TEER) and fluorescent dextran permeability assays measured cytotoxicity as barrier loss post-toxin exposure. Leaky epithelial barriers were induced with diclofenac. ResultsscRNAseq demonstrated broad and variable toxin receptor expression across the human gut lineages. Absorptive colonocytes displayed generally enhanced toxin receptor, Rho GTPase, and cell junction expression. 21-day differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes. hCE monolayers exhibited high barrier function after 1-day differentiation. Basal TcdA/B application to monolayers caused more toxicity and apoptosis than apical exposure. Diclofenac induced leaky hCE monolayers and enhanced toxicity of apical TcdB exposure. ConclusionsApical/basal toxicities are uncoupled with more rapid onset and increased magnitude of basal toxicity. Leaky paracellular junctions enhance toxicity of apical TcdB exposure. hCE monolayers represent a physiologically relevant and sensitive culture system to evaluate the impact of microbial toxins on gut epithelium.

cell biology↗

bioRxiv2021

A proximal-to-distal survey of healthy adult human small intestine and colon epithelium by single-cell transcriptomics

Background and AimsSingle-cell transcriptomics offer unprecedented resolution of tissue function at the cellular level, yet studies analyzing healthy adult human small intestine and colon are sparse. Here, we present single-cell transcriptomics covering the duodenum, jejunum, ileum, and ascending, transverse, and descending colon from 3 humans. Methods12,590 single epithelial cells from three independently processed organ donors were evaluated for organ-specific lineage biomarkers, differentially regulated genes, receptors, and drug targets. Analyses focused on intrinsic cell properties and capacity for response to extrinsic signals along the gut axis across different humans. ResultCells were assigned to 25 epithelial lineage clusters. Human intestinal stem cells (ISCs) are not specifically marked by many murine ISC markers. Lysozyme expression is not unique to human Paneth cells (PCs), and PCs lack expression of expected niche-factors. BEST4+ cells express NPY and show maturational differences between SI and colon. Tuft cells possess a broad ability to interact with the innate and adaptive immune systems through previously unreported receptors. Some classes of mucins, hormones, cell-junction, and nutrient absorption genes show unappreciated regional expression differences across lineages. Differential expression of receptors and drug targets across lineages reveals biological variation and potential for variegated responses. ConclusionsOur study identifies novel lineage marker genes; covers regional differences; shows important differences between mouse and human gut epithelium; and reveals insight into how the epithelium responds to the environment and drugs. This comprehensive cell atlas of the healthy adult human intestinal epithelium resolves likely functional differences across anatomical regions along the gastrointestinal tract and advances our understanding of human intestinal physiology.

physiology↗

bioRxiv2021

Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells

Background & Aims2D monolayers of primary intestinal and colonic epithelial cells represent next-generation in vitro models of the gut. Efficient transgenesis and gene-editing in human intestinal stem cells (hISCs) would significantly improve utility of these models by enabling generation of reporter and loss/gain-of-function hISCs, but no published methods exist for transfecting 2D hISC monolayers. Electroporation has proven effective in other difficult-to-transfect cells; thus we applied this method to hISCs. MethodsTwenty-four electroporation parameters were tested, and the optimal condition for efficiency and viability was validated on hISCs from six anatomical regions along the small intestine and colon. PiggyBac transposase and Cas9 ribonucleoprotein (RNP) complexes were used for stable genomic integration of reporter genes. High-throughput methods for clone isolation, expansion, and screening were developed. An hISC OLFM4-emGFP reporter was generated and validated by qPCR, organoid assays, and hISC compartmentalization on a planar crypt-microarray (PCM) device. ResultsMaximum electroporation efficiency was 79.9% with a mean survival of 65%. Transfection of 105 hISCs produced [~]142 (0.14%) stable transposase-mediated clones. Transfection of OLFM4-targetting RNPs yielded [~]35% editing and 99/220 (45%) of antibiotic-resistant colonies analyzed expressed emGFP. OLFM4-emGFP hISCs applied to PCMs remained emGFP+ and proliferative in high-Wnt3a/R-spondin3/Noggin zones yet differentiated to emGFP-/KRT20+ cells outside engineered crypt zones. OLFM4-emGFP levels correlated with endogenous OLFM4. Olfm4-emGFPhigh cells were LGR5high/KRT20low, and demonstrated high organoid-forming potential. ConclusionsElectroporation of hISCs is highly efficient for stable transgenesis and transgenic lines can be generated in 3-4 weeks. Workflows mirror conventional culture methods, facilitating rapid integration into established tissue-culture operations. OLFM4high is a robust hISC marker with functional properties in culture.

molecular biology↗