Biology subjects

Shroyer, N. F.

Publications and source records attributed to Shroyer, N. F..

4 recordsLinked to original sources

bioRxiv2021

Effect of Substrate Stiffness on Human Intestinal Enteroids Infectivity by Enteroaggregative Escherichia coli

Human intestinal enteroids (HIE) models have contributed significantly to our understanding of diarrheal diseases and other intestinal infections, but their routine culture conditions fail to mimic the mechanical environment of the native intestinal wall. Because the mechanical characteristics of the intestine significantly alter how pathogens interact with the intestinal epithelium, we used different concentrations of polyethylene glycol (PEG) to generate soft ([~]2 kPa), medium ([~]10 kPa), and stiff ([~]100 kPa) hydrogel biomaterial scaffolds. The height of HIEs cultured in monolayers atop these hydrogels was 18 {micro}m whereas HIEs grown on rigid tissue culture surfaces (with stiffness in the GPa range) were 10 {micro}m. Substrate stiffness also influenced the amount of enteroaggregative E. coli (EAEC strain 042) adhered to the HIEs. We quantified a striking difference in adherence pattern; on the medium and soft gels, the bacteria formed clusters of >100 and even >1000 on both duodenal and jejunal HIEs (such as would be found in biofilms), but did not on glass slides and stiff hydrogels. All hydrogel cultured HIEs showed significant enrichment for gene and signaling pathways related to epithelial differentiation, cell junctions and adhesions, extracellular matrix, mucins, and cell signaling compared to the HIEs cultured on rigid tissue culture surfaces. Collectively, these results indicate that the HIE monolayers cultured on the hydrogels are primed for a robust engagement with their mechanical environment, and that the soft hydrogels promote the formation of larger EAEC aggregates, likely through an indirect differential effect on mucus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/448533v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@f9c7b3org.highwire.dtl.DTLVardef@a5d5feorg.highwire.dtl.DTLVardef@184224forg.highwire.dtl.DTLVardef@1e8a28d_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

bioengineering↗

bioRxiv2021

Drivers of Transcriptional Variance in Human Intestinal Epithelial Organoids

Background & AimsHuman intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. MethodsRNA-sequencing datasets from 251 experiments performed on 35 human enteroid and colonoid lines from 28 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and Gene Set Enrichment Analysis (GSEA) was used to identify differentially expressed genes and enriched of pathways. ResultsPERMANOVA, Pearson correlations, and dendrogram analysis of all data indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3D, transwell, and monolayer) had the largest effect (7,271-1,305 differentially expressed genes-DEGs); segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect (5,977-420 DEGs), and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell vs. monolayer cultures, and cholesterol biosynthesis genes enriched in Matrigel vs. collagen cultures. ConclusionsSurprisingly large differences in organoid transcriptome were driven by variations in culture methods such as format and substrate, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal organoids.

molecular biology↗

bioRxiv2020

Ontogeny and function of the circadian clock in intestinal organoids

Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed molecular mechanisms regulating circadian physiology of the intestine remain largely unknown. The lack of appropriate human model systems that enable organ- and/or diseasespecific interrogation of clock functions is a major obstacle hindering advancements of translational applications using chronotherapy. In this report, we show that both pluripotent stem cell-derived human intestinal organoids engrafted into mice and patient-derived human intestinal enteroids (HIEs) possess robust circadian rhythms, and demonstrate circadian phase-dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB). Intriguingly, mouse and human enteroids demonstrate anti-phasic necrotic cell death responses. RNA-Seq data show ~4% of genes are rhythmically expressed in HIEs. Remarkably, we observe anti-phasic gene expression of Rac1, a small GTPase directly inactivated by TcdB, between mouse and human enteroids. Importantly, the observed circadian time-dependent necrotic cell death response is abolished in both mouse enteroids and human intestinal organoids (HIOs) lacking robust circadian rhythms. Our findings uncover robust functions of circadian rhythms regulating critical clock-controlled genes (CCGs) in human enteroids governing organism-specific, circadian phasedependent necrotic cell death responses. Our data highlight unique differences between mouse and human enteroids, and lay a foundation for human organ- and disease-specific investigation of clock functions using human organoids for translational applications.

molecular biology↗

bioRxiv2017

Sox4 drives intestinal secretory differentiation toward tuft and enteroendocrine fates

Background & AimsThe intestinal epithelium is maintained by intestinal stem cells (ISCs), which produce post-mitotic absorptive and secretory epithelial cells. Initial fate specification toward enteroendocrine, goblet, and Paneth cell lineages is dependent on Atoh1, a master regulator of secretory differentiation. However, the origin of tuft cells, which participate in Type II immune responses to parasitic infection, is less clear and appears to occur in an Atoh1-independent manner. Here we examine the role of Sox4 in ISC proliferation and differentiation.\n\nMethodsWe used mice with intestinal epithelial-specific conditional knockout of Sox4 (Sox4fl/fl:vilCre; Sox4cKO) to study the role of Sox4 in the small intestine. Crypt- and single cell-derived organoids were used to assay proliferation and ISC potency between control and Sox4cKO mice. Lineage allocation and genetic consequences of Sox4 ablation were studied by immunofluorescence, RT-qPCR, and RNA-seq. In vivo infection with helminths and in vitro cytokine treatment in primary intestinal organoids were used to assess tuft cell hyperplasia in control and Sox4cKO samples. Atoh1GFP reporter mice and single cell RNA-seq (scRNA-seq) were used to determine co-localization of SOX4 and Atoh1. Wild-type and inducible Atoh1 knockout (Atoh1fl/fl:vilCreER; Atoh1iKO) organoids carrying an inducible Sox4 overexpression vector (Sox4OE) were used to determine the role of Atoh1 in Sox4 driven secretory differentiation.\n\nResultsLoss of Sox4 impairs ISC function and secretory differentiation, resulting in decreased numbers of enteroendocrine and tuft cells. In wild-type mice, SOX4+ cells are significantly upregulated following helminth infection coincident with tuft cell hyperplasia. Sox4 is activated by IL13 in vitro and Sox4cKO knockout mice demonstrate impaired tuft cell hyperplasia and parasite clearance following infection with helminths. A subset of Sox4-expressing cells colocalize with Atoh1 and enteroendocrine markers by scRNA-seq, while Sox4+/Atoh1-cells correlate strongly with tuft cell populations. Gain-of-function studies in primary organoids demonstrate that Sox4 is sufficient to drive both enteroendocrine and tuft cell differentiation, and can do so in the absence of Atoh1.\n\nConclusionOur data demonstrate that Sox4 promotes enteroendocrine and tuft cell lineage allocation independently of Atoh1. These results challenge long-standing views of Atoh1 as the sole regulator of secretory differentiation in the intestine and are relevant for understanding host epithelial responses to parasitic infection.

cell biology↗