A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable

The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base. However, the relative functions of these two pools and their interrelationship are not understood. Here we specifically ablated Lgr5-expressing cells in mice using a human diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus. We found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for the elimination of this population. After ablation of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, indicating that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium. Our results demonstrate that Lgr5-expressing cells are dispensable for normal intestinal homeostasis, and that in the absence of these cells, Bmi1-expressing cells can serve as an alternative stem cell pool. These data provide the first experimental evidence for the interrelationship between these populations. The Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under non-pathological conditions.     

Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts

Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, which are small cycling cells located at crypt bottoms. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells. Here we find a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro. CD24(+) Paneth cells express EGF, TGF-α, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24(+) cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.     

Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling

The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues.     

猕猴大肠5-羟色胺免疫活性细胞的免疫组织化学研究

以免疫组织化学ＳＰ法显示猕猴大肠含５－ ＨＴ的细胞,并用Ｗｅｉｂｅｌ体视学方法对其进行了定量分析．结果表明,猕猴大肠５－ ＨＴ免疫活性内分泌细胞的密度在结肠离心段最高,直肠密度中等,盲肠和结肠向心段最低．５－ ＨＴ免疫反应阳性细胞多分布在肠腺处,其形态多样,大多为锥形、梭形、圆形等．有些细胞的基底部有突起,突起的末端含有５－ ＨＴ阳性物质;有些细胞的５－ ＨＴ阳性物质释放到腺腔或肠腔面．     

Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism

Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter, but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with submicrometre resolution. Here we apply MIMS to diverse organisms, including Drosophila, mice and humans. We test the 'immortal strand hypothesis', which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labelling mice with (15)N-thymidine from gestation until post-natal week 8, we find no (15)N label retention by dividing small intestinal crypt cells after a four-week chase. In adult mice administered (15)N-thymidine pulse-chase, we find that proliferating crypt cells dilute the (15)N label, consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human haematopoietic system. These studies show that MIMS provides high-resolution quantification of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research.     

Microenvironmental regulation of stem cells in intestinal homeostasis and cancer

The identification of intestinal stem cells as well as their malignant counterparts, colon cancer stem cells, has undergone rapid development in recent years. Under physiological conditions, intestinal homeostasis is a carefully balanced and efficient interplay between stem cells, their progeny and the microenvironment. These interactions regulate the astonishingly rapid renewal of the intestinal epithelial layer, which consequently puts us at serious risk of developing cancer. Here we highlight the microenvironment-derived signals that regulate stem-cell fate and epithelial differentiation. As our understanding of normal intestinal crypt homeostasis grows, these developments may point towards new insights into the origin of cancer and the maintenance and regulation of cancer stem cells.     

The behaviour of Drosophila adult hindgut stem cells is controlled by Wnt and Hh signalling

The intestinal tract maintains proper function by replacing aged cells with freshly produced cells that arise from a population of self-renewing intestinal stem cells (ISCs). In the mammalian intestine, ISC self renewal, amplification and differentiation take place along the crypt-villus axis, and are controlled by the Wnt and hedgehog (Hh) signalling pathways. However, little is known about the mechanisms that specify ISCs within the developing intestinal epithelium, or about the signalling centres that help maintain them in their self-renewing stem cell state. Here we show that in adult Drosophila melanogaster, ISCs of the posterior intestine (hindgut) are confined to an anterior narrow segment, which we name the hindgut proliferation zone (HPZ). Within the HPZ, self renewal of ISCs, as well as subsequent proliferation and differentiation of ISC descendants, are controlled by locally emanating Wingless (Wg, a Drosophila Wnt homologue) and Hh signals. The anteriorly restricted expression of Wg in the HPZ acts as a niche signal that maintains cells in a slow-cycling, self-renewing mode. As cells divide and move posteriorly away from the Wg source, they enter a phase of rapid proliferation. During this phase, Hh signal is required for exiting the cell cycle and the onset of differentiation. The HPZ, with its characteristic proliferation dynamics and signalling properties, is set up during the embryonic phase and becomes active in the larva, where it generates all adult hindgut cells including ISCs. The mechanism and genetic control of cell renewal in the Drosophila HPZ exhibits a large degree of similarity with what is seen in the mammalian intestine. Our analysis of the Drosophila HPZ provides an insight into the specification and control of stem cells, highlighting the way in which the spatial pattern of signals that promote self renewal, growth and differentiation is set up within a genetically tractable model system.     

Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors

LGR5+ stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals. Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in LGR5+ stem cells. Simultaneous deletion of the two genes encoding these proteins in the intestinal epithelium of mice induces rapidly growing adenomas containing high numbers of Paneth and LGR5+ stem cells. In vitro, growth of organoids derived from these adenomas is arrested when Wnt secretion is inhibited, indicating a dependence of the adenoma stem cells on Wnt produced by adenoma Paneth cells. In the HEK293T human cancer cell line, expression of RNF43 blocks Wnt responses and targets surface-expressed frizzled receptors to lysosomes. In the RNF43-mutant colorectal cancer cell line HCT116, reconstitution of RNF43 expression removes its response to exogenous Wnt. We conclude that RNF43 and ZNRF3 reduce Wnt signals by selectively ubiquitinating frizzled receptors, thereby targeting these Wnt receptors for degradation.     

Notch signals control the fate of immature progenitor cells in the intestine

The Notch signalling pathway plays a crucial role in specifying cellular fates in metazoan development by regulating communication between adjacent cells. Correlative studies suggested an involvement of Notch in intestinal development. Here, by modulating Notch activity in the mouse intestine, we directly implicate Notch signals in intestinal cell lineage specification. We also show that Notch activation is capable of amplifying the intestinal progenitor pool while inhibiting cell differentiation. We conclude that Notch activity is required for the maintenance of proliferating crypt cells in the intestinal epithelium.     

FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation

Intestinal immune homeostasis depends on a tightly regulated cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). Epithelial barrier disruption is considered to be a potential cause of inflammatory bowel disease; however, the mechanisms regulating intestinal epithelial integrity are poorly understood. Here we show that mice with IEC-specific knockout of FADD (FADD(IEC-KO)), an adaptor protein required for death-receptor-induced apoptosis, spontaneously developed epithelial cell necrosis, loss of Paneth cells, enteritis and severe erosive colitis. Genetic deficiency in RIP3, a critical regulator of programmed necrosis, prevented the development of spontaneous pathology in both the small intestine and colon of FADD(IEC-KO) mice, demonstrating that intestinal inflammation is triggered by RIP3-dependent death of FADD-deficient IECs. Epithelial-specific inhibition of CYLD, a deubiquitinase that regulates cellular necrosis, prevented colitis development in FADD(IEC-KO) but not in NEMO(IEC-KO) mice, showing that different mechanisms mediated death of colonic epithelial cells in these two models. In FADD(IEC-KO) mice, TNF deficiency ameliorated colon inflammation, whereas MYD88 deficiency and also elimination of the microbiota prevented colon inflammation, indicating that bacteria-mediated Toll-like-receptor signalling drives colitis by inducing the expression of TNF and other cytokines. However, neither CYLD, TNF or MYD88 deficiency nor elimination of the microbiota could prevent Paneth cell loss and enteritis in FADD(IEC-KO) mice, showing that different mechanisms drive RIP3-dependent necrosis of FADD-deficient IECs in the small and large bowel. Therefore, by inhibiting RIP3-mediated IEC necrosis, FADD preserves epithelial barrier integrity and antibacterial defence, maintains homeostasis and prevents chronic intestinal inflammation. Collectively, these results show that mechanisms preventing RIP3-mediated epithelial cell death are critical for the maintenance of intestinal homeostasis and indicate that programmed necrosis of IECs might be implicated in the pathogenesis of inflammatory bowel disease, in which Paneth cell and barrier defects are thought to contribute to intestinal inflammation.     

Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells

Epidermal growth factor, and its human homologue urogastrone (EGF/URO), are secreted by the gut-associated salivary and Brunner's glands. Recombinant EGF/URO is a powerful stimulator of cell proliferation and differentiation in the rodent and neonatal human intestine. But EGF/URO is not absorbed from the adult gut and has no action when given through the gut lumen; thus the role of secreted EGF/URO is unknown. We now report that ulceration of the epithelium anywhere in the human gastrointestinal tract induces the development of a novel cell lineage from gastrointestinal stem cells. This lineage initially appears as a bud from the base of intestinal crypts, adjacent to the ulcer, and grows locally as a tubule, ramifying to form a new small gland, and ultimately emerges onto the mucosal surface. The lineage produces neutral mucin, shows a unique lectin-binding profile and immunophenotype, is nonproliferative, and contains and secretes abundant immunoreactive EGF/URO. We propose that all gastrointestinal stem cells can produce this cell lineage after mucosal ulceration, secreting EGF/URO to stimulate cell proliferation, regeneration and ulcer healing. This cell lineage is very commonly associated with gastrointestinal mucosal ulceration, and we conclude that a principal in vivo role for EGF/URO is to stimulate ulcer healing throughout the gut through induction of this cell lineage in the adjacent mucosa.     

小鼠胃肠道5-羟色胺免疫反应阳性肥大细胞

利用免疫组化技术和甲苯胺蓝(TB)染色技术,对小鼠胃肠道5-羟色胺免疫反应阳性细胞(5-HT+细胞)、肥大细胞(MC)和5-羟色胺免疫反应阳性肥大细胞(5-HT+MC)进行了研究.结果表明:小鼠胃肠中5-HT+细胞多分布于黏膜上皮细胞之间、腺泡上皮细胞之间和固有层内;MC多分布于黏膜层和黏膜下层结缔组织之中;小鼠胃和小肠中分别有49%和47%的MC与免疫组化染色的邻片中5-HT+细胞在位置上相对应,该部分MC为5-HT+MC;小鼠胃和小肠中分别有24%和20%的5-HT+细胞为MC.本文从形态学的角度证明了小鼠胃肠道MC可分泌5-HT,MC是消化道5-HT的来源之一.     

中华蟾蜍消化道组织学及组织化学研究

采用组织学，组织化学方法研究中华蟾蜍消化道，结果表明：舌有粘膜腺，口咽腔具有皱褶，其粘膜为复层柱状纤毛上皮，口咽腔及食道粘膜中有巨型杯状细胞，食道至胃的粘膜层有丰富的粘液细胞，食道－贲门区具绒毛。这些是中华蟾蜍与摄食有关的重要适应性特征。整个消化道有5类粘液细胞－大型柱状粘液细胞，I型矮柱状粘液细胞，Ⅱ型矮柱状粘液细胞，巨型杯状细胞，普通杯状细胞。     

扬子鳄胚胎十二指肠的组织发生

本文观察了１８例不同时间扬子鳄胚胎十二指肠的组织发生过程。孵化第１０天十二指肠出现形态分化。１６天,上皮细胞增殖为３￣４层,．肠腔扩大．３０天,上皮局部出现突起,多具２层细胞。３４天,间充质已分化为固有膜、粘膜下层、环行肌层及纵行肌层,环行肌层较厚,平均约为２８μｍ。４０天,上皮为单层柱状,纹状缘明显,开始出现原始小肠腺。５２天,杯状细胞较多。６２天,各层结构与成体时相似;扫描电镜下观察,绒毛为指     

Demonstration of somatic mutation and colonic crypt clonality by X-linked enzyme histochemistry

Cellular mosaicism resulting from X-chromosome inactivation in heterozygous females can be shown histochemically; using this approach we have demonstrated age-related gene reactivation and tumour clonality. We now show in female mice heterozygous for reduced expression of glucose-6-phosphate dehydrogenase (G6PD) activity that colonic epithelial cells express either normal or low enzyme activity, and form patches composed of multiple crypts of uniform phenotype. We also show that a low-enzyme colonic epithelial cell phenotype can be induced in normal mice by carcinogen treatment, these cells again occur in patches, but are restricted to scattered single crypts, the frequency of which is related to treatment. A small proportion of colonic tumours in carcinogen treated normal mice are also of low-enzyme phenotype. We conclude that we have visualized the effects of a sporadic carcinogen induced somatic mutation in the G6PD gene of crypt stem cells and that a single stem cell maintains each colonic crypt. This inducible defective activity of a ubiquitous 'housekeeping' enzyme provides a somatic clonal marker system of wide potential application.     

家兔派尹尔氏(Peyer's Patches)淋巴集结圆顶上皮内微褶细胞的来源与分化

对不同发育时期家兔Peyer's Patches圆顶的显微和亚显微结构系统观察表明,圆顶上皮内的微褶细胞来源于中胚层,而不是由未分化的肠上皮细胞分化而来,可能同巨噬细胞是同一组细胞系来源。     

Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation

How tissues generate and maintain the correct number of cells is a fundamental problem in biology. In principle, tissue turnover can occur by the differentiation of stem cells, as is well documented for blood, skin and intestine, or by the duplication of existing differentiated cells. Recent work on adult stem cells has highlighted their potential contribution to organ maintenance and repair. However, the extent to which stem cells actually participate in these processes in vivo is not clear. Here we introduce a method for genetic lineage tracing to determine the contribution of stem cells to a tissue of interest. We focus on pancreatic beta-cells, whose postnatal origins remain controversial. Our analysis shows that pre-existing beta-cells, rather than pluripotent stem cells, are the major source of new beta-cells during adult life and after pancreatectomy in mice. These results suggest that terminally differentiated beta-cells retain a significant proliferative capacity in vivo and cast doubt on the idea that adult stem cells have a significant role in beta-cell replenishment.