Small Intestinal Stem Cells Research Articles

Shenqi Pill decreases the goblet cell differentiation of small intestinal stem cells and reactivates Notch signaling pathway in the duodenum of aging rats

Shenqi Pill decreases the goblet cell differentiation of small intestinal stem cells and reactivates Notch signaling pathway in the duodenum of aging rats

Clinically Approved Carbon Nanoparticles with Oral Administration for Intestinal Radioprotection via Protecting the Small Intestinal Crypt Stem Cells and Maintaining the Balance of Intestinal Flora.

The exploration of an old drug for new biomedical applications has an absolute predominance in shortening the clinical conversion time of drugs for clinical application. In this work, carbon nanoparticles suspension injection (CNSI), the first clinically approved carbon nanoparticles in China, is explored as a new nano-radioprotective agent for potent intestinal radioprotection. CNSI shows powerful radioprotective performance in the intestine under oral administration, including efficient free radical scavenging ability, good biosafety, high chemical stability, and relatively long retention time. For example, CNSI shows high reactive oxygen species (ROS) scavenging activities, which effectively alleviates the mitochondrial dysfunction and DNA double-strand breaks to protect the cells against radiation-induced damage. Most importantly, this efficient ROS scavenging ability greatly helps restrain the apoptosis of the small intestinal epithelial and crypt stem cells, which decreases the damage of the mechanical barrier and thus relieves radiation enteritis. Moreover, CNSI helps remove the free radicals in the intestinal microenvironment and thus maintain the balance of intestinal flora so as to mitigate the radiation enteritis. The finding suggests a new application of clinically approved carbon nanoparticles, which not only promotes the development of new intestinal radioprotector, but also has a great potential for clinical transformation.

Downregulation of lncRNA MALAT1 suppresses abnormal proliferation of small intestinal epithelial stem cells through miR‑129‑5p expression in diabetic mice.

The problems caused by diabetes mellitus (DM) and its related complications are gaining increasing attention. In our previous study, the abnormal proliferation of small intestinal epithelial cells (IECs) were observed in diabetic mice. However, little is known regarding the potential underlying mechanism. In the present study, the abnormal proliferation of IECs in DM and the marked upregulation of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was observed. Additionally, knockdown of MALAT1 significantly reduced abnormal IESC proliferation in DM mice. Bioinformatics analysis and luciferase reporter assays revealed that microRNA (miR)‑129‑5p was directly targeted by MALAT1. Moreover, the results of the bioinformatics prediction and luciferase assays demonstrated that MALAT1 directly interacted with SRY‑box 9 (SOX9). Furthermore, MALAT1 silencing was observed to attenuate the abnormal proliferation of IESCs through the SOX9‑mediated WNT/β‑catenin signaling pathway. Knockdown of MALAT1 downregulated SOX9 expression by binding to miR‑129‑5p, thereby inhibiting the abnormal proliferation of IESCs via the WNT/β‑catenin signaling pathway.

Increased Mutagenesis during Fetal Hematopoietic Development in Down Syndrome

Children with Down syndrome are predisposed to leukemia during the first years of their life. 5-10% of newborns with Down syndrome are born with transient myeloproliferative disorder (TMD), which often spontaneously disappears. The majority of these patients achieves complete remission. However, in 20-30% of all TMD patients the disease progress in acute megakaryoblastic leukemia. In addition, they have a 20 fold higher risk of developing B-lymphocyte acute leukemia (B-ALL). Leukemic development in Down syndrome is initiated during fetal development. However, it is unclear why fetuses with a trisomy of chromosome 21 have an increased risk of developing leukemia. Previously, we have developed a method to study somatic mutations in single cells using clonal cultures. Here, we applied this method to human fetal hematopoietic stem and progenitor cells (HSPCs) from liver and bone marrow of Down syndrome human fetuses and control fetuses with two copies of chromosome 21 (D21). In addition, we characterized somatic mutation accumulation in not affected small intestine stem cells. Subsequently, we performed in depth mutational analyses to characterize active processes using mutational signatures in fetal stem cells, which potentially can drive leukemic development during early life. Recently, we have shown that that healthy adult HSPCs gradually accumulate somatic mutations in a linear fashion with an annual mutation rate of 14.2 base substitutions per year. Whereas the somatic mutation rate is significantly higher during fetal development. Subsequently, in Down syndrome fetuses the overall somatic mutation rate of fetal stem cells is significantly increased compared to D21 fetal stem cells (P-value: 0,024). We performed phylogenetic analysis to study relatedness of the cells and observed an higher somatic mutation rate in the first cell divisions. This elevated mutation rate can be explained by increased contribution of mutational process signature 1 and 5, which are already present in fetal stem cells. Therefore, Down syndrome fetal stem cells show enhanced activity or increased sensitivity to mutational processes that are normally active during development. The same mutational signatures are present in TMD blast cells, indicating that these processes can cause cancer driver mutations and subsequently contribute to leukemic development. Interestingly, some Down syndrome fetal stem cells showed very high mutation numbers that could partly be attributed to mutational signature 18, which likely reflect oxidative-stress induced mutagenesis. These findings, show increased mutagenesis in Down syndrome during fetal development in hematopoietic stem cells and small intestine stem cells. This increased mutagenesis can potentially explain why children with Down syndrome have an increased risk of developing leukemia in early life. Disclosures No relevant conflicts of interest to declare.

Cellular Composition and Differentiation Signaling in Chicken Small Intestinal Epithelium.

Simple SummaryThe small intestine is the major place for chickens to digest and absorb the nutrients. The intestinal epithelium is a single layer of cells that are generated by the stem cells in the crypt. This review mainly summarized the recent findings of molecular signaling pathways that control the proliferation and differentiation of chicken small intestinal stem cells, and the biological functions of chicken small intestinal stem cells, and the biological functions of chicken small intestinal epithelium corresponding to different cell types. We also provided some novel in vitro models for studying chicken small intestinal stem cells. With the understanding of cellular composition and differentiation in chicken small intestinal epithelium, it may bring the new ideas for the poultry industry to improve the small intestinal health and nutrition.The small intestine plays an important role for animals to digest and absorb nutrients. The epithelial lining of the intestine develops from the embryonic endoderm of the embryo. The mature intestinal epithelium is composed of different types of functional epithelial cells that are derived from stem cells, which are located in the crypts. Chickens have been widely used as an animal model for researching vertebrate embryonic development. However, little is known about the molecular basis of development and differentiation within the chicken small intestinal epithelium. This review introduces processes of development and growth in the chicken gut, and compares the cellular characteristics and signaling pathways between chicken and mammals, including Notch and Wnt signaling that control the differentiation in the small intestinal epithelium. There is evidence that the chicken intestinal epithelium has a distinct cellular architecture and proliferation zone compared to mammals. The establishment of an in vitro cell culture model for chickens will provide a novel tool to explore molecular regulation of the chicken intestinal development and differentiation.

Human Intestinal Enteroids Model MHC-II in the Gut Epithelium.

The role of intestinal epithelial cells (IECs) in mucosal tolerance and immunity remains poorly understood. We present a method for inducing MHC class II (MHC-II) in human enteroids, “mini-guts” derived from small intestinal crypt stem cells, and show that the intracellular MHC-II peptide-pathway is intact and functional in IECs. Our approach enables human enteroids to be used for novel in vitro studies into IEC MHC-II regulation and function during health and disease.

Abstract 3746: Fasting in mice enables abdominal radiation dose escalation in the setting of pancreatic cancer by mitigating small intestinal toxicity

Abstract Surgical resection is the only potentially curative treatment for pancreatic cancer, but only 15-20% of patients have resectable tumors. In unresectable cases, stereotactic body radiotherapy (SBRT) may be used to give tumor-directed radiotherapy (RT). Unfortunately, this can cause severe gastrointestinal (GI) toxicity due to proximity of the pancreatic head to the duodenum. Protecting the intestine from the toxic side-effects of radiation may enable dose-escalation that could achieve more effective local control of disease. We and others have previously shown that a fast of 24 hours protects mice from lethal doses of the DNA-damaging agent etoposide. In this study, we demonstrate that a 24 hour fast also protects mice from lethal doses of total-abdominal radiation. Histologic analyses using the Withers-Elkind microcolony assay show that fasting protected small intestinal (SI) stem cells from radiation damage and promoted early regeneration. To show a proof-of-principle for the use of this radioporotective maneuver in cancer therapy, we used an orthotopic model of pancreatic cancer using KPC tumor cells syngeneic to C57BL/6. Here, we show that fasting-mediated intestinal protection enabled dose escalated SBRT for treatment of these orthotopic tumors. RT with fasting-mediated radioprotection delayed tumor growth and improved survival compared to controls. Given this robust phenotype, we developed a 3D culture ex vivo assay using intestinal stem cell-enriched epithelial spheroid cultures. We modified these intestinal spheroids with a bioluminescent reporter and used these cells to develop a modified clonogenic assay for 3D culture that can be used to identify novel radioprotectors, such as a fasting mimetic. Taken together, these results suggest that fasting protects small intestinal stem cells, allowing animals to receive potentially curative doses of abdominal radiation that would otherwise be lethal. Future work will aim to identifying the mechanisms by which fasting confers intestinal protection and drug candidates that can be used to mimic this fasting-mediated protection. Citation Format: Marimar de la Cruz Bonilla, Kristina M. Stemler, Sabrina Jeter-Jones, Tara N. Fujimoto, Jessica Molkentine, Gabriela M. Asencio Torres, Cullen M. Taniguchi, Helen Piwnica-Worms. Fasting in mice enables abdominal radiation dose escalation in the setting of pancreatic cancer by mitigating small intestinal toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3746.

Integrative analysis of Paneth cell proteomic and transcriptomic data from intestinal organoids reveals functional processes dependent on autophagy.

ABSTRACTPaneth cells are key epithelial cells that provide an antimicrobial barrier and maintain integrity of the small-intestinal stem cell niche. Paneth cell abnormalities are unfortunately detrimental to gut health and are often associated with digestive pathologies such as Crohn's disease or infections. Similar alterations are observed in individuals with impaired autophagy, a process that recycles cellular components. The direct effect of autophagy impairment on Paneth cells has not been analysed. To investigate this, we generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells, making these cells impaired in autophagy. Using three-dimensional intestinal organoids enriched for Paneth cells, we compared the proteomic profiles of wild-type and autophagy-impaired organoids. We used an integrated computational approach combining protein-protein interaction networks, autophagy-targeted proteins and functional information to identify the mechanistic link between autophagy impairment and disrupted pathways. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids, suggesting reduced protein degradation. Interestingly, these differentially abundant proteins comprised 116 proteins (41%) that are predicted targets of the selective autophagy proteins p62, LC3 and ATG16L1. Our integrative analysis revealed autophagy-mediated mechanisms that degrade key proteins in Paneth cell functions, such as exocytosis, apoptosis and DNA damage repair. Transcriptomic profiling of additional organoids confirmed that 90% of the observed changes upon autophagy alteration have effects at the protein level, not on gene expression. We performed further validation experiments showing differential lysozyme secretion, confirming our computationally inferred downregulation of exocytosis. Our observations could explain how protein-level alterations affect Paneth cell homeostatic functions upon autophagy impairment.This article has an associated First Person interview with the joint first authors of the paper.

Abstract 4164: Fasting protects mice from lethal radiation by promoting small intestinal stem cell survival

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths in the US. Surgical resection is the only potentially curative treatment; however, only 15%-20% of patients present with tumors that can be resected. There is no consensus regarding standard of care in unresectable cases, however many academic centers use stereotactic body radiotherapy (SBRT) to give tumor-directed radiotherapy (RT). Unfortunately, even this conformal technique can still cause severe gastrointestinal (GI) toxic effects caused by the proximity of the pancreatic head to the duodenum. Protecting the intestine from the toxic effects of radiation may enable dose escalation that could achieve more effective local control of disease. We and others have previously shown that a prolonged fast of 24 hours protects mice from lethal doses of etoposide. In this study, we extend and build on our previous finding to demonstrate that a similar 24 hour fast also protects from lethal doses of total abdominal radiation. Histologic analyses, using the Withers-Elkind microcolony assay, show that fasting protected small intestinal (SI) stem cells from radiation damage and promoted early regeneration. To show a proof-of-principle for the use of this radioporotective maneuver in cancer therapy, we developed an orthotopic model of pancreatic cancer using KPC tumor cells syngeneic to C57BL/6. Here, we show that fasting-mediated intestinal protection enabled dose escalated SBRT for treatment of these orthotopic tumors. RT with fasting radioprotection delayed tumor growth and improved survival compared to controls. Given this robust phenotype, we developed a 3D culture ex vivo assay using intestinal stem cell enriched epithelial spheroid cultures. We modified these intestinal spheroids with a bioluminescent reporter and used these cells to develop a modified clonogenic assay for 3D culture that can be used to identify novel radioprotectors, such as a fasting mimetic. Taken together, these results suggest that fasting protects small intestinal stem cells sufficiently to allow animals to receive potentially curative doses of abdominal radiation that would other wise be lethal. Future work will aim to identifying the mechanism by which fasting confers intestinal protection and drug candidates that can be used to mimic this fasting-mediated protection. Citation Format: Marimar de la Cruz Bonilla, Kristina M. Stemler, Tara N. Fujimoto, Sabrina Jeter-Jones, Jessica M. Molkentine, Gabriela M. Asencio Torres, Cullen M. Taniguchi, Helen Piwnica-Worms. Fasting protects mice from lethal radiation by promoting small intestinal stem cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4164.

Colon organoid formation and cryptogenesis are stimulated by growth factors secreted from myofibroblasts.

Although small intestinal epithelial stem cells form crypts when using intestinal culture conditions, colon stem cells usually form colonospheres. Colon mesenchymal cell feeder layers can stimulate colon crypts to form organoids and produce crypts. We have investigated whether conditioned medium from colon mesenchymal cells can also stimulate colonosphere and organoid cryptogenesis. We prepared conditioned medium (CM) from WEHI-YH2 cells (mouse colon myofibroblasts); the CM stimulated both colonosphere formation and organoid cryptogenesis in vitro. The colon organoid-stimulating factors in WEHI-YH2 CM are inactivated by heating and trypsin digestion and proteins can be concentrated by ultrafiltration. Both the colonosphere- and organoid cryptogenesis- stimulatory effects of the CM are independent of canonical Wnt and Notch signaling. In contrast, bone morphogenetic protein 4 (BMP4) abolishes colonosphere formation and organoid cryptogenesis. The Transforming Growth Factor beta (TGFβ) Type I receptor kinase inhibitor (A83-01) stimulates colonosphere formation, whereas the Epidermal Growth Factor receptor (EGFR) kinase inhibitor (AG1478) reduces the formation of colonospheres, but in the presence of EGF, a “just-right” concentration of AG1478 increases colon organoid cryptogenesis.

598 - Single-Cell Level Analysis of Organoids Derived from CD Patients Reveals Disease-Status Related Modifications of Small Intestinal Stem Cells

598 - Single-Cell Level Analysis of Organoids Derived from CD Patients Reveals Disease-Status Related Modifications of Small Intestinal Stem Cells

Enteric Nervous System Regulation of Intestinal Stem Cell Differentiation and Epithelial Monolayer Function

The Enteric Nervous System (ENS) is a complex network of neurons and glia, which regulates sensorimotor function throughout the gastroinestinal tract (GI). Here we investigated the role of the ENS and intestinal myofibroblasts in the maintenance of a primary intestinal epithelial barrier through regulation of monolayer permeability, cytokine production, and differentiation of intestinal stem cells. Utilizing a novel, in vitro, transwell-based coculture system, murine small intestinal stem cells were isolated and cultured with ENS neurons and glia or subepithelial myofibroblasts. Results show that the ENS contributes to regulation of intestinal stem cell fate, promoting differentiation into chemosensory enteroendocrine cells, with 0.9% of cells expressing chromogranin A when cultured with ENS versus 0.6% in cocultures with myofibroblasts and 0.3% in epithelial cultures alone. Additionally, enteric neurons and myofibroblasts differentially release cytokines Macrophage Inflammatory Protein 2 (MIP-2), Transforming Growth Factor beta 1 (TGF-β1), and Interleukin 10 (IL-10) when cultured with intestinal epithelial cells, with a 1.5 fold increase of IL-10 and a 3 fold increase in MIP-2 in ENS cocultures compared to coculture with myofibroblasts. These results indicate the importance of enteric populations in the regulation of intestinal barrier function.

Single cell analysis of Crohn\u2019s disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties

BackgroundIntestinal stem cells (ISCs) play indispensable roles in the maintenance of homeostasis, and also in the regeneration of the damaged intestinal epithelia. However, whether the inflammatory environment of Crohn’s disease (CD) affects properties of resident small intestinal stem cells remain uncertain.MethodsCD patient-derived small intestinal organoids were established from enteroscopic biopsy specimens taken from active lesions (aCD-SIO), or from mucosa under remission (rCD-SIO). Expression of ISC-marker genes in those organoids was examined by immunohistochemistry, and also by microfluid-based single-cell multiplex gene expression analysis. The ISC-specific function of organoid cells was evaluated using a single-cell organoid reformation assay.ResultsISC-marker genes, OLFM4 and SLC12A2, were expressed by an increased number of small intestinal epithelial cells in the active lesion of CD. aCD-SIOs, rCD-SIOs or those of non-IBD controls (NI-SIOs) were successfully established from 9 patients. Immunohistochemistry showed a comparable level of OLFM4 and SLC12A2 expression in all organoids. Single-cell gene expression data of 12 ISC-markers were acquired from a total of 1215 cells. t-distributed stochastic neighbor embedding analysis identified clusters of candidate ISCs, and also revealed a distinct expression pattern of SMOC2 and LGR5 in ISC-cluster classified cells derived from aCD-SIOs. Single-cell organoid reformation assays showed significantly higher reformation efficiency by the cells of the aCD-SIOs compared with that of cells from NI-SIOs.ConclusionsaCD-SIOs harbor ISCs with modified marker expression profiles, and also with high organoid reformation ability. Results suggest modification of small intestinal stem cell properties by unidentified factors in the inflammatory environment of CD.

Advanced three-dimensional culture of equine intestinal epithelial stem cells.

Intestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine. To describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities. Descriptive study. Intestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts were cryopreserved immediately following collection and viability assessed. Intestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Previously frozen crypts were successfully cultured following a freeze-thaw cycle. Tissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time. The successful culture of equine intestinal crypts into 3D "mini-guts" allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease (colic).

Highly conserved type 1 pili promote enterotoxigenic E. coli pathogen-host interactions.

Enterotoxigenic Escherichia coli (ETEC), defined by their elaboration of heat-labile (LT) and/or heat-stable (ST) enterotoxins, are a common cause of diarrheal illness in developing countries. Efficient delivery of these toxins requires ETEC to engage target host enterocytes. This engagement is accomplished using a variety of pathovar-specific and conserved E. coli adhesin molecules as well as plasmid encoded colonization factors. Some of these adhesins undergo significant transcriptional modulation as ETEC encounter intestinal epithelia, perhaps suggesting that they cooperatively facilitate interaction with the host. Among genes significantly upregulated on cell contact are those encoding type 1 pili. We therefore investigated the role played by these pili in facilitating ETEC adhesion, and toxin delivery to model intestinal epithelia. We demonstrate that type 1 pili, encoded in the E. coli core genome, play an essential role in ETEC virulence, acting in concert with plasmid-encoded pathovar specific colonization factor (CF) fimbriae to promote optimal bacterial adhesion to cultured intestinal epithelium (CIE) and to epithelial monolayers differentiated from human small intestinal stem cells. Type 1 pili are tipped with the FimH adhesin which recognizes mannose with stereochemical specificity. Thus, enhanced production of highly mannosylated proteins on intestinal epithelia promoted FimH-mediated ETEC adhesion, while conversely, interruption of FimH lectin-epithelial interactions with soluble mannose, anti-FimH antibodies or mutagenesis of fimH effectively blocked ETEC adhesion. Moreover, fimH mutants were significantly impaired in delivery of both heat-stable and heat-labile toxins to the target epithelial cells in vitro, and these mutants were substantially less virulent in rabbit ileal loop assays, a classical model of ETEC pathogenesis. Collectively, our data suggest that these highly conserved pili play an essential role in virulence of these diverse pathogens.

Distinct Levels of Radioresistance in Lgr5+ Colonic Epithelial Stem Cells versus Lgr5+ Small Intestinal Stem Cells.

Although small and large intestines possess seemingly similar Wnt-driven leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)+ adult epithelial stem cells, we report here that the two organs exhibit distinct mechanisms of tissue response to ionizing radiation. Employing Lgr5-lacZ transgenic mice and Lgr5 in situ hybridization, we found colonic epithelial stem cells (CESC) markedly more radioresistant in vivo than small intestinal crypt base columnar stem cells (CBC; D0 = 6.0 ± 0.3 Gy vs. 1.3 ± 0.1, respectively; P < 0.01). Accordingly, CESCs survived 30 Gy exposure, while CBCs were completely depleted after 15 Gy. EdU incorporation studies indicated that after 19 Gy, CBCs exited growth arrest at 12 hours, resuming normal mitotic activity despite 60% of this population displaying residual γH2AX foci, indicative of persistent unrepaired DNA damage. Checkpoint recovery before complete double-strand break (DSB) repair represents the sine qua non of a newly defined potentially lethal pathophysiology termed checkpoint adaptation. In the small intestinal mucosa, checkpoint adaptation resulted in CBCs succumbing to an 8-fold increase in the incidence of highly lethal chromosomal aberrations and mitotic catastrophe by 48 hours postradiation. In contrast, Lgr5+ CESCs displayed delayed checkpoint recovery at 48 hours post-19 Gy, coordinated with complete DSB repair and regeneration of colonic mucosa originating, at least in part, from surviving CESCs. The discovery that small intestinal CBCs succumb to checkpoint adaptation is the first demonstration that this aberrant cell-cycle response may drive mammalian tissue radiosensitivity. Cancer Res; 77(8); 2124-33. ©2017 AACR.

Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells

The apical membranes of intestinal cells are exposed to luminal food contents, and the cells' ability to perceive and adapt to these environmental cues is critical so that appropriate endocrine signals are released and that specific types as well as numbers of enzymes and transporters are synthesized to match the variety and fluctuating concentrations of dietary constituents. Since enteroendocrine and enterocyte cells represent secretory and absorptive lineages, respectively, and have previously been shown to demonstrate nutrient sensing capacity, we tested the hypothesis that progenitor intestinal stem cells (ISCs) possess nutrient sensing ability which is subsequently inherited by their progenies. We directed via modulators of Wnt and Notch signaling, the primary three‐dimensional culture of mouse intestinal crypts into ISC, enterocyte, goblet or Paneth organoids. Each specialized organoid contained mostly a single specific cell type comprised primarily of ISC (~90%), enterocytes (~90%), goblet (~85%) or Paneth cells (~65%) at relative proportions much higher than in situ as determined by mRNA expression and immunocytochemical detection of cell type biomarkers. We identified nutrient sensing cell type(s) by examining responses of fructose responsive genes after a fructose challenge. Typical (control) organoids comprised of all cell types as well as enterocyte, but not ISC, organoids responded specifically to fructose without altering expression of nonfructolytic genes. Surprisingly, Paneth and goblet organoids responded dramatically to fructose, suggesting that each of the three major secretory cell types has nutrient sensing ability. Responses of all fructose‐sensing organoids increased with higher fructose concentrations. Typical organoids generated from fructose transporter GLUT5‐KO and fructokinase KHK‐KO mice did not respond to fructose, suggesting that transport and metabolism of fructose are required for fructose sensing, and providing a likely mechanism underlying absence of fructose sensing in ISCs that do not significantly express GLUT5 and KHK. More mature absorptive and secretory organoids tended to exhibit stronger fructose responses, suggesting that sensing improves with increased differentiation. Remarkably, mature enterocytes, upon forced dedifferentiation to reacquire ISC characteristics, retained fructose sensing ability. Fructose response was independent of either Wnt or Notch modulators used to direct organoid formation, and of glucose concentrations in the organoid culture medium. Using an innovative experimental approach, we determined that nutrient sensing is likely repressed in progenitor ISCs, then derepressed prior to specification between absorptive and secretory lineages. This study increases our understanding of the role of differentiation in regulating nutrient sensing during development of intestinal epithelial cells.Support or Funding InformationSupported by NSF Grants No. IOS‐1121049 and 1456673 (RPF). RPF also received support from NIH R01‐DK‐102934 (NG).

Ex vivo Culture of Adult Mouse Antral Glands.

The tri-dimensional culture, initially described by Sato et al. (2009) in order to isolate and characterize epithelial stem cells of the adult small intestine, has been subsequently adapted to many different organs. One of the first examples was the isolation and culture of antral stem cells by Barker et al. (2010), who efficiently generated organoids that recapitulate the mature pyloric epithelium in vitro. This ex vivo approach is suitable and promising to study gastric function in homeostasis as well as in disease. We have adapted Barker's protocol to compare homeostatic and regenerating tissues and here, we meticulously describe, step by step, the isolation and culture of antral glands as well as the isolation of single cells from antral glands that might be useful for culture after cell sorting as an example (Fernandez Vallone et al., 2016 ).

A new scaffold containing small intestinal submucosa and mesenchymal stem cells improves pancreatic islet function and survival invitro and invivo.

It is unknown whether a scaffold containing both small intestinal submucosa (SIS) and mesenchymal stem cells (MSCs) for transplantation may improve pancreatic islet function and survival. In this study, we examined the effects of a SIS-MSC scaffold on islet function and survival in vitro and in vivo. MSCs and pancreatic islets were isolated from Sprague-Dawley rats, and SIS was isolated from Bamei pigs. The islets were apportioned among 3 experimental groups as follows: SIS-islets, SIS-MSC-islets and control-islets. In vitro, islet function was measured by a glucose-stimulated insulin secretion test; cytokines in cultured supernatants were assessed by enzyme-linked immunosorbent assay; and gene expression was analyzed by reverse transcription-quantitative PCR. In vivo, islet transplantation was performed in rats, and graft function and survival were monitored by measuring the blood glucose levels. In vitro, the SIS-MSC scaffold was associated with improved islet viability and enhanced insulin secretion compared with the controls, as well as with the increased the expression of insulin 1 (Ins1), pancreatic and duodenal homeobox 1 (Pdx1), platelet endothelial cell adhesion molecule 1 [Pecam1; also known as cluster of differentiation 31 (CD31)] and vascular endothelial growth factor A (Vegfa) in the islets, increased growth factor secretion, and decreased tumor necrosis factor (TNF) secretion. In vivo, the SIS-MSC scaffold was associated with improved islet function and graft survival compared with the SIS and control groups. On the whole, our findings demonstrate that the SIS-MSC scaffold significantly improved pancreatic islet function and survival in vitro and in vivo. This improvement may be associated with the upregulation of insulin expression, the improvement of islet microcirculation and the secretion of cytokines.

Paradoxical Roles of Elongation Factor-2 Kinase in Stem Cell Survival

Protein synthesis inhibition is an immediate response during stress to switch the composition of protein pool in order to adapt to the new environment. It was reported that this response could be either protective or deleterious. However, how cells choose to live or die upon protein synthesis inhibition is largely unknown. Previously, we have shown that elongation factor-2 kinase (eEF2K), a protein kinase that suppresses protein synthesis during elongation phase, is a positive regulator of apoptosis both in vivo and in vitro Consistently, here we report that knock-out of eEF2K protects mice from a lethal dose of whole-body ionizing radiation at 8 Gy by reducing apoptosis levels in both bone marrow and gastrointestinal tracts. Surprisingly, similar to the loss of p53, eEF2K deficiency results in more severe damage to the gastrointestinal tract at 20 Gy with the increased mitotic cell death in small intestinal stem cells. Furthermore, using epithelial cell lines, we showed that eEF2K is required for G2/M arrest induced by radiation to prevent mitotic catastrophe in a p53-independent manner. Specifically, we observed the elevation of Akt/ERK activity as well as the reduction of p21 expression in Eef2k(-/-) cells. Therefore, eEF2K also provides a protective strategy to maintain genomic integrity by arresting cell cycle in response to stress. Our results suggest that protective versus pro-apoptotic roles of eEF2K depend on the type of cells: eEF2K is protective in highly proliferative cells, such as small intestinal stem cells and cancer cells, which are more susceptible to mitotic catastrophe.