Enteroendocrine Cell Differentiation Research Articles

Congenital enteropathies in children: An algorithm for differential diagnosis and therapeutic management. Case report

Infant diarrhea may be caused by infectious, allergic, or surgical diseases. A distinct group comprises congenital enteropathies, a heterogeneous group of genetically determined diseases, including defects in the absorption and transport of nutrients and electrolytes, impaired differentiation of enterocytes and enteroendocrine cells, and defects in the modulation of the intestinal immune response. The final diagnosis of enteropathies is based on the results of molecular genetic tests; however, a detailed assessment of clinical and medical history data and the results of laboratory and instrumental studies enables quick diagnostic search direction and choice of the correct therapeutic tactics. The article considers the algorithm for the differential diagnosis of congenital diarrhea, presents a clinical case of congenital osmotic diarrhea due to disaccharidase insufficiency, and considers the difficulties of the diagnostic search and approaches to diet therapy. The prospective of using a fructose-based formula, Galactomin-19, in the diet therapy of glucose-galactose malabsorption, is presented.

LSD1 and CoREST2 Potentiate STAT3 Activity to Promote Enteroendocrine Cell Differentiation in Mucinous Colorectal Cancer.

Neuroendocrine cells have been implicated in therapeutic resistance and worse overall survival in many cancer types. Mucinous colorectal cancer (mCRC) is uniquely enriched for enteroendocrine cells (EECs), the neuroendocrine cell of the normal colon epithelium, as compared to non-mCRC. Therefore, targeting EEC differentiation may have clinical value in mCRC. Here, single cell multi-omics uncovered epigenetic alterations that accompany EEC differentiation, identified STAT3 as a regulator of EEC specification, and discovered a rare cancer-specific cell type with enteric neuron-like characteristics. Furthermore, LSD1 and CoREST2 mediated STAT3 demethylation and enhanced STAT3 chromatin binding. Knockdown of CoREST2 in an orthotopic xenograft mouse model resulted in decreased primary tumor growth and lung metastases. Collectively, these results provide rationale for developing LSD1 inhibitors that target the interaction between LSD1 and STAT3 or CoREST2, which may improve clinical outcomes for patients with mCRC.

Wuliangye strong aroma baijiu promotes intestinal homeostasis by improving gut microbiota and regulating intestinal stem cell proliferation and differentiation.

Wuliangye strong aroma baijiu (hereafter, Wuliangye baijiu) is a traditional Chinese grain liquor containing short-chain fatty acids, ethyl caproate, ethyl lactate, other trace components, and a large proportion of ethanol. The effects of Wuliangye baijiu on intestinal stem cells and intestinal epithelial development have not been elucidated. Here, the role of Wuliangye baijiu in intestinal epithelial regeneration and gut microbiota modulation was investigated by administering a Lieber-DeCarli chronic ethanol liquid diet in a mouse model to mimic long-term (8 weeks') light/moderate alcohol consumption (1.6 g kg-1 day-1) in healthy human adults. Wuliangye baijiu promoted colonic crypt proliferation in mice. According to immunofluorescence and reverse transcription-quantitative polymerase chain reaction analyses, compared with the ethanol-only treatment, Wuliangye baijiu increased the number of intestinal stem cells and goblet cells and the expression of enteroendocrine cell differentiation markers in the mouse colon. Furthermore, gut microbiota analysis showed an increase in the relative abundance of microbiota related to intestinal homeostasis following Wuliangye baijiu administration. Notably, increased abundance of Bacteroidota, Faecalibaculum, Lachnospiraceae, and Blautia may play an essential role in promoting stem-cell-mediated intestinal epithelial development and maintaining intestinal homeostasis. In summary, these findings suggest that Wuliangye baijiu can be used to regulate intestinal stem cell proliferation and differentiation in mice and to alter gut microbiota distributions, thereby promoting intestinal homeostasis. This research elucidates the mechanism by which Wuliangye baijiu promotes intestinal health. © 2024 Society of Chemical Industry.

Transcription factor dynamics, oscillation, and functions in human enteroendocrine cell differentiation

Enteroendocrine cells (EECs) secrete serotonin (enterochromaffin [EC] cells) or specific peptide hormones (non-EC cells) that serve vital metabolic functions. The basis for terminal EEC diversity remains obscure. By forcing activity of the transcription factor (TF) NEUROG3 in 2D cultures of human intestinal stem cells, we replicated physiologic EEC differentiation and examined transcriptional and cis-regulatory dynamics that culminate in discrete cell types. Abundant EEC precursors expressed stage-specific genes and TFs. Before expressing pre-terminal NEUROD1, post-mitotic precursors oscillated between transcriptionally distinct ASCL1+ and HES6hi cell states. Loss of either factor accelerated EEC differentiation substantially and disrupted EEC individuality; ASCL1 or NEUROD1 deficiency had opposing consequences on EC and non-EC cell features. These TFs mainly bind cis-elements that are accessible in undifferentiated stem cells, and they tailor subsequent expression of TF combinations that underlie discrete EEC identities. Thus, early TF oscillations retard EEC maturation to enable accurate diversity within a medically important cell lineage.

Aloe emodin promotes mucosal healing by modifying the differentiation fate of enteroendocrine cells via regulating cellular free fatty acid sensitivity

The proper differentiation and reorganization of the intestinal epithelial cell population is critical to mucosal regeneration post injury. Label retaining cells (LRCs) expressing SRY-box transcription factor 9 (SOX9) promote epithelial repair by replenishing LGR5+ intestinal stem cells (ISCs). While, LRCs are also considered precursor cells for enteroendocrine cells (EECs) which exacerbate mucosal damage in inflammatory bowel disease (IBD). The factors that determine LRC-EEC differentiation and the effect of intervening in LRC-EEC differentiation on IBD remain unclear. In this study, we investigated the effects of a natural anthraquinone called aloe emodin (derived from the Chinese herb rhubarb) on mucosal healing in IBD models. Our findings demonstrated that aloe emodin effectively interfered with the differentiation to EECs and preserved a higher number of SOX9+ LRCs, thereby promoting mucosal healing. Furthermore, we discovered that aloe emodin acted as an antagonist of free fatty acid receptors (FFAR1), suppressing the FFAR1-mediated Gβγ/serine/threonine-protein kinase (AKT) pathway and promoting the translocation of forkhead box protein O1 (FOXO1) into the nucleus, ultimately resulting in the intervention of differentiation fate. These findings reveal the effect of free fatty acid accessibility on EEC differentiation and introduce a strategy for promoting mucosal healing in IBD by regulating the FFAR1/AKT/FOXO1 signaling pathway.

Insights into core molecular changes associated with metamorphosis in gilthead seabream larvae across diverse hatcheries

Early development is a critical period in fish aquaculture and is influenced by biotic and abiotic factors (e.g., temperature, feed) that can vary significantly between hatcheries, making it difficult to identify core factors determining quality. Many of the existing larval transcriptome studies are small-scale and occur under specific rearing conditions that do not mirror the diversity of larviculture practices at an industrial level. In the present transcriptome study, gilthead seabream at the larval to juvenile transition (metamorphosis) from several hatcheries in Europe (Greece, Italy, and France) were analysed in a large-scale RNA-seq study. The aim was to uncover the most significant molecular modifications occurring during metamorphosis, irrespective of differences in biotic or abiotic factors, to address knowledge gaps associated with critical early developmental stages under industrial hatchery conditions. Commonly modified gene transcripts between larval stages were identified based on the clustering of gene expression profiles of 25 gilthead seabream libraries from different hatcheries in a PCA analysis. When larvae at flexion were compared to larvae at mid-metamorphosis, 2243 differentially expressed genes (DEGs) were identified, and when larvae at early to mid-metamorphosis were compared to mid to late-metamorphosis, 2299 DEGs were identified. Comparative analysis across the developmental stages of gilthead seabream revealed genes of importance for the metamorphic transition and adaptation to rearing conditions, including genes related to the nervous system at flexion (24 days post hatch), enteroendocrine cell differentiation, and lipid homeostasis at early to mid-metamorphosis (46 dph), and enrichment of genes indicative of immune competence at mid to late-metamorphosis (51–54 dph). The differential expression of some endocrine-associated genes, dio1, dio2, cldn1, ing4, Pou3f4, and fgf22, highlights their importance in metamorphosis. Meta-analysis of the transcriptomes from two species, the gilthead seabream and Senegalese sole, that have differing symmetry and ecology uncovered common molecular expression patterns that underlie larvae maturation during metamorphosis, and we propose that these represent core gene markers of metamorphosis in these two fish species.

Abstract 1690: LSD1 and CoREST2 demethylate STAT3 to promote enteroendocrine cell differentiation in mucinous colorectal cancer

Abstract Across different cancer types, tumor heterogeneity has been shown to drive tumor progression, metastasis, and therapeutic resistance. Adenocarcinoma to neuroendocrine lineage transition is an emergent mechanism of targeted therapy resistance in several cancer types, including lung and prostate cancer. Therefore, understanding the dynamics and mechanisms driving neuroendocrine cell fates in cancer is critical. Mucinous colorectal cancer accounts for upwards of 20% of colorectal cancer cases and is characterized by tumors with mucous accounting for at least 50% of the tumor volume. The normal colon epithelium consists of several specialized cell types, including hormone secreting enteroendocrine cells (EECs), which are the neuroendocrine cell of the intestine. We have previously shown that EEC progenitors are enriched in mucinous colorectal cancer and promote cancer cell survival via secreted factors. Additionally, we have shown that lysine specific demethylase 1 (LSD1) promotes EEC differentiation in these tumors; however, the mechanism by which LSD1 promotes EEC differentiation has remained unknown. Typically to carry out its enzymatic function, LSD1 must be a part of a transcriptional regulatory complex. One such complex is the CoREST complex, which contains LSD1, HDAC1, and one of three CoREST protein family members. Here we report that LSD1 and CoREST2 promote EEC differentiation by demethylating the transcription factor signal transducer and activator of transcription 3 (STAT3) to promote STAT3 chromatin binding. Additionally, we demonstrate that knocking down CoREST2 decreases tumor growth and lung metastases of mucinous colorectal cancer cells injected orthotopically into the colons of immunocompromised mice. Furthermore, we utilized single cell multi-omics that combines single cell RNA sequencing with single cell ATAC sequencing to show that during EEC differentiation there is an increase in chromatin accessibility at regulatory regions of known EEC-promoting transcription factors. Finally, through our single cell multi-omics analysis we identify a new rare cell type with neuron-like but not secretory characteristics. Mucinous colorectal cancer is an aggressive and chemotherapeutically intractable form of CRC that is enriched for EEC progenitors. Our data demonstrates that LSD1 and STAT3 promote EEC differentiation, suggesting that inhibiting LSD1 and or STAT3 may have benefits for patients with mucinous colorectal cancer. Citation Format: Christopher A. Ladaika, Ahmed H. Ghobashi, William C. Boulton, Heather M. O'Hagan. LSD1 and CoREST2 demethylate STAT3 to promote enteroendocrine cell differentiation in mucinous colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1690.

A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium

Epithelial damage due to gastrointestinal disorders frequently causes severe disease in horses. To study the underlying pathophysiological processes, we aimed to establish equine jejunum and colon enteroids (eqJE, eqCE) mimicking the in vivo epithelium. Therefore, enteroids were cultivated in four different media for differentiation and subsequently characterized histomorphologically, on mRNA and on protein level in comparison to the native epithelium of the same donor horses to identify ideal culture conditions for an in vitro model system. With increasing enterocyte differentiation, the enteroids showed a reduced growth rate as well as a predominantly spherical morphology and less budding compared to enteroids in proliferation medium. Combined or individual withdrawal of stem cell niche pathway components resulted in lower mRNA expression levels of stem cell markers and concomitant differentiation of enterocytes, goblet cells and enteroendocrine cells. For eqCE, withdrawal of Wnt alone was sufficient for the generation of differentiated enterocytes with a close resemblance to the in vivo epithelium. Combined removal of Wnt, R-spondin and Noggin and the addition of DAPT stimulated differentiation of eqJE at a similar level as the in vivo epithelium, particularly with regard to enterocytes. In summary, we successfully defined a medium composition that promotes the formation of eqJE and eqCE consisting of multiple cell types and resembling the in vivo epithelium. Our findings emphasize the importance of adapting culture conditions to the respective species and the intestinal segment. This in vitro model will be used to investigate the pathological mechanisms underlying equine gastrointestinal disorders in future studies.

Stem cell mTOR signaling directs region-specific cell fate decisions during intestinal nutrient adaptation.

The adult intestine is a regionalized organ, whose size and cellular composition are adjusted in response to nutrient status. This involves dynamic regulation of intestinal stem cell (ISC) proliferation and differentiation. How nutrient signaling controls cell fate decisions to drive regional changes in cell-type composition remains unclear. Here, we show that intestinal nutrient adaptation involves region-specific control of cell size, cell number, and differentiation. We uncovered that activation of mTOR complex 1 (mTORC1) increases ISC size in a region-specific manner. mTORC1 activity promotes Delta expression to direct cell fate toward the absorptive enteroblast lineage while inhibiting secretory enteroendocrine cell differentiation. In aged flies, the ISC mTORC1 signaling is deregulated, being constitutively high and unresponsive to diet, which can be mitigated through lifelong intermittent fasting. In conclusion, mTORC1 signaling contributes to the ISC fate decision, enabling regional control of intestinal cell differentiation in response to nutrition.

The anti-aging effect of vitamin D and vitamin D receptor in Drosophila midgut.

Adult stem cells are pivotal for maintaining tissue homeostasis, and their functional decline is linked to aging and its associated diseases, influenced by the niche cells' environment. Age- and cancer-related reduction of vitamin D and its receptor levels are well documented in human clinical studies. However, the mechanisms through which the vitamin D/vitamin D receptor pathway contributes to anti-aging and extends life expectancy are not well understood. In this study, we aimed to determine the protective role of the vitamin D/vitamin D receptor pathway in differentiated enterocytes (ECs) during intestinal stem cell (ISC) aging. By utilizing a well- established Drosophila midgut model for stem cell aging biology, we revealed that vitamin D receptor knockdown in ECs induced ISC proliferation, EC death, ISC aging, and enteroendocrine cell differentiation. Additionally, age- and oxidative stress-induced increases in ISC proliferation and centrosome amplification were reduced by vitamin D treatment. Our findings suggest a direct evidence of the anti-aging role of the vitamin D/vitamin D receptor pathway and provides insights into the molecular mechanisms underlying healthy aging in Drosophila.

PROTEIN ARGININE METHYLTRANSFERASE 1 REGULATES ADULT INTESTINAL CELL PROLIFERATION AND ENTEROENDOCRINE CELLS DIFFERENTIATION

Abstract The adult intestinal epithelium is a complex, self-renewing tissue composed of specialized cell types with diverse functions. Intestinal stem cells (ISCs) at the base of crypts divide and differentiate into absorptive and secretory cells. Enteroendocrine cells (EEC), one type of secretory cells, are the most abundant hormone-producing cells in mammals and involved in the control of energy homeostasis. Many studies investigated the mechanisms that control cell fate determination; however, regulation of EEC development and function are still unclear or controversial. Here, we found that Protein arginine methyltransferase 1(PRMT1), a major arginine methyltransferase, is highly expressed in the proliferating transit-amplifying (TA) cells and ISCs of adult mouse intestinal crypts. By using tamoxifen-induced intestinal epithelial cell-specific deletion of PRMT1 in adult mice, we observed the number of EEC dramatically increased. Transcription analyses showed the expression levels of Enteroendocrine-specific hormone and transcription factors are upregulated in PRMT1-deficient small intestine. In addition, the top enriched upregulated pathways were all associated with EEC functions. Concomitantly, Neurogenin 3-expressing progenitor cells accumulate in the mutant small intestine, and its target genes such as Neuod1, pax4, insm1 are upregulated of mutant crypts by RT-PCR. Furthermore, intestinal epithelium of the mutant mice showed elongated crypts in the small intestine, while increased cell proliferation in TA cells. Additionally, inducible PRMT1 deletion led to increased cell death, which compensated for increased cell proliferation in the crypts to maintain overall intestinal morphology and intestinal homeostasis. Together, our results revealed that the loss of PRMT1 in the adult intestinal epithelium altered TA cell proliferation and EEC differentiation, which was probably via Neurogenin 3-mediated pathway. Thus, our results may provide potential roles of PRMT1 as an essential transcriptional regulator of EECs specification and strategy for metabolic disorder.

A hydrolyzed casein diet promotes Ngn3 controlling enteroendocrine cell differentiation to increase gastrointestinal motility in mice.

Gut hormones are produced by enteroendocrine cells (EECs) found along the intestinal epithelium, and these cells play a crucial role in regulating intestinal function, nutrient absorption and food intake. A hydrolyzed casein diet has been reported to promote the secretion of gut hormones through the regulation of EEC development, but the underlying mechanism remains unclear. Therefore, this study was conducted to investigate whether the hydrolyzed casein diet can regulate EEC differentiation by employing mouse and organoid models. Mice were fed diets containing either casein (casein group) or hydrolyzed casein (hydrolyzed casein group) as the sole protein source. The hydrolyzed casein diet upregulated the expression of transcription factors, induced EEC differentiation, increased fasting serum ghrelin concentrations and promoted gastrointestinal (GI) motility in the duodenum compared to the casein diet. Interestingly, these differences could be abolished when there is addition of antibiotics to the drinking water, suggesting a significant role of gut microbiota in the hydrolyzed casein-mediated EEC function. Further investigation showed that the hydrolyzed casein diet led to reduced microbial diversity, especially the abundance of Akkermansia muciniphila (A. muciniphila) on the duodenal mucosa. In contrast, gavage with A. muciniphila impaired EEC differentiation through attenuated neurog3 transcription factor (Ngn3) expression, mediated through the promotion of Notch signaling. Moreover, pasteurized A. muciniphila showed similar effects to enter organoids in vitro. Overall, we found that a hydrolyzed casein diet reduced the abundance of A. muciniphila and promoted Ngn3 controlling EEC differentiation and this pathway is associated with increased GI motility in mice. The findings provide new insights into the role of hydrolyzed casein in gut transit and guidelines for using hydrolyzed casein in safe formula milk.

Unbiased transcription factor CRISPR screen identifies ZNF800 as master repressor of enteroendocrine differentiation.

Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI), and colon. EECs regulate aspects of metabolic activity, including insulin levels, satiety, gastrointestinal secretion, and motility. The generation of different EEC lineages is not completely understood. In this work, we report a CRISPR knockout screen of the entire repertoire of transcription factors (TFs) in adult human SI organoids to identify dominant TFs controlling EEC differentiation. We discovered ZNF800 as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional CRISPR screens for genes that program cell fate.

Vertical sleeve gastrectomy induces enteroendocrine cell differentiation of intestinal stem cells through bile acid signaling.

Vertical sleeve gastrectomy (VSG) results in an increase in the number of hormone-secreting enteroendocrine cells (EECs) in the intestinal epithelium; however, the mechanism remains unclear. Notably, the beneficial effects of VSG are lost in a mouse model lacking the nuclear bile acid receptor farnesoid X receptor (FXR). FXR is a nuclear transcription factor that has been shown to regulate intestinal stem cell (ISC) function in cancer models. Therefore, we hypothesized that the VSG-induced increase in EECs is due to changes in intestinal differentiation driven by an increase in bile acid signaling through FXR. To test this, we performed VSG in mice that express EGFP in ISC/progenitor cells and performed RNA-Seq on GFP-positive cells sorted from the intestinal epithelia. We also assessed changes in EEC number (marked by glucagon-like peptide-1, GLP-1) in mouse intestinal organoids following treatment with bile acids, an FXR agonist, and an FXR antagonist. RNA-Seq of ISCs revealed that bile acid receptors are expressed in ISCs and that VSG explicitly alters expression of several genes that regulate EEC differentiation. Mouse intestinal organoids treated with bile acids and 2 different FXR agonists increased GLP-1–positive cell numbers, and administration of an FXR antagonist blocked these effects. Taken together, these data indicate that VSG drives ISC fate toward EEC differentiation through bile acid signaling.

Immune regulation of intestinal-stem-cell function in Drosophila.

SummaryIntestinal progenitor cells integrate signals from their niche, and the gut lumen, to divide and differentiate at a rate that maintains an epithelial barrier to microbial invasion of the host interior. Despite the importance of evolutionarily conserved innate immune defenses to maintain stable host-microbe relationships, we know little about contributions of stem-cell immunity to gut homeostasis. We used Drosophila to determine the consequences of intestinal-stem-cell immune activity for epithelial homeostasis. We showed that loss of stem-cell immunity greatly impacted growth and renewal in the adult gut. In particular, we found that inhibition of stem-cell immunity impeded progenitor-cell growth and differentiation, leading to a gradual loss of stem-cell numbers with age and an impaired differentiation of mature enteroendocrine cells. Our results highlight the importance of immune signaling in stem cells for epithelial function in the adult gut.

Enteroendocrine cell differentiation and function in the intestine.

The intestinal enteroendocrine cells (EECs) are specialized hormone-secreting cells that respond to both circulating and luminal cues. Collectively, EECs constitute the largest endocrine organ of the body and signal to a multitude of targets including locally to neighboring intestinal cells, enteric neurons, as well as systemically to other organs, such as the pancreas and brain. To accomplish their wide range of downstream signaling effects, EECs secrete multiple hormones; however, the mechanisms that influence EEC development in the embryo and differentiation in adults are not well defined. This review highlights the recent discoveries in EEC differentiation and function while also discussing newly revealed roles of transcription factors and signaling networks involved in the allocation of EEC subtypes that were discovered using a combination of novel intestinal model systems and genetic sequencing. We also discuss the potential of these new experimental models that study the mechanisms regulating EEC function and development both to uncover novel therapeutic targets. Several EEC hormones are being used to treat various metabolic disorders, such as type 2 diabetes and obesity. Therefore, understanding the signaling pathways and gene regulatory networks that facilitate EEC formation is paramount to the development of novel therapies.

ID2 controls differentiation of enteroendocrine cells in mouse small intestine.

The mammalian gut is the largest endocrine organ. Dozens of hormones secreted by enteroendocrine cells regulate a variety of physiological functions of the gut but also of the pancreas and brain. Here, we examined the role of the helix-loop-helix transcription factor ID2 during the differentiation of intestinal stem cells along the enteroendocrine lineage. To assess the functions of ID2 in the adult mouse small intestine, we used single-cell RNA sequencing, genetically modified mice, and organoid assays. We found that in the adult intestinal epithelium Id2 is predominantly expressed in enterochromaffin and peptidergic enteroendocrine cells. Consistently, the loss of Id2 leads to the reduction of Chromogranin A-positive enteroendocrine cells. In contrast, the numbers of tuft cells are increased in Id2 mutant small intestine. Moreover, ablation of Id2 elevates the numbers of Serotonin+ enterochromaffin cells and Ghrelin+ X-cells in the posterior part of the small intestine. Finally, ID2 acts downstream of BMP signalling during the differentiation of Glucagon-like peptide-1+ L-cells and Cholecystokinin+ I-cells towards Neurotensin+ PYY+ N-cells. ID2 plays an important role in cell fate decisions in the adult small intestine. First, ID2 is essential for establishing a differentiation gradient for enterochromaffin and X-cells along the anterior-posterior axis of the gut. Next, ID2 is necessary for the differentiation of N-cells thus ensuring a differentiation gradient along the crypt-villi axis. Finally, ID2 suppresses the commitment of secretory intestinal epithelial progenitors towards tuft cell lineage and thus controls host immune response to commensal and parasitic microbiota.

Robust differentiation of human enteroendocrine cells from intestinal stem cells

Enteroendocrine (EE) cells are the most abundant hormone-producing cells in humans and are critical regulators of energy homeostasis and gastrointestinal function. Challenges in converting human intestinal stem cells (ISCs) into functional EE cells, ex vivo, have limited progress in elucidating their role in disease pathogenesis and in harnessing their therapeutic potential. To address this, we employed small molecule targeting of the endocannabinoid receptor signaling pathway, JNK, and FOXO1, known to mediate endodermal development and/or hormone production, together with directed differentiation of human ISCs from the duodenum and rectum. We observed marked induction of EE cell differentiation and gut-derived expression and secretion of SST, 5HT, GIP, CCK, GLP-1 and PYY upon treatment with various combinations of three small molecules: rimonabant, SP600125 and AS1842856. Robust differentiation strategies capable of driving human EE cell differentiation is a critical step towards understanding these essential cells and the development of cell-based therapeutics.

Col6a1+/CD201+ mesenchymal cells regulate intestinal morphogenesis and homeostasis.

Intestinal mesenchymal cells encompass multiple subsets, whose origins, functions, and pathophysiological importance are still not clear. Here, we used the Col6a1Cre mouse, which targets distinct fibroblast subsets and perivascular cells that can be further distinguished by the combination of the CD201, PDGFRα and αSMA markers. Developmental studies revealed that the Col6a1Cre mouse also targets mesenchymal aggregates that are crucial for intestinal morphogenesis and patterning, suggesting an ontogenic relationship between them and homeostatic PDGFRαhi telocytes. Cell depletion experiments in adulthood showed that Col6a1+/CD201+ mesenchymal cells regulate homeostatic enteroendocrine cell differentiation and epithelial proliferation. During acute colitis, they expressed an inflammatory and extracellular matrix remodelling gene signature, but they also retained their properties and topology. Notably, both in homeostasis and tissue regeneration, they were dispensable for normal organ architecture, while CD34+ mesenchymal cells expanded, localised at the top of the crypts, and showed increased expression of villous-associated morphogenetic factors, providing thus evidence for the plasticity potential of intestinal mesenchymal cells. Our results provide a comprehensive analysis of the identities, origin, and functional significance of distinct mesenchymal populations in the intestine.

Methionine deficiency and its hydroxy analogue influence chicken intestinal 3-dimensional organoid development.

Methionine and its hydroxy analogue (MHA) have been shown to benefit mouse intestinal regeneration. The intestinal organoid is a good model that directly reflects the impact of certain nutrients or chemicals on intestinal development. Here, we aimed to establish a chicken intestinal organoid culture method first and then use the model to explore the influence of methionine deficiency and MHA on intestinal organoid development. The results showed that 125-μm cell strainer exhibited the highest efficiency for chicken embryo crypt harvesting. We found that transforming growth factor-β inhibitor (A8301) supplementation promoted enterocyte differentiation at the expense of the proliferation of intestinal stem cells (ISC). The mitogen-activated protein kinase p38 inhibitor (SB202190) promoted intestinal organoid formation and enterocyte differentiation but suppressed the differentiation of enteroendocrine cells, goblet cells and Paneth cells. However, the suppression of enteroendocrine cell and Paneth cell differentiation by SB202190 was alleviated at the presence of A8301. The glycogen synthase kinase 3 inhibitor (CHIR99021), valproic acid (VPA) alone and their combination promoted chicken intestinal organoid formation and enterocyte differentiation at the expense of the expression of Paneth cells and goblet cells. Chicken serum significantly improved organoid formation, especially in the presence of A8301, SB202190, CHIR99021, and VPA, but inhibited the differentiation of Paneth cells and enteroendocrine cells. Chicken serum at a concentration of 0.25% meets the requirement of chicken intestinal organoid development, and the beneficial effect of chicken serum on chicken intestinal organoid culture could not be replaced by fetal bovine serum and insulin-like growth factor-1. Moreover, commercial mouse organoid culture medium supplemented with A8301, SB202190, CHIR99021, VPA, and chicken serum promotes chicken organoid budding. Based on the chicken intestinal organoid model, we found that methionine deficiency mimicked by cycloleucine suppressed organoid formation and organoid size, and this effect was reinforced with increased cycloleucine concentrations. Methionine hydroxy analogue promoted regeneration of ISC but decreased cell differentiation compared with the results obtained with L-methionine. In conclusion, our results provide a potentially excellent guideline for chicken intestinal organoid culture and insights into methionine function in crypt development.