Key regulators of intestinal stem cells:diet,microbiota,and microbial metabolites

Interactions between diet and the intestinal microbiome play an important role in human health and disease development.It is well known that such interactions,whether direct or indirect,trigger a series of metabolic reactions in the body.Evidence suggests that intestinal stem cells(ISCs),which are phenotypic precursors of various intestinal epithelial cells,play a significant role in the regulation of intestinal barrier function and homeostasis.The advent and evolution of intestinal organoid culture techniques have presented a key opportunity to study the association between the intestinal microenvironment and ISCs.As a result,the effects exerted by dietary factors,intestinal microbiomes,and their metabolites on the metabolic regulation of ISCs and the potential mechanisms underlying such effects are being gradually revealed.This review summarises the effects of different dietary patterns on the behaviour and functioning of ISCs and focuses on the crosstalk between intestinal microbiota,related metabolites,and ISCs,with the aim of fully understanding the relationship between these three factors and providing further insights into the complex mechanisms associated with ISCs in the human body.Gaining an understanding of these mechanisms may lead to the development of novel dietary interventions or drugs conducive to intestinal health.

The response of intestinal stem cells and epithelium after alemtuzumab administration

Qiurong Li1,Qiang Zhang1,Chenyang Wang1,Shaojun Jiang2,Ning Li1,Jieshou Li1 Intestinal stem cells may have important roles in the maintenance of epithelial integrity during tissue repair.Alemtuzumab is a humanized anti-CD52 lymphocytic antibody that is increasingly being used to induce immunosuppression;intestinal barrier function is impaired during treatment with alemtuzumab.We investigated the response of intestinal stem cells to epithelial damage resulting from alemtuzumab treatment.Intestinal epithelial cell loss and abnormal Paneth cell morphology were found following a single dose of alemtuzumab.The animals receiving alemtuzumab exhibited increased apoptosis in the villi 3 days after alemtuzumab treatment and in the crypt on day 9,but apoptosis was scarce on day 35.We assessed expression of Musashi-1-and Lgr5-positive stem cells following alemtuzumab treatment.Increased numbers of cells staining positive for both Musashi-1 and Lgr5 were found in the stem cell zone after alemtuzumab treatment for 3 and 9 days.These data indicated that the epithelial cells were injured following alemtuzumab treatment,with the associated expansion of intestinal stem cells.After alemtuzumab treatment for 35 days,the numbers of intestinal epithelial cells and intestinal stem cells returned to normal.This study suggests that alemtuzumab treatment induced the increase in stem cells,resulting in the availability of more enterocytes for repair.

The cellular niche for intestinal stem cells: ateam effort

The rapidly self-renewing epithelium in the mammalian intestine is maintained by multipotent intestinal stem cells(ISCs) located at the bottom of the intestinal crypt that are interspersed with Paneth cells in the small intestine andPaneth-like cells in the colon. The ISC compartment is also closely associated with a sub-epithelial compartmentthat contains multiple types of mesenchymal stromal cells. With the advances in single cell and gene editingtechnologies, rapid progress has been made for the identification and characterization of the cellular componentsof the niche microenvironment that is essential for self-renewal and differentiation of ISCs. It has becomeincreasingly clear that a heterogeneous population of mesenchymal cells as well as the Paneth cells collectivelyprovide multiple secreted niche signals to promote ISC self-renewal. Here we review and summarize recentadvances in the regulation of ISCs with a main focus on the definition of niche cells that sustain ISCs.

N-Acetyl-D-glucosamine improves the intestinal development and nutrient absorption of weaned piglets via regulating the activity of intestinal stem cells

Early weaning in piglets can cause a series of negative effects.This causes serious losses to the livestock industry.N-Acetyl-D-glucosamine(D-GlcNAc)plays an important role in regulating the homeostasis of the intestine.This study aimed to investigate the effects of D-GlcNAc on the growth performance and intestinal function of weaned piglets.Twenty-four weaned piglets([Yorkshire×Landrace]Duroc,6.58±0.15 kg,n=8)at 21 d old were fed 3 diets supplemented with 0(control),1 and 3 g/kg D-GlcNAc.The intestinal organoid model was used to verify the regulatory mechanism of D-GlcNAc on intestinal epithelial cells.On the whole,supplementation of D-GlcNAc in the piglet diet has no significant effect on the growth performance and diarrhoea of weaned piglets(P>0.05).The apparent digestibility of nutrients and mRNA abundance of nutrient transporters in the 1 g/kg D-GlcNAc group were increased significantly(P<0.05).D-GlcNAc did not affect villus height(VH)and crypt depth(CD)but resulted in a numerically shorter VH and shallower CD,which lead to an increase in ileal VH:CD ratio(P<0.05).Cell shedding rates in the ileum villi increased(P<0.05).The relative length and weight of the small intestine of weaned piglets increased(P<0.05).In vitro studies found that the budding rates of organoids treated with 0.1 mmol/L D-GlcNAc increased on the d 3 and 5(P<0.05).The average budding numbers per budding organoid treated with 0.1 and 10 mmol/L D-GlcNAc increased on d 3(P<0.05).D-GlcNAc upregulated leucine rich repeat containing G protein-coupled receptor 5(Lgr5^(+))and Chromogranin A mRNA abundance in organoids(P<0.05).Mucin 2(Muc2)expression increased when treated with 1 and 10 mmol/L D-GlcNAc(P<0.05).In conclusion,dietary D-GlcNAc cannot improve the growth performance of weaned piglets.However,it can promote the growth and development of the intestinal tract and improve the digestion and absorption capacity of the intestine,which is achieved by affecting the activity of intestinal stem cells.

Intestinal stem cells and intestinal organoids

The intestinal epithelium is one of the most rapidly renewing tissues,which is fueled by stem cells at the base of the crypts.Strategies of genetic lineage tracing and organoids,which capture major features of original tissues,are powerful avenues for exploring the biology of intestinal stem cells in vivo and in vitro,respectively.The combination of intestinal organoideculturing system and genetic modification approaches provides an attractive platform to uncover the mechanism of colorectal cancer and genetic disorders in the human minigut.Here,we will provide a comprehensive overview of studies on intestinal epithelium and intestinal stem cells.We will also review the applications of organoids and genetic markers in intestinal research studies.Furthermore,we will discuss the advantages and drawbacks of organoids as disease models compared with mice models and cell lines.

Modulation of stem cell fate in intestinal homeostasis,injury and repair

The mammalian intestinal epithelium constitutes the largest barrier against the external environment and makes flexible responses to various types of stimuli.Epithelial cells are fast-renewed to counteract constant damage and disrupted barrier function to maintain their integrity.The homeostatic repair and regeneration of the intestinal epithelium are governed by the Lgr5+intestinal stem cells(ISCs)located at the base of crypts,which fuel rapid renewal and give rise to the different epithelial cell types.Protracted biological and physicochemical stress may challenge epithelial integrity and the function of ISCs.The field of ISCs is thus of interest for complete mucosal healing,given its relevance to diseases of intestinal injury and inflammation such as inflammatory bowel diseases.Here,we review the current understanding of the signals and mechanisms that control homeostasis and regeneration of the intestinal epithelium.We focus on recent insights into the intrinsic and extrinsic elements involved in the process of intestinal homeostasis,injury,and repair,which fine-tune the balance between self-renewal and cell fate specification in ISCs.Deciphering the regulatory machinery that modulates stem cell fate would aid in the development of novel therapeutics that facilitate mucosal healing and restore epithelial barrier function.

Intestinal Wnt in the transition from physiology to oncology

Adult stem cells are necessary for self-renewal tissues and regeneration after damage.Especially in the intestine,which self-renews every few days,they play a key role in tissue homeostasis.Therefore,complex regulatory mechanisms are needed to prevent hyperproliferation,which can lead in the worst case to carcinogenesis or under-activation of stem cells,which can result in dysfunctional epithelial.One main regulatory signaling pathway is the Wnt/β-catenin signaling pathway.It is a highly conserved pathway,withβ-catenin,a transcription factor,as target protein.Translocation ofβ-catenin from cytoplasm to nucleus activates the transcription of numerous genes involved in regulating stem cell pluripotency,proliferation,cell differentiation and regulation of cell death.This review presents a brief overview of the Wnt/β-catenin signaling pathway,the regulatory mechanism of this pathway and its role in intestinal homeostasis.Additionally,this review highlights the molecular mechanisms and the histomorphological features of Wnt hyperactivation.Furthermore,the central role of the Wnt signaling pathway in intestinal carcinogenesis as well as its clinical relevance in colorectal carcinoma are discussed.

Leucine-rich repeat-containing G protein-coupled receptor 5 marks different cancer stem cell compartments in human Caco-2 and LoVo colon cancer lines

BACKGROUND Colon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers.It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeatcontaining G protein-coupled receptor 5(LGR5)-expressing stem cells,these socalled“tumour-initiating”cells,reminiscent in their properties of the normal intestinal stem cells(ISCs),may explain the apparent heterogeneity of colon cancer cell lines.Also,colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5.Furthermore,in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell.AIM To investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells.METHODS A portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines(Caco-2 and LoVo).These cells were then characterized according to their proliferation capacity,gene expression signatures of ISC markers,and their tumorigenic properties in vivo and in vitro.RESULTS The data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2,but not from the LoVo,cell lines,in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog(+4 crypt)stem cells.This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny.Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein(EYFP)from this cell line exhibit tumorigenic-like properties in vivo and in vitro.In contrast,cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties.Thus,cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells,but not LoVo cells.CONCLUSION Our findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines.Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data.We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines.

Monogenic deficiency in murine intestinal Cdc42 leads to mucosal inflammation that induces crypt dysplasia

CDC42 controls intestinal epithelial(IEC)stem cell(IESC)division.How aberrant CDC42 initiates intestinal inflammation or neoplasia is unclear.We utilized models of inflam-matory bowel diseases(IBD),colorectal cancer,aging,and IESC injury to determine the loss of intestinal Cdc42 upon inflammation and neoplasia.Intestinal specimens were collected to determine the levels of CDC42 in IBD or colorectal cancer.Cdc42 floxed mice were crossed with Villin-Cre,Villin-CreERT2 and/or Lgr5-eGFP-IRES-CreERT2,or Bmi1-CreERT2 mice to generate Cdc42 deficient mice.Irradiation,colitis,aging,and intestinal organoid were used to evaluate CDC42 upon mucosal inflammation,IESC/progenitor regenerative capacity,and IEC repair.Our studies revealed that increased CDC42 in colorectal cancer correlated with lower survival;in contrast,lower levels of CDC42 were found in the inflamed IBD colon.Colonic Cdc42 depletion significantly reduced Lgr5+IEsCs,increased progenitors'hyperplasia,and induced mucosal inflammation,which led to crypt dysplasia.Colonic Cdc42 depletion markedly enhanced irra-diation-or chemical-induced colitis.Depletion or inhibition of Cdc42 reduced colonic Lgr5+IESC regeneration.In conclusion,depletion of Cdc42 reduces the IESC regeneration and IEC repair,leading to prolonged mucosal inflammation.Constitutive monogenic loss of Cdc42 in-duces mucosal inflammation,which could result in intestinal neoplasia in the context of aging.

REGγ drives Lgr5^(+) stem cells to potentiate radiation induced intestinal regeneration

Leucine-rich repeat containing G protein-coupled receptor 5(Lgr5), a marker of intestinal stem cells(ISCs), is considered to play key roles in tissue homoeostasis and regeneration after acute radiation injury. However, the activation of Lgr5 by integrated signaling pathways upon radiation remains poorly understood. Here, we show that irradiation of mice with whole-body depletion or conditional ablation of REGγ in Lgr5^(+) stem cell impairs proliferation of intestinal crypts, delaying regeneration of intestine epithelial cells. Mechanistically, REGγ enhances transcriptional activation of Lgr5 via the potentiation of both Wnt and Hippo signal pathways. TEAD4 alone or cooperates with TCF4, a transcription factor mediating Wnt signaling, to enhance the expression of Lgr5. Silencing TEAD4 drastically attenuated β-catenin/TCF4 dependent expression of Lgr5. Together, our study reveals how REGγ controls Lgr5 expression and expansion of Lgr5+stem cells in the regeneration of intestinal epithelial cells.Thus, REGγ proteasome appears to be a potential therapeutic target for radiation-induced gastrointestinal disorders.

Recent advances in tissue stem cells

Stem cells are undifferentiated cells capable of self-renewal and differentiation,giving rise to specialized functional cells.Stem cells are of pivotal importance for organ and tissue development,homeostasis,and injury and disease repair.Tissue-specific stem cells are a rare population residing in specific tissues and present powerful potential for regeneration when required.They are usually named based on the resident tissue,such as hematopoietic stem cells and germline stem cells.This review discusses the recent advances in stem cells of various tissues,including neural stem cells,muscle stem cells,liver progenitors,pancreatic islet stem/progenitor cells,intestinal stem cells,and prostate stem cells,and the future perspectives for tissue stem cell research.

No Intestinal Stem Cell Regeneration after Complete Progenitor Ablation in Drosophila Adult Midgut

Adult stem cells or progenitors are essential for maintaining the normal structure and function of adult tissues （i.e., ho- meostasis）. One of the best examples is the adult intestinal epithelium which is constantly renewed by the progeny of intestinal stem cells （ISCs）. The proliferation and differentiation of adult stem cells must be tightly controlled in order to maintain resident tissue homeostasis. Mis-regulation of stem cell proliferation and differentiation leads to depletion or excessive proliferation of stem cells, eventually resulting in severe diseases such as cancer （Radtke and Clevers, 2005; Morrison and Spradling, 2008）. Understanding the detailed regulatory mechanisms of stem cell proliferation and differ- entiation will shed insights into the causes of related human diseases.

Hippo pathway in intestinal homeostasis and tumorigenesis

The Hippo pathway plays a crucial role in controlling organ size by inhibiting cell proliferation and promoting cell death.Recent findings implicate that this pathway is involved in the process of intestinal regeneration and tumorigenesis.Here we summarize current studies for the function of the Hippo signaling pathway in intestinal homeostasis,regeneration and tumorigenesis,and the crosstalk between the Hippo signaling pathway and other major signaling pathways,i.e.Wnt,Notch and Jak/Stat signaling pathways in intestinal compartment.

Organoid models of gastrointestinal Neoplasms:Origin,current status and future applications in personalized medicine

The in vitro organoid model is a major technological breakthrough that has been established as an important tool in many basic biological and clinical applications.This near-physiological 3D culture system accurately models various biological processes,including tissue renewal,stem cell/niche functions and tissue responses to drugs,mutations or damage.Organoids have the potential value of being an accurate model for disease predictions or drug screening applications and to identify the ideal treatment for that patient.Carcinogenesis can be modeled by mutating specific cancer genes in wild-type organoids;and patient-derived organoids provide an important resource in the development of personalized cancer treatment.Organoids from cancer patients could be used to identify the ideal treatment for a specific patient by growing matched healthy and diseased organoids from human cancer patients which additionally enables clinical screens for drug combinations.Organoids could also provide autologous cells ordin the futuredtissue for transplantation.In this review,we discuss the current advances,challenges and potential applications of this technique in gastrointestinal neoplasms.

Changes in progenitors and differentiated epithelial cells of neonatal piglets

This study aimed to assess the changes of small intestinal morphology,progenitors,differentiated epithelial cells,and potential mechanisms in neonatal piglets.Hematoxylin and eosin staining of samples from 36 piglets suggested that dramatic changes were observed in the jejunum crypts depth and crypt fission index of neonatal piglets(P<0.001).The number of intestinal stem cells(ISC)tended to increase(P<0.10),and a decreased number of enteroendocrine cells appeared in the jejunal crypt on d 7(P<0.05).Furthermore,the mRNA expression of jejunal chromogranin A(ChgA)was down-regulated in d 7 piglets(P<0.05).There was an up-regulation of the adult ISC marker gene of SPARC related modular calcium binding 2(Smoc2),and Wnt/b-catenin target genes on d 7(P<0.05).These results were further verified in vitro enteroid culture experiments.A mass of hollow spheroids was cultured from the fetal intestine of 0-d-old piglets(P<0.001),whereas substantial organoids with budding and branching structures were cultured from the intestine of 7-d-old piglets(P<0.001).The difference was reflected by the organoid budding efficiency,crypt domains per organoid,and the surface area of the organoid.Furthermore,spheroids on d 0 had more Ki67-positive cells and enteroendocrine cells(P<0.05)and showed a decreasing trend in the ISC and goblet cells(P<0.10).Moreover,the mRNA expression of spheroids differed markedly from that of organoids,with low expression of intestinal differentiation gene(Lysozyme;P<0.05),epithelial-specific markers(Villin,E-cadherin;P<0.05),and adult ISC markers(leucine-rich repeat-containing G protein-coupled receptor 5[Lgr5],Smoc2;P<0.001),and upregulation of fetal marker(connexin 43[Cnx43];P<0.05).The mRNA expression of relevant genes was up-regulated,and involved in Wnt/b-catenin,epidermal growth factor(EGF),Notch,and bone morphogenetic protein(BMP)signaling on d 7 organoids(P<0.05).Spheroids displayed low differentiated phenotype and high proliferation,while organoids exhibited strong differentiation potential.These results indicated that the conversion from the fetal progenitors(spheroids)to adult ISC(normal organoids)might largely be responsible for the fast development of intestinal epithelial cells in neonatal piglets.