Crypt Base Columnar Stem Cells Research Articles

Glycans that regulate Notch signaling in the intestine.

Intestinal homeostasis is key to the maintenance of good health. The small intestine plays important roles in absorption, digestion, hormonal and immune functions. Crypt base columnar (CBC) stem cells residing at the bottom of crypts are nurtured by Paneth cells, and together create the stem cell niche, the foundation of intestinal homeostasis. CBC stem cells replicate to replenish their number, or differentiate into a variety of epithelial cells with specialized functions. Notch signaling is a cell-cell signaling pathway that regulates both the proliferation and differentiation of CBC stem cells. NOTCH1 and NOTCH2 stimulated by canonical Notch ligands DLL1 and DLL4 mediate Notch signaling in the intestine that, in concert with other signaling pathways including the WNT and BMP pathways, determines cell fates. Importantly, interactions between Notch receptors and canonical Notch ligands are regulated by O-glycans linked to Ser/Thr in epidermal growth factor-like (EGF) repeats of the Notch receptor extracellular domain (NECD). The O-glycans attached to NECD are key regulators of the strength of Notch signaling. Imbalances in Notch signaling result in altered cell fate decisions and may lead to cancer in the intestine. In this review, we summarize the impacts of mutations in Notch pathway members on intestinal development and homeostasis, with a focus on the glycosyltransferases that transfer O-glycans to EGF repeats of NOTCH1, NOTCH2, DLL1 and DLL4.

Limitations to Understanding Intestinal Stem Cell Activity via Cre-Lox-Based Lineage Tracing.

The intestinal epithelium is a highly proliferative tissue with robust regenerative potential, governed by the intestinal stem cell (ISC) compartment. Our understanding of the cellular hierarchy within this compartment is drawn from Cre-lox–based lineage tracing studies, initially via the identification of crypt base columnar stem cells (CBCs) using an Lgr5eGFP-IRES-CreER allele and the R26LSL-LacZ reporter,1 showing that CBCs reconstitute all epithelial cell types during homeostasis.2 Despite their proliferative capacity, CBCs are exquisitely sensitive to injury, requiring facultative stem cells for postinjury regeneration.

NF-κB determines Paneth versus goblet cell fate decision in the small intestine.

ABSTRACTAlthough the role of the transcription factor NF-κB in intestinal inflammation and tumor formation has been investigated extensively, a physiological function of NF-κB in sustaining intestinal epithelial homeostasis beyond inflammation has not been demonstrated. Using NF-κB reporter mice, we detected strong NF-κB activity in Paneth cells, in ‘+4/+5’ secretory progenitors and in scattered Lgr5+ crypt base columnar stem cells of small intestinal (SI) crypts. To examine NF–κB functions in SI epithelial self-renewal, mice or SI crypt organoids (‘mini-guts’) with ubiquitously suppressed NF-κB activity were used. We show that NF-κB activity is dispensable for maintaining SI epithelial proliferation, but is essential for ex vivo organoid growth. Furthermore, we demonstrate a dramatic reduction of Paneth cells in the absence of NF-κB activity, concomitant with a significant increase in goblet cells and immature intermediate cells. This indicates that NF-κB is required for proper Paneth versus goblet cell differentiation and for SI epithelial homeostasis, which occurs via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The current study thus presents evidence for an important role for NF-κB in intestinal epithelial self-renewal.

Cycling Stem Cells Are Radioresistant and Regenerate the Intestine

SUMMARYA certain number of epithelial cells in intestinal crypts are DNA damage resistant and contribute to regeneration. However, the cellular mechanism underlying intestinal regeneration remains unclear. Using lineage tracing, we show that cells marked by an Msi1 reporter (Msi1+) are right above Lgr5high cells in intestinal crypts and exhibit DNA damage resistance. Single-cell RNA sequencing reveals that the Msi1+ cells are heterogeneous with the majority being intestinal stem cells (ISCs). The DNA damage-resistant subpopulation of Msi1+ cells is characterized by low-to-negative Lgr5 expression and is more rapidly cycling than Lgr5high radiosensitive crypt base columnar stem cells (CBCs). This enables an efficient repopulation of the intestinal epithelium at early stage when Lgr5high cells are not emerging. Furthermore, relative to CBCs, Msi1+ cells preferentially produce Paneth cells during homeostasis and upon radiation repair. Together, we demonstrate that the DNA damage-resistant Msi1+ cells are cycling ISCs that maintain and regenerate the intestinal epithelium.

A83 INTESTINAL EPITHELIAL CELL STEMNESS AND DIFFERENTIATION ARE REGULATED BY THE HIPPO PATHWAY EFFECTOR YAP1

Abstract Background The high rate of cell turnover in the intestinal epithelium is supported by the LGR5+ crypt base columnar (CBC) stem cells, which are located at the lower part of the gland. Among of the various factors and signals like Wnt and Notch, YAP1 (yes associated protein) also plays an important role in stemness of CBC stem cells. YAP1 is the effector of the Hippo pathway. Hippo Pathway restricts the cells proliferation, tissues overgrowth and cancer formation through the phosphorylation and inactivation of the YAP1 protein. When active, YAP1 transfers into nucleus, forms the complex with TEADs transcription factors and promotes the transcription of genes involved in cell growth and proliferation. Aims In the present study, we investigated the role of the YAP1 in the colorectal cancer multipotent HT29 cell line, which contain cancer stem cells (CSC). Methods For approaching to this goal, YAP1 expression was knocked down using shRNAs in HT29 cells. Then stem cells and intestinal cell lineages (secretory goblet, Paneth and enteroendocrine and absorptive) markers expression was analyzed using qPCR and Western blot. Results The results showed the reduction of the expression of stem cells markers including LGR5 in YAP1 knockdown HT29 cells compare with control. Expression of the goblet cells markers (MUC2 and trefoil factor 3) and absorptive cells markers (sucrase-isomaltase and dipeptidylpeptidase IV) were significantly increased in YAP1 knockdown cells but Paneth (DEFA5 and lysozyme) and enteroendocrine (CHGA) were not detected. Finally, examination of the main transcription factors for intestinal differentiation revealed an increase in CDX2 expression. Conclusions These results suggest that YAP1 is involved in the maintenance of colorectal cancer stem cells while preventing intestinal differentiation in both secretory and absorptive lineages through the repression of CDX2. Funding Agencies CIHR

Genome Toxicity and Impaired Stem Cell Function after Conditional Activation of CreERT2 in the Intestine.

SummaryWith the tamoxifen-inducible CreERT2 system, genetic recombination can be temporally controlled in a cell-type-specific manner in intact animals, permitting dissection of the molecular underpinnings of mammalian physiology. Here we present a significant drawback to CreERT2 technology for analysis of intestinal stem cells. Using the intestine-specific Villin-CreERT2 mouse strain, we observed delayed intestinal regeneration post irradiation. Villin-CreERT2 activation was associated with DNA damage and cryptic loxP site cleavage. Analysis of stem cell-specific CreERT2 strains showed that the genome toxicity impairs function of crypt base columnar stem cells, resulting in loss of organoid initiating activity. Importantly, the stem cell impairment is short-lived, with return to normal by 7 days post tamoxifen treatment. Our findings demonstrate that mouse genetic experiments that utilize CreERT2 should consider the confounding effects of enhanced stem cell sensitivity to genome toxicity resulting from CreERT2 activation.

Demystifying the Differences Between Tumor-Initiating Cells and Cancer Stem Cells in Colon Cancer

Tumor-initiating cells and cancer stem cells refer to a subpopulation of self-renewing cells involved in tumor initiation and tumor maintenance, respectively. With this review, we aimed to define the functional and molecular differences between both cell types in the context of colon cancer. Recent evidence suggests that the two major stem cell populations in the normal intestinal crypt have tumor-initiating capacity and could be the cell-of-origin in colon cancer. Activation of the Wnt/β-catenin pathway (an early event in mouse intestinal carcinogenesis) in the crypt base columnar stem cells and reserve stem cells leads to adenoma formation, supporting a role in tumor initiation. On the other hand, colon cancer stem cells express several membrane markers facilitating their isolation by FACS and are associated with treatment resistance and higher metastatic potential. Tumor-initiating cells and cancer stem cells express distinct markers, display discrete biological functions, and can be studied using different molecular and cellular approaches. While cancer stem cells may be derived from tumor-initiating cells, these terms are not necessarily interchangeable and more likely reflect a specific cell state.

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.

Mutual reinforcement between telomere capping and canonical Wnt signalling in the intestinal stem cell niche

Critical telomere shortening (for example, secondary to partial telomerase deficiency in the rare disease dyskeratosis congenita) causes tissue pathology, but underlying mechanisms are not fully understood. Mice lacking telomerase (for example, mTR−/− telomerase RNA template mutants) provide a model for investigating pathogenesis. In such mice, after several generations of telomerase deficiency telomeres shorten to the point of uncapping, causing defects most pronounced in high-turnover tissues including intestinal epithelium. Here we show that late-generation mTR−/− mutants experience marked downregulation of Wnt pathway genes in intestinal crypt epithelia, including crypt base columnar stem cells and Paneth cells, and in underlying stroma. The importance of these changes was revealed by rescue of crypt apoptosis and Wnt pathway gene expression upon treatment with Wnt pathway agonists. Rescue was associated with reduced telomere-dysfunction-induced foci and anaphase bridges, indicating improved telomere capping. Thus a mutually reinforcing feedback loop exists between telomere capping and Wnt signalling, and telomere capping can be impacted by extracellular cues in a fashion independent of telomerase.

Orphan Gpr182 suppresses ERK-mediated intestinal proliferation during regeneration and adenoma formation.

Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the Gpr182 during development and adulthood. We observed that Gpr182 is expressed at the crypt base throughout the small intestine, where it is enriched in crypt base columnar stem cells, one of the most active stem cell populations in the body. Gpr182 knockdown had no effect on homeostatic intestinal proliferation in vivo, but led to marked increases in proliferation during intestinal regeneration following irradiation-induced injury. In the ApcMin mouse model, which forms spontaneous intestinal adenomas, reductions in Gpr182 led to more adenomas and decreased survival. Loss of Gpr182 enhanced organoid growth efficiency ex vivo in an EGF-dependent manner. Gpr182 reduction led to increased activation of ERK1/2 in basal and challenge models, demonstrating a potential role for this orphan GPCR in regulating the proliferative capacity of the intestine. Importantly, GPR182 expression was profoundly reduced in numerous human carcinomas, including colon adenocarcinoma. Together, these results implicate Gpr182 as a negative regulator of intestinal MAPK signaling-induced proliferation, particularly during regeneration and adenoma formation.

Heterogeneity in readouts of canonical wnt pathway activity within intestinal crypts

Canonical Wnt pathway signaling is necessary for maintaining the proliferative capacity of mammalian intestinal crypt base columnar stem cells (CBCs). Furthermore, dysregulation of the Wnt pathway is a major contributor to disease, including oncogenic transformation of the intestinal epithelium. Given the critical importance of this pathway, numerous tools have been used as proxy measures for Wnt pathway activity, yet the relationship between Wnt target gene expression and reporter allele activity within individual cells at the crypt base remains unclear. Here, we describe a novel Axin2-CreERT2-tdTomato allele that efficiently marks both Wnt(High) CBCs and radioresistant reserve intestinal stem cells. We analyze the molecular and functional identity of Axin2-CreERT2-tdTomato-marked cells using single cell gene expression profiling and tissue regeneration assays and find that Axin2 reporter activity does not necessarily correlate with expression of Wnt target genes and, furthermore, that Wnt target genes themselves vary in their expression patterns at the crypt base. Wnt target genes and reporter alleles can vary greatly in their cell-type specificity, demonstrating that these proxies cannot be used interchangeably. Furthermore, Axin2-CreERT2-tdTomato is a robust marker of both active and reserve intestinal stem cells and is thus useful for understanding the intestinal stem cell compartment. Developmental Dynamics 245:822-833, 2016. © 2016 Wiley Periodicals, Inc.

Agonists of the TRAIL Death Receptor DR5 Sensitize Intestinal Stem Cells to Chemotherapy-Induced Cell Death and Trigger Gastrointestinal Toxicity.

The combination of TRAIL death receptor agonists and radiochemotherapy to treat advanced cancers continues to be investigated in clinical trials. We previously showed that normal cells with a functional DNA damage response (DDR) upregulate the expression of death-inducing receptor DR5/TRAILR2/TNFRSF10B in a p53-dependent manner that sensitizes them to treatment with DR5 agonists. However, it is unclear if targeting DR5 selectively sensitizes cancer cells to agonist treatment following exposure to DNA-damaging chemotherapy, and to what extent normal tissues are targeted. Here, we show that the combined administration of the DR5 agonistic monoclonal antibody (mAb) and chemotherapy to wild-type mice triggered synergistic gastrointestinal toxicities (GIT) that were associated with the death of Lgr5(+) crypt base columnar stem cells in a p53- and DR5-dependent manner. Furthermore, we confirmed that normal human epithelial cells treated with the human DR5-agonistic mAb and chemotherapeutic agents were also greatly sensitized to cell death. Interestingly, our data also indicated that genetic or pharmacologic targeting of Chk2 may counteract GIT without negatively affecting the antitumor responses of combined DR5 agonist/chemotherapy treatment, further linking the DDR to TRAIL death receptor signaling in normal cells. In conclusion, the combination of DR5-targeting agonistic mAbs with DNA damaging chemotherapy may pose a risk of developing toxicity-induced conditions, and the effects of mAb-based strategies on the dose-limiting toxicity of chemotherapy must be considered when establishing new combination therapies.

Role of Krüppel-like factor 5 in the maintenance of the stem cell niche in the intestinal crypt

The intestinal epithelium is a tissue that undergoes continuous self-renewal initiated at the bottom of the crypts, which harbor the intestinal stem cell (ISC) pool. The ISC pool is sub-divided into crypt base columnar (CBC) cells at the crypt bottom and label retention cells (LRC) at position +4 from the crypt bottom. CBC cells are marked by Leucine-rich repeat-containing G-protein coupled receptor (Lgr5) while LRC cells are identified by several markers including Bmi1, mTert, Hopx, Lrig1, and Sox9. Krüppel-like factors (KLFs) belong to a family of transcription factors that exert important physiological function in various tissues. In the intestine, KLF4 is predominantly expressed in the terminally differentiated, non-proliferating cells lining the villus. Its deletion in the adult mouse intestine results in perturbed homeostasis. In contrast, KLF5 is expressed in actively proliferating cells of the intestinal crypt, including CBC cells and transit amplifying (TA) cells. We recently investigated the effect of Klf5 deletion specifically from the Lgr5-expressing CBC cells in adult mouse intestine using an inducible Cre recombinase system. Shortly (3-5 days) after Cre induction, proliferation of both CBC and TA cells ceased, which was accompanied by an increase in apoptosis in the crypt. Beginning at two weeks following Cre induction, both Klf5 expression and proliferation re-appeared but without the re-emergence of Lgr5-positive CBC cells, which were eventually depleted by four months following induction. These findings indicate that KLF5 plays an important role in regulating proliferation and survival of CBC stem cells in the intestine.

Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium.

Snail family members regulate epithelial-to-mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation-induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.

Temozolomide Increases the Number of Mismatch Repair–Deficient Intestinal Crypts and Accelerates Tumorigenesis in a Mouse Model of Lynch Syndrome

Lynch syndrome, a nonpolyposis form of hereditary colorectal cancer, is caused by inherited defects in DNA mismatch repair (MMR) genes. Most patients carry a germline mutation in 1 allele of the MMR genes MSH2 or MLH1. With spontaneous loss of the wild-type allele, cells with defects in MMR exist among MMR-proficient cells, as observed in healthy intestinal tissues from patients with Lynch syndrome. We aimed to create a mouse model of this situation to aid in identification of environmental factors that affect MMR-defective cells and their propensity for oncogenic transformation. We created mice in which the MMR gene Msh2 can be inactivated in a defined fraction of crypt base columnar stem cells to generate MSH2-deficient intestinal crypts among an excess of wild-type crypts (Lgr5-CreERT2;Msh2(flox/-) mice). Intestinal tissues were collected; immunohistochemical analyses were performed for MSH2, along with allele-specific PCR assays. We traced the fate of MSH2-deficient crypts under the influence of different external factors. Lgr5-CreERT2;Msh2(flox/-) mice developed more adenomas and adenocarcinomas than control mice; all tumors were MSH2 deficient. Exposure of Lgr5-CreERT2;Msh2(flox/-) mice to the methylating agent temozolomide caused MSH2-deficient intestinal stem cells to proliferate more rapidly than wild-type stem cells. The MSH2-deficient intestinal stem cells were able to colonize the intestinal epithelium and many underwent oncogenic transformation, forming intestinal neoplasias. We developed a mouse model of Lynch syndrome (Lgr5-CreERT2;Msh2(flox/-) mice) and found that environmental factors can modify the number and mutability of the MMR-deficient stem cells. These findings provide evidence that environmental factors can promote development of neoplasias and tumors in patients with Lynch syndrome.

302 Dynamic Regulation of Intestinal Crypt Base Columnar Stem Cells by Notch Signaling

Background/Aim: The intestinal epithelium is a rapidly renewed tissue, requiring continual replenishment by intestinal stem cells (ISCs) in order to sustain life. The crypt base columnar cell (CBC) is an actively dividing ISC population marked by the expression of Lgr5, Ascl2, and Olfm4. Our recent studies show that Notch signaling regulates epithelial cell homeostasis by directing differentiated cell fate and promoting stem cell maintenance. In this study we examined Notch regulation of CBCs by characterizing Olfm4 expression and function. Methods: Gamma-secretase inhibitors (GSIs) were used to block Notch signaling in C57BL/ 6 mice (30μmol/kg dibenzazepine; DBZ) or in the human colon cancer cell line LS174T (40μM DAPT). Tissue was analyzed at various time points for progenitor and differentiated marker expression and epithelial proliferation by histological staining and qRT-PCR. LS174T cells were used to investigate the mechanism of Notch regulation of Olfm4 expression. Genetically engineered Olfm4-deficient mouse intestine was studied to examine the function of this CBC marker for intestinal epithelial cell homeostasis. Results: Chronic Notch inhibition for 6 days resulted in marked secretory cell hyperplasia, decreased cellular proliferation, and a striking decrease in Olfm4 mRNA, showing that Notch is critical for many different aspects of cellular renewal. Surprisingly, acute block of Notch with a single dose of DBZ was sufficient to cause a rapid and transient loss of Olfm4, in addition to a delayed, yet sustained surge in secretory cells and cellular proliferation. This surge in proliferation was in stark contrast to the loss of proliferation observed with chronic DBZ treatment. The acute DBZ model revealed exquisite Olfm4 regulation: mice treated with one dose showed a significant decrease in Olfm4 mRNA within 12 hours, but recovery to baseline within 2 days. Similarly, LS174T colon cancer cells showed decreased Olfm4 mRNA 4 hours after DAPT administration. To test whether changes in Olfm4 expression may mediate Notch effects in the intestine we examinedOlfm4-/mice. This analysis showed normal proliferation, cell fate, and CBC marker gene expression patterns, demonstrating that Olfm4 is not required for CBC function. However, we observed expression of other Olfactomedin family members in intestine, suggesting functional redundancy.Conclusions:Olfm4 is dynamically regulated by Notch signaling, with rapid loss of transcripts observed upon acute Notch inhibition. Thus this CBC marker is a sensitive read out of Notch activity in the intestine. Olfm4-/mice did not exhibit an intestinal phenotype, although related family members may play a compensatory role, suggesting compound mouse mutants may be needed to understand the function of Notch regulation of Olfm4 in the CBC.

Notch signaling regulates proliferation and differentiation of the intestinal crypt base columnar (CBC) stem cell

This study examined the importance of the Notch signaling pathway for homeostasis of the CBC stem cell in the intestine. The Lgr5‐GFP reporter mouse strain was used to identify and track CBC stem cells after Notch inhibition with DBZ. Global Notch disruption in adult mice induced rapid CBC cell loss, with reduced proliferation and apoptotic cell death. Furthermore, expression of the CBC stem cell‐specific marker Olfactomedin 4 (Olfm4) was observed to be directly dependent on Notch signaling, with transcription activated through RBP‐Jκ binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitor cells into secretory cell types, including goblet, enteroendocrine and tuft cells, as well as an unusual secretory cell type that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1‐deficient mouse intestine demonstrated that the generalized secretory cell hyperplasia induced by Notch inhibition was dependent on this transcription factor, whereas Notch regulation of Olfm4 gene expression was Atoh1‐independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice in order to control epithelial cell proliferation and differentiation.

Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells

Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells

Paneth cells and stem cells in the intestinal stem cell niche and their association with inflammatory bowel disease

The intestinal epithelial cells are replaced in every three to four days. This system is maintained by self-reproduction, differentiation of the intestinal stem cells. The continuous monolayer of intestinal epithelial cells functions in innate immunity as the primary barrier against microbial colonization. Paneth cells secrete α-defensins, and they are actively involved in the innate immunity and maintain the intestinal homeostasis by controlling intestinal microbiota. In addition, recent analyses of epithelial renewal in the intestine illustrate that Paneth cells provide survival signals to crypt intestinal stem cells. Two types of intestinal stem cells, crypt base columnar (CBC) stem cell and +4 stem cell, have been identified. Lgr5 positive CBC stem cells, Bmi1 positive +4 stem cells and Paneth cells create the stem cell niche in the small intestine. In this review, recent advances in understanding the roles of Paneth cells and stem cells which involve stem cell niche, and their association of inflammatory bowel disease were discussed and summarized.

Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells

Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-Jκ binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis.