Intestinal Progenitor Research Articles

Identification of Novel Molecules Targeting Cancer Stem Cells.

Multiple studies focused on tumor heterogeneity and cellular hierarchies have demonstrated the role of cancer stem cells (CSC) in tumor initiation and recurrence. Colorectal cancer is one of the leading causes of cancer-related death and is hierarchically organized, with the majority of tumor cells descending from a small population of colon cancer stem cells (CCSCs). Such a rare self-renewing population is marked by the acquisition of distinct chromatin regulation and transcriptional programs. Fundamental molecular deviations between CCSCs and bulk tumor cells as well as normal tissues represent a unique therapeutic access to develop novel, selective anticancer therapies.In this chapter, we describe a methodological pipeline to identify novel molecules to selectively target human CCSC. We present a point-by-point description of a typical phenotypic molecular screening experiment, aiming to identify selective modulators of human CCSCs vs. normal intestinal progenitor cells.

Leucine-rich Repeat-containing G-protein Coupled Receptor 5 Gene Overexpression of the Rat Small Intestinal Progenitor Cells in Response to Orally Administered Grape Exosome-like Nanovesicles.

Background:Grape exosome-like nanovesicles (GELNs) have the advantage of inherent biocompatibility and biodegradability, the potential to be used as oral delivery vehicles. The objective of this research was to evaluate the efficiency of Syrah GELN purification and their effects on the intestinal stem cells when orally administrated to the rats.Materials and Methods:In this experimental study, Syrah GELN isolated by differential centrifugation and sucrose gradient ultracentrifugation method, then the concentration of protein, size, and zeta potential were measured as well as nanoparticles morphology. The stability of nanoparticles was investigated in the solution that mimicked the condition encountered in the stomach and intestine. To demonstrate transfection efficiency of intestinal stem cells, real-time PCR was carried out using rat leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)-specific primers on cDNA derived from total RNA extracted from the upper part of the small intestine of GELN-treated rats and their controls.Results:The mean size, zeta potential, and concentration of nanoparticles were 205.1 nm, −12.5 mV, and 250 μg/ml, respectively. The result of stability test demonstrated that Syrah GELN were resistant to the harsh environment of the stomach. Lgr5 gene expression was increased by tenfold in GELN-treated rats compared with the controls.Conclusions:As intestinal stem cells are poorly accessible by common exogenous agents in vivo, oral delivery of GELNs provides a new approach to modulate the stem cell microenvironment for intestinal remodeling. This novel and effective method would help to overcome conditions such as inflammatory bowel disease, colorectal cancer, and applicable in regenerative medicine.

Protein biomarker of cell proliferation determines survival to discharge in cases of equine large colon volvulus

Progenitor cells play critical roles in epithelial repair following ischaemic injury. Protein biomarkers have been used to identify intestinal progenitor cell subpopulations. This study aims to determine if a critical number of intestinal progenitor cells can predict tissue viability and survival to discharge of large colon volvulus (LCV) cases. The objectives were to 1) identify intestinal progenitor cell subpopulations using biomarkers: proliferating cell nuclear antigen (PCNA), sex determining region Y box 9 (SOX9), phospho-histone H3 (PHH3) and Ki-67, 2) define cut-off values for critical numbers of positive cells and 3) determine if survival to discharge is associated with cut-off values. Retrospective cohort study. Adult horses admitted to the Farm and Equine Veterinary Medical Center at NC State's Veterinary Hospital and Peterson and Smith Equine Hospital between 2006 and 2016 that underwent an exploratory coeliotomy with a diagnosis of LCV of ≥360 degrees, had pelvic flexure biopsy and that recovered from general anaesthesia were selected for inclusion in the study. Immunohistochemical analyses were performed and positive cells were counted. Optimal cut-off values were determined using receiver operator curves. A Fisher's exact test was used to associate cut-off values with survival to discharge. In this study, 23 cases of LCV ≥360° were included. Of 23 horses, 13 (57%) survived to discharge. A cut-off value of <2.1 PHH3 positive cells per crypt correctly predicted death with 100% sensitivity (95% CI; 69.15-100%) and 84.62% specificity (95% CI; 54.55-98.08%). LCV cases with <2.1 PHH3 positive cells per crypt were 96.6 times more likely to die (95% CI; 4.14-2255 and P<0.0001). Biomarkers PCNA, SOX9 and Ki-67 did not predict short-term survival. The population size was small. PHH3 immunohistochemical analysis may assist in more accurate prediction of survival to hospital discharge of LCV cases. The summary is available in Spanish - see Supporting Information.

Intestinal grafts grown from stem cells

Gastrointestinal disorders such as Crohn’s disease can lead to a condition known as short bowel syndrome (SBS) in which a significant portion of a person’s small intestine is no longer capable of absorbing nutrients. Intestinal grafts to supplement the remaining small intestine could help restore this lost function. A team led by Harald C. Ott of Massachusetts General Hospital has grown functional intestinal tissue by seeding scaffolds formed from rat small intestines with human pluripotent stem cells (Nat. Commun. 2017, DOI: 10.1038/s41467-017-00779-y). The researchers remove all cells from a segment of rat small intestine to form a scaffold that retains the extracellular architecture of the original tissue. They direct the human stem cells to differentiate into intestinal progenitor cells and then use them to seed the scaffold. After two weeks in culture, the progenitor cells produce many of the cell types usually found in intestinal tissue. The researchers also add

Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage.

Corrigendum2 October 2017free access Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage Si Tao Si Tao Search for more papers by this author Duozhuang Tang Duozhuang Tang Search for more papers by this author Yohei Morita Yohei Morita Search for more papers by this author Tobias Sperka Tobias Sperka Search for more papers by this author Omid Omrani Omid Omrani Search for more papers by this author André Lechel André Lechel Search for more papers by this author Vadim Sakk Vadim Sakk Search for more papers by this author Johann Kraus Johann Kraus Search for more papers by this author Hans A Kestler Hans A Kestler Search for more papers by this author Michael Kühl Michael Kühl Search for more papers by this author Karl Lenhard Rudolph Karl Lenhard Rudolph Search for more papers by this author Si Tao Si Tao Search for more papers by this author Duozhuang Tang Duozhuang Tang Search for more papers by this author Yohei Morita Yohei Morita Search for more papers by this author Tobias Sperka Tobias Sperka Search for more papers by this author Omid Omrani Omid Omrani Search for more papers by this author André Lechel André Lechel Search for more papers by this author Vadim Sakk Vadim Sakk Search for more papers by this author Johann Kraus Johann Kraus Search for more papers by this author Hans A Kestler Hans A Kestler Search for more papers by this author Michael Kühl Michael Kühl Search for more papers by this author Karl Lenhard Rudolph Karl Lenhard Rudolph Search for more papers by this author Author Information Si Tao, Duozhuang Tang, Yohei Morita, Tobias Sperka, Omid Omrani, André Lechel, Vadim Sakk, Johann Kraus, Hans A Kestler, Michael Kühl and Karl Lenhard Rudolph The EMBO Journal (2017)36:2920-2921https://doi.org/10.15252/embj.201797813 This article corrects the following: Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage21 January 2015 ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The authors state that it came to their attention that the figure legends of two figures were not sufficiently detailed in the original version of the manuscript. The figure legend of Fig 1E and F should read: A single representative FACS plot was chosen to illustrate (E) the gating of LGR5hi and LGR5lo populations within the population of LGR5+ (GFP-positive) intestinal cells and (F) the gating of LGR5hi-high, LGR5hi-low, LGR5lo-high, and LGR5lo-low populations. The same FACS plot is displayed in (E) and (F). The figure legend of Fig 6C should read: Representative Western blots of cell lysates for the expression of phospho-p53 and cleaved caspase-3 (each n = 3; see Source Data for this figure). Samples from a single experiment were divided into identical portions and probed for phospho-p53 or cleaved casp3. The expression control of beta-actin was conducted on the second aliquot of the samples and was run in parallel on a separate gel. The corrections do not affect the original conclusions presented. We apologize for the lack of detail and any inconvenience it may have caused. Editorial Note These changes were based on the results of an investigation of the Leibniz Association. All authors agree with this correction. Source data for Figure 6C. Previous ArticleNext Article Read MoreAbout the coverClose modalView large imageVolume 36,Issue 19,02 October 2017Cover: Deregulation of the actin cytoskeleton regulator Formin 2 is a common denominator of post-traumatic stress disorders and age-dependent Alzheimer’s disease phenotypes, possibly explaining their clinical connection. From Roberto Carlos Agís-Balboa, Andre Fischer, Farahnaz Sananbenesi and colleagues: Formin 2 links neuropsychiatric phenotypes at young age to an increased risk for dementia. or details, see the Article on p 2815, also highlighted by Johannes Gräff on p 2809. (Illustration by Uta Mackensen) Volume 36Issue 192 October 2017In this issue RelatedDetailsLoading ...

Generation of Mouse and Human Organoid-Forming Intestinal Progenitor Cells by Direct Lineage Reprogramming

Intestinal organoids hold great promise as a valuable tool for studying and treating intestinal diseases. The currently available sources of human intestinal organoids, tissue fragments or pluripotent stem cells, involve invasive procedures or complex differentiation protocols, respectively. Here, we show that a set of four transcription factors, Hnf4α, Foxa3, Gata6, and Cdx2, can directly reprogram mouse fibroblasts to acquire the identity of fetal intestine-derived progenitor cells (FIPCs). These induced FIPCs (iFIPCs) form spherical organoids that develop into adult-type budding organoids containing cells with intestinal stem cell properties. The resulting stem cells produce all intestinal epithelial cell lineages and undergo self-renewing cell divisions. After transplantation, the induced spherical and budding organoids can reconstitute colonic and intestinal epithelia, respectively. The same combination of four defined transcription factors can also induce human iFIPCs. This alternative approach for producing intestinal organoids may well facilitate application for disease analysis and therapy development.

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss‐of‐function disorders such as Hirschsprung disease.

Epithelial TNF Receptor Signaling Promotes Mucosal Repair in Inflammatory Bowel Disease.

TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn's disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/β-catenin-mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/β-catenin signaling correlates with inflammation status. TNF-deficient (Tnf-/-) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM-derived cells and TNFR expression by radioresistant IECs. Wild-type→Tnfr1/2-/- BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/β-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/β-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced β-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/β-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.

Cover Image, Volume 6, Issue 5

The cover image, by Ryan J. Smith et al., is based on the Advanced Review Signaling and epigenetic mechanisms of intestinal stem cells and progenitors: insight into crypt homeostasis, plasticity, and niches, DOI: 10.1002/wdev.281. Image Credit: Wasi Rizvi and Abilasha Rao‐Bhatia. image

Constitutive Immune Activity Promotes Tumorigenesis in Drosophila Intestinal Progenitor Cells

Gut innate immune defenses control bacterial populations and protect the host interior from invasion. Although excess intestinal immune activity frequently promotes inflammatory illnesses, we know little about the consequences of chronic innate immune activity exclusively in endodermal gut cells of an otherwise normal animal. To address this question, we examined the consequences of persistent inflammatory signals in adult fly intestinal progenitor cells. We found that constitutive immune activity disrupts expression of homeostatic regulators such as Notch pathway components and induces hyperplasia throughout the gut. Consistent with these observations, we found that persistent immune signals interfere with progenitor cell differentiation and exacerbate the formation of Notch-dependent intestinal tumors. These findings uncover a link between constitutive immune activity and tumorigenesis in intestinal stem cells.

Signaling and epigenetic mechanisms of intestinal stem cells and progenitors: insight into crypt homeostasis, plasticity, and niches.

The rapid turnover of intestinal epithelial cells is maintained by a small number of stem cells located in pocket-like gland structures called crypts. While our understanding of the identity and function of intestinal stem cells (ISCs) has rapidly progressed, epigenetic and transcriptional regulation in crypt stem cell and progenitor pools remains an active field of investigation. Surrounded by various types of cells in the stroma, crypt progenitors display high levels of plasticity, harboring the ability to interconvert in the face of epithelial damage. Recent studies analyzing epigenetic patterns of intestinal epithelial cells have provided evidence that plasticity is maintained by a broadly permissive epigenomic state, wherein cell-lineage specification is directed through activation of signaling pathways and transcription factor (TF) expression. New studies also have shown that the ISC niche, which is comprised of surrounding epithelial and mesenchymal tissues, plays a crucial role in supporting the maintenance and differentiation of stem cells by providing contextual information in the form of signaling cascades, such as Wnt, Notch, and Hippo. These cascades ultimately govern TF expression to promote early cell-lineage decisions in both crypt stem cells and progenitors. Highlighting recent studies investigating signaling, transcriptional, and epigenetic mechanisms of intestinal epithelial cells, we will discuss the mechanisms underlying crypt homeostasis, plasticity, and niches. WIREs Dev Biol 2017, 6:e281. doi: 10.1002/wdev.281 For further resources related to this article, please visit the WIREs website.

Sa1241 - High-Fat Feeding Enhances Stemness of Intestinal Progenitors and Stimulates Epithelial Proliferation via Upregulation of R-Spondin 3 and its Receptors LGR4 and 5

Sa1241 - High-Fat Feeding Enhances Stemness of Intestinal Progenitors and Stimulates Epithelial Proliferation via Upregulation of R-Spondin 3 and its Receptors LGR4 and 5

Sedentary lifestyle related exosomal release of Hotair from gluteal-femoral fat promotes intestinal cell proliferation

Pioneering epidemiological work has established strong association of sedentary lifestyle and obesity with the risk of colorectal cancer, while the detailed underlying mechanism remains unknown. Here we show that Hotair (HOX transcript antisense RNA) is a pro-adipogenic long non-coding RNA highly expressed in gluteal-femoral fat over other fat depots. Hotair knockout in adipose tissue results in gluteal-femoral fat defect. Squeeze of the gluteal-femoral fat induces intestinal proliferation in wildtype mice, while not in Hotair knockout mice. Mechanistically, squeeze of the gluteal-femoral fat induces exosomal Hotair secretion mainly by transcriptional upregulation of Hotair via NFκB. And increased exosomal Hotair in turn circulates in the blood and is partially endocytosed by the intestine, finally promoting the stemness and proliferation of intestinal stem/progenitor cells via Wnt activation. Clinically, obese subjects with sedentary lifestyle have much higher exosomal HOTAIR expression in the serum. These findings establish that sedentary lifestyle promotes exosomal Hotair release from the gluteal-femoral fat, which in turn facilitates intestinal stem and/or progenitor proliferation, raising a possible link between sedentary lifestyle with colorectal tumorigenesis.

The ErbB3 receptor tyrosine kinase negatively regulates Paneth cells by PI3K-dependent suppression of Atoh1.

Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing antimicrobial peptides, including lysozyme and defensins. PC dysfunction is associated with disorders such as Crohn's disease and necrotizing enterocolitis, but the specific pathways regulating PC development and function are not fully understood. Here we tested the role of the neuregulin receptor ErbB3 in control of PC differentiation and the ISC niche. Intestinal epithelial ErbB3 knockout caused precocious appearance of PCs as early as postnatal day 7, and substantially increased the number of mature PCs in adult mouse ileum. ErbB3 loss had no effect on other secretory lineages, but increased expression of the ISC marker Lgr5. ErbB3-null intestines had elevated levels of the Atoh1 transcription factor, which is required for secretory fate determination, while Atoh1+ cells had reduced ErbB3, suggesting reciprocal negative regulation. ErbB3-null intestinal progenitor cells showed reduced activation of the PI3K-Akt and ERK MAPK pathways. Inhibiting these pathways in HT29 cells increased levels of ATOH1 and the PC marker LYZ. Conversely, ErbB3 activation suppressed LYZ and ATOH1 in a PI3K-dependent manner. Expansion of the PC compartment in ErbB3-null intestines was accompanied with elevated ER stress and inflammation markers, raising the possibility that negative regulation of PCs by ErbB3 is necessary to maintain homeostasis. Taken together, our data suggest that ErbB3 restricts PC numbers through PI3K-mediated suppression of Atoh1 levels leading to inhibition of PC differentiation, with important implications for regulation of the ISC niche.

Dynamic changes in chromatin states during specification and differentiation of adult intestinal stem cells.

Epigenetic mechanisms, including chromatin structure, chromatin dynamics and histone modifications play an important role for maintenance and differentiation of pluripotent embryonic stem cells. However, little is known about the molecular mechanisms of adult stem cell specification and differentiation. Here, we used intestinal stem cells (ISCs) as a model system to reveal the epigenetic changes coordinating gene expression programs during these processes. We found that two distinct epigenetic mechanisms participate in establishing the transcriptional program promoting ISC specification from embryonic progenitors. A large number of adult ISC signature genes are targets of repressive DNA methylation in embryonic intestinal epithelial progenitors. On the other hand, genes essential for embryonic development acquire H3K27me3 and are silenced during ISC specification. We also show that the repression of ISC signature genes as well as the activation of enterocyte specific genes is accompanied by a global loss of H2A.Z during ISCs differentiation. Our results reveal that, already during ISC specification, an extensive remodeling of chromatin both at promoters and distal regulatory elements organizes transcriptional landscapes operating in differentiated enterocytes, thus explaining similar chromatin modification patterns in the adult gut epithelium.

Apoptosis restores cellular density by eliminating a physiologically or genetically induced excess of enterocytes in the Drosophila midgut.

Using pathogens or high levels of opportunistic bacteria to damage the gut, studies in Drosophila have identified many signaling pathways involved in gut regeneration. Dying cells emit signaling molecules that accelerate intestinal stem cell proliferation and progenitor differentiation to replace the dying cells quickly. This process has been named 'regenerative cell death'. Here, mimicking environmental conditions, we show that the ingestion of low levels of opportunistic bacteria was sufficient to launch an accelerated cellular renewal program despite the brief passage of bacteria in the gut and the absence of cell death and this is is due to the moderate induction of the JNK pathway that stimulates stem cell proliferation. Consequently, the addition of new differentiated cells to the gut epithelium, without preceding cell loss, leads to enterocyte overcrowding. Finally, we show that a couple of days later, the correct density of enterocytes is promptly restored by means of a wave of apoptosis involving Hippo signaling and preferential removal of old enterocytes.

Dietary Regulation of Adult Stem Cells.

Dietary intake is a critical regulator of organismal physiology and health. Tissue homeostasis and regeneration are dependent on adult tissue stem cells that self-renew and differentiate into the specialized cell types. As stem cells respond to cues from their environment, dietary signals and nutrients influence tissue biology by altering the function and activity of adult stem cells. In this review, we highlight recent studies that illustrate how diverse diets such as caloric restriction, fasting, high fat diets, and ketogenic diets impact stem cell function and their microenvironments. Caloric restriction generally exerts positive effects on adult stem cells, notably increasing stem cell functionality in the intestine and skeletal muscle as well as increasing hematopoietic stem cell quiescence. Similarly, fasting confers protection of intestinal, hematopoietic, and neuronal stem cells against injury. High fat diets induce intestinal stem cell niche independence and stem-like properties in intestinal progenitors, while high fat diets impair hematopoiesis and neurogenesis. Caloric restriction and fasting are generally beneficial to adult stem cell function, while high fat diets impair stem cell function or create opportunities for tumorigenesis. However, the effects of each diet on stem cell biology are complex and vary greatly between tissues. Given the recent interest in developing dietary interventions or mimetics as therapeutics, further studies, including on ketogenic diets, will be essential to understand how adult stem cells respond to diet-induced signals and physiology.

Id2 controls specification of Lgr5+ intestinal stem cell progenitors during gut development.

The adult intestinal stem cells (ISCs), their hierarchies, mechanisms of maintenance and differentiation have been extensively studied. However, when and how ISCs are established during embryogenesis remains unknown. We show here that the transcription regulator Id2 controls the specification of embryonic Lgr5+ progenitors in the developing murine small intestine. Cell fate mapping analysis revealed that Lgr5+ progenitors emerge at E13.5 in wild-type embryos and differ from the rest on the intestinal epithelium by a characteristic ISC signature. In the absence of Id2, the intestinal epithelium differentiates into Lgr5+ cells already at E9.5. Furthermore, the size of the Lgr5+ cell pool is significantly increased. We show that Id2 restricts the activity of the Wnt signalling pathway at early stages and prevents precocious differentiation of the embryonic intestinal epithelium. Id2-deficient embryonic epithelial cells cultured exvivo strongly activate Wnt target genes as well as markers of neoplastic transformation and form fast growing undifferentiated spheroids. Furthermore, adult ISCs from Id2-deficient mice display a distinct transcriptional signature, supporting an essential role for Id2 in the correct specification of ISCs.

Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors.

Isolation and tridimensional culture of murine fetal progenitors from the digestive tract represents a new approach to study the nature and the biological characteristics of these epithelial cells that are present before the onset of the cytodifferentiation process during development. In 2013, Mustata et al. described the isolation of intestinal fetal progenitors growing as spheroids in the ex vivo culture system initially implemented by Sato et al. (2009) to grow adult intestinal stem cells. Noteworthy, fetal-derived spheroids have high self-renewal capacity making easy their indefinite maintenance in culture. Here, we report an adapted protocol for isolation and ex vivo culture and maintenance of fetal epithelial progenitors from distal pre-glandular stomach growing as gastric spheroids (Fernandez Vallone et al., 2016 ).

Wnt couples the cell cycle to the circadian cycle

Wnt-releasing Paneth cells synchronize the division of intestinal stem cells and progenitor cells with the daily circadian cycle.