Tissue geometry drives deterministic organoid patterning.

Epithelial organoids are stem cell­derived tissues that approximate aspects of real organs, and thus they have potential as powerful tools in basic and translational research. By definition, they self-organize, but the structures formed are often heterogeneous and irreproducible, which limits their use in the lab and clinic. We describe methodologies for spatially and temporally controlling organoid formation, thereby rendering a stochastic process more deterministic. Bioengineered stem cell microenvironments are used to specify the initial geometry of intestinal organoids, which in turn controls their patterning and crypt formation. We leveraged the reproducibility and predictability of the culture to identify the underlying mechanisms of epithelial patterning, which may contribute to reinforcing intestinal regionalization in vivo. By controlling organoid culture, we demonstrate how these structures can be used to answer questions not readily addressable with the standard, more variable, organoid models.

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

Although 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.

Cdk5rap3 is essential for intestinal Paneth cell development and maintenance.

Intestinal Paneth cells are professional exocrine cells that play crucial roles in maintenance of homeostatic microbiome, modulation of mucosal immunity, and support for stem cell self-renewal. Dysfunction of these cells may lead to the pathogenesis of human diseases such as inflammatory bowel disease (IBD). Cdk5 activator binding protein Cdk5rap3 (also known as C53 and LZAP) was originally identified as a binding protein of Cdk5 activator p35. Although previous studies have indicated its involvement in a wide range of signaling pathways, the physiological function of Cdk5rap3 remains largely undefined. In this study, we found that Cdk5rap3 deficiency resulted in very early embryonic lethality, indicating its indispensable role in embryogenesis. To further investigate its function in the adult tissues and organs, we generated intestinal epithelial cell (IEC)-specific knockout mouse model to examine its role in intestinal development and tissue homeostasis. IEC-specific deletion of Cdk5rap3 led to nearly complete loss of Paneth cells and increased susceptibility to experimentally induced colitis. Interestingly, Cdk5rap3 deficiency resulted in downregulation of key transcription factors Gfi1 and Sox9, indicating its crucial role in Paneth cell fate specification. Furthermore, Cdk5rap3 is highly expressed in mature Paneth cells. Paneth cell-specific knockout of Cdk5rap3 caused partial loss of Paneth cells, while inducible acute deletion of Cdk5rap3 resulted in disassembly of the rough endoplasmic reticulum (RER) and abnormal zymogen granules in the mature Paneth cells, as well as loss of Paneth cells. Together, our results provide definitive evidence for the essential role of Cdk5rap3 in Paneth cell development and maintenance.

Direct On-Chip Differentiation of Intestinal Tubules from Induced Pluripotent Stem Cells.

Intestinal organoids have emerged as the new paradigm for modelling the healthy and diseased intestine with patient-relevant properties. In this study, we show directed differentiation of induced pluripotent stem cells towards intestinal-like phenotype within a microfluidic device. iPSCs are cultured against a gel in microfluidic chips of the OrganoPlate, in which they undergo stepwise differentiation. Cells form a tubular structure, lose their stem cell markers and start expressing mature intestinal markers, including markers for Paneth cells, enterocytes and neuroendocrine cells. Tubes develop barrier properties as confirmed by transepithelial electrical resistance (TEER). Lastly, we show that tubules respond to pro-inflammatory cytokine triggers. The whole procedure for differentiation lasts 14 days, making it an efficient process to make patient-specific organoid tubules. We anticipate the usage of the platform for disease modelling and drug candidate screening.

Lactobacillus reuteri maintains intestinal epithelial regeneration and repairs damaged intestinal mucosa.

Little is known about the regulatory effect of microbiota on the proliferation and regeneration of ISCs. Here, we found that L. reuteri stimulated the proliferation of intestinal epithelia by increasing the expression of R-spondins and thus activating the Wnt/ß-catenin pathway. The proliferation-stimulating effect of Lactobacillus on repair is further enhanced under TNF -induced intestinal mucosal damage, and the number of Lgr5+ cells is maintained. Moreover, compared to the effects of C. rodentium on the induction of intestinal inflammation and crypt hyperplasia in mice, L. reuteri protected the intestinal mucosal barrier integrity by moderately modulating the Wnt/ß-catenin signaling pathway to avoid overactivation. L. reuteri had the ability to maintain the number of Lgr5+ cells and stimulate intestinal epithelial proliferation to repair epithelial damage and reduce proinflammatory cytokine secretion in the intestine and the LPS concentration in serum. Moreover, activation of the Wnt/ß-catenin pathway also induced differentiation toward Paneth cells and increased antimicrobial peptide expression to inhibit C. rodentium colonization. The protective effect of Lactobacillus against C. rodentium infection disappeared upon application of the Wnt antagonist Wnt-C59 in both mice and intestinal organoids. This study demonstrates that Lactobacillus is effective at maintaining intestinal epithelial regeneration and homeostasis as well as at repairing intestinal damage after pathological injury and is thus a promising alternative therapeutic method for intestinal inflammation.

Cell-type-specific signaling networks in heterocellular organoids.

Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simultaneous analysis by mass cytometry of 28 PTMs in >1 million single cells derived from small intestinal organoids reveals cell-type- and cell-state-specific signaling networks in stem, Paneth, enteroendocrine, tuft and goblet cells, as well as enterocytes. Integrating single-cell PTM analysis with thiol-reactive organoid barcoding in situ (TOBis) enables high-throughput comparison of signaling networks between organoid cultures. Cell-type-specific PTM analysis of colorectal cancer organoid cocultures reveals that shApc, KrasG12D and Trp53R172H cell-autonomously mimic signaling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type-specific signaling analysis of healthy and cancerous organoids.

Enteroids expressing a disease-associated mutant of EpCAM are a model for congenital tufting enteropathy.

Congenital tufting enteropathy (CTE) is an autosomal recessive disease characterized by severe intestinal failure in infancy and mutations in the epithelial cell adhesion molecule (EPCAM) gene. Previous studies of CTE in mice expressing mutant EpCAM show neonatal lethality. Hence, to study the cellular, molecular, and physiological alterations that result from EpCAM mutation, a tamoxifen-inducible mutant EpCAM enteroid model has been generated. The presence of mutant EpCAM in the model was confirmed at both mRNA and protein levels. Immunofluorescence microscopy demonstrated the reduced expression of mutant EpCAM. Mutant enteroids had reduced budding potential as well as significantly decreased mRNA expression for epithelial lineage markers (Mucin 2, lysozyme, sucrase-isomaltase), proliferation marker Ki67, and secretory pathway transcription factors (Atoh1, Hnf1b). Significantly decreased numbers of Paneth and goblet cells were confirmed by staining. These findings were correlated with intestinal tissue from CTE patients and the mutant mice model that had significantly fewer Paneth and goblet cells than in healthy counterparts. FITC-dextran studies demonstrated significantly impaired barrier function in monolayers derived from mutant enteroids compared with control monolayers. In conclusion, we have established an ex vivo CTE model. The role of EpCAM in the budding potential, differentiation, and barrier function of enteroids is noted. Our study establishes new facets of EpCAM biology that will aid in understanding the pathophysiology of CTE and role of EpCAM in health and disease.NEW & NOTEWORTHY Here, we develop a novel ex vivo enteroid model for congenital tufting enteropathy (CTE) based on epithelial cell adhesion molecule (EPCAM) gene mutations found in patients. With this model we demonstrate the role of EpCAM in maintaining the functional homeostasis of the intestinal epithelium, including differentiation, proliferation, and barrier integrity. This study further establishes a new direction in EpCAM biology that will help in understanding the detailed pathophysiology of CTE and role of EpCAM.

SH3BP4 Regulates Intestinal Stem Cells and Tumorigenesis by Modulating ß-Catenin Nuclear Localization.

Wnt signals at the base of mammalian crypts play a pivotal role in intestinal stem cell (ISC) homeostasis, whereas aberrant Wnt activation causes colon cancer. Precise control of Wnt signal strength is governed by a number of negative inhibitory mechanisms acting at distinct levels of the cascade. Here, we identify the Wnt negative regulatory role of Sh3bp4 in the intestinal crypt. We show that the loss of Sh3bp4 increases ISC and Paneth cell numbers in murine intestine and accelerates adenoma development in Apcmin mice. Mechanistically, human SH3BP4 inhibits Wnt signaling downstream of ß-catenin phosphorylation and ubiquitination. This Wnt inhibitory role is dependent on the ZU5 domain of SH3BP4. We further demonstrate that SH3BP4 is expressed at the perinuclear region to restrict nuclear localization of ß-catenin. Our data uncover the tumor-suppressive role of SH3BP4 that functions as a negative feedback regulator of Wnt signaling through modulating ß-catenin's subcellular localization.

Impact of Autophagy of Innate Immune Cells on Inflammatory Bowel Disease.

Autophagy, an intracellular degradation mechanism, has many immunological functions and is a constitutive process necessary for maintaining cellular homeostasis and organ structure. One of the functions of autophagy is to control the innate immune response. Many studies conducted in recent years have revealed the contribution of autophagy to the innate immune response, and relationships between this process and various diseases have been reported. Inflammatory bowel disease is an intractable disorder with unknown etiology; however, immunological abnormalities in the intestines are known to be involved in the pathology of inflammatory bowel disease, as is dysfunction of autophagy. In Crohn's disease, many associations with autophagy-related genes, such as ATG16L1, IRGM, NOD2, and others, have been reported. Abnormalities in the ATG16L1 gene, in particular, have been reported to cause autophagic dysfunction, resulting in enhanced production of inflammatory cytokines by macrophages as well as abnormal function of Paneth cells, which are important in intestinal innate immunity. In this review, we provide an overview of the autophagy mechanism in innate immune cells in inflammatory bowel disease.

Proteomic Profiling of Enteroid Cultures Skewed toward Development of Specific Epithelial Lineages.

Recently, 3D small intestinal organoids (enteroids) have been developed from cultures of intestinal stem cells which differentiate in vitro to generate all the differentiated epithelial cell types associated with the intestine and mimic the structural properties of the intestine observed in vivo. Small-molecule drug treatment can skew organoid epithelial cell differentiation toward particular lineages, and these skewed enteroids may provide useful tools to study specific epithelial cell populations, such as goblet and Paneth cells. However, the extent to which differentiated epithelial cell populations in these skewed enteroids represent their in vivo counterparts is not fully understood. This study utilises label-free quantitative proteomics to determine whether skewing murine enteroid cultures toward the goblet or Paneth cell lineages results in changes in abundance of proteins associated with these cell lineages in vivo. Here, proteomics data confirms that skewed enteroids recapitulate important features of the in vivo gut environment, demonstrating that they can serve as useful models for the investigation of normal and disease processes in the intestine. Furthermore, comparison of mass spectrometry data with histology data contained within the Human Protein Atlas identifies putative novel markers for goblet and Paneth cells.

Mouse Intestinal Krt15+ Crypt Cells Are Radio-Resistant and Tumor Initiating.

Two principal stem cell pools orchestrate the rapid cell turnover in the intestinal epithelium. Rapidly cycling Lgr5+ stem cells are intercalated between the Paneth cells at the crypt base (CBCs) and injury-resistant reserve stem cells reside above the crypt base. The intermediate filament Keratin 15 (Krt15) marks either stem cells or long-lived progenitor cells that contribute to tissue repair in the hair follicle or the esophageal epithelium. Herein, we demonstrate that Krt15 labels long-lived and multipotent cells in the small intestinal crypt by lineage tracing. Krt15+ crypt cells display self-renewal potential in vivo and in 3D organoid cultures. Krt15+ crypt cells are resistant to high-dose radiation and contribute to epithelial regeneration following injury. Notably, loss of the tumor suppressor Apc in Krt15+ cells leads to adenoma and adenocarcinoma formation. These results indicate that Krt15 marks long-lived, multipotent, and injury-resistant crypt cells that may function as a cell of origin in intestinal cancer.

Gastrointestinal mucosal development and injury in premature lambs supported by the artificial placenta.

BACKGROUND: An Artificial Placenta (AP) utilizing extracorporeal life support (ECLS) could revolutionize care of extremely premature newborns, but its effects on gastrointestinal morphology and injury need investigation. METHODS: Lambs (116-121days GA, term=145; n=5) were delivered by C-section, cannulated for ECLS, had total parenteral nutrition (TPN) provided, and were supported for 7days before euthanasia. Early and Late Tissue Controls (ETC, n=5 and LTC, n=5) delivered at 115-121days and 125-131days, respectively, were immediately sacrificed. Standardized jejunal samples were formalin-fixed for histology. Crypt depth (CD), villus height (VH), and VH:CD ratios were measured. Measurements also included enterocyte proliferation (Ki-67), Paneth cell count (Lysozyme), and injury scores (H&E). ANOVA and Chi Square were used with p<0.05 considered significant. RESULTS: CD, VH, and VH:CD were similar between groups (p>0.05). AP demonstrated more enterocyte proliferation (95.7±21.8) than ETC (49.4±23.4; p=0.003) and LTC (66.1+11.8; p=0.04), and more Paneth cells (81.7±17.5) than ETC (41.6±7.0; p=0.0005) and LTC (40.7±8.2, p=0.0004). Presence of epithelial injury and congestion in the bowel of all groups were not statistically different. No villus atrophy or inflammation was present in any group. CONCLUSIONS: This suggests preserved small bowel mucosal architecture, high cellular turnover, and minimal evidence of injury. STUDY TYPE: Research paper/therapeutic potential. LEVEL OF EVIDENCE: N/A.

The role of CSF1R-dependent macrophages in control of the intestinal stem-cell niche.

Colony-stimulating factor 1 (CSF1) controls the growth and differentiation of macrophages.CSF1R signaling has been implicated in the maintenance of the intestinal stem cell niche and differentiation of Paneth cells, but evidence of expression of CSF1R within the crypt is equivocal. Here we show that CSF1R-dependent macrophages influence intestinal epithelial differentiation and homeostasis. In the intestinal lamina propria CSF1R mRNA expression is restricted to macrophages which are intimately associated with the crypt epithelium, and is undetectable in Paneth cells. Macrophage ablation following CSF1R blockade affects Paneth cell differentiation and leads to a reduction of Lgr5+ intestinal stem cells. The disturbances to the crypt caused by macrophage depletion adversely affect the subsequent differentiation of intestinal epithelial cell lineages. Goblet cell density is enhanced, whereas the development of M cells in Peyer's patches is impeded. We suggest that modification of the phenotype or abundance of macrophages in the gut wall alters the development of the intestinal epithelium and the ability to sample gut antigens.

Paneth and intestinal stem cells preserve their functional integrity during worsening of acute cellular rejection in small bowel transplantation.

Graft survival after small bowel transplantation remains impaired due to acute cellular rejection (ACR), the leading cause of graft loss. Although it was shown that the number of enteroendocrine progenitor cells in intestinal crypts was reduced during mild ACR, no results of Paneth and intestinal stem cells localized at the crypt bottom have been shown so far. Therefore, we wanted to elucidate integrity and functionality of the Paneth and stem cells during different degrees of ACR, and to assess whether these cells are the primary targets of the rejection process. We compared biopsies from ITx patients with no, mild, or moderate ACR by immunohistochemistry and quantitative PCR. Our results show that numbers of Paneth and stem cells remain constant in all study groups, whereas the transit-amplifying zone is the most impaired zone during ACR. We detected an unchanged level of antimicrobial peptides in Paneth cells and similar numbers of Ki-67+ IL-22R+ stem cells revealing cell functionality in moderate ACR samples. We conclude that Paneth and stem cells are not primary target cells during ACR. IL-22R+ Ki-67+ stem cells might be an interesting target cell population for protection and regeneration of the epithelial monolayer during/after a severe ACR in ITx patients.

HMGA1 amplifies Wnt signalling and expands the intestinal stem cell compartment and Paneth cell niche.

High-mobility group A1 (Hmga1) chromatin remodelling proteins are enriched in intestinal stem cells (ISCs), although their function in this setting was unknown. Prior studies showed that Hmga1 drives hyperproliferation, aberrant crypt formation and polyposis in transgenic mice. Here we demonstrate that Hmga1 amplifies Wnt/ß-catenin signalling to enhance self-renewal and expand the ISC compartment. Hmga1 upregulates genes encoding both Wnt agonist receptors and downstream Wnt effectors. Hmga1 also helps to 'build' an ISC niche by expanding the Paneth cell compartment and directly inducing Sox9, which is required for Paneth cell differentiation. In human intestine, HMGA1 and SOX9 are positively correlated, and both become upregulated in colorectal cancer. Our results define a unique role for Hmga1 in intestinal homeostasis by maintaining the stem cell pool and fostering terminal differentiation to establish an epithelial stem cell niche. This work also suggests that deregulated Hmga1 perturbs this equilibrium during intestinal carcinogenesis.

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.

LRRK2 but not ATG16L1 is associated with Paneth cell defect in Japanese Crohn's disease patients.

BACKGROUND. Morphological patterns of Paneth cells are a prognostic biomarker in Western Crohn's disease (CD) patients, and are associated with autophagy-associated ATG16L1 and NOD2 variants. We hypothesized that genetic determinants of Paneth cell phenotype in other ethnic CD cohorts are distinct but also involved in autophagy. METHODS. We performed a hypothesis-driven analysis of 56 single nucleotide polymorphisms (SNPs) associated with CD susceptibility or known to affect Paneth cell function in 110 Japanese CD patients who underwent ileal resection. We subsequently performed a genome-wide association analysis. Paneth cell phenotype was determined by defensin-5 immunofluorescence. Selected genotype-Paneth cell defect correlations were compared to a Western CD cohort (n = 164). RESULTS. The average percentage of abnormal Paneth cells in Japanese CD was similar to Western CD (P = 0.87), and abnormal Paneth cell phenotype was also associated with early recurrence (P = 0.013). In contrast to Western CD, ATG16L1 T300A was not associated with Paneth cell defect in Japanese CD (P = 0.20). Among the 56 selected SNPs, only LRRK2 M2397T showed significant association with Paneth cell defect (P = 3.62 × 10-4), whereas in the Western CD cohort it was not (P = 0.76). Pathway analysis of LRRK2 and other candidate genes with P less than 5 × 10-4 showed connections with known CD susceptibility genes and links to autophagy and TNF-α networks. CONCLUSIONS. We found dichotomous effects of ATG16L1 and LRRK2 on Paneth cell defect between Japanese and Western CD. Genes affecting Paneth cell phenotype in Japanese CD were also associated with autophagy. Paneth cell phenotype also predicted prognosis in Japanese CD. FUNDING. Helmsley Charitable Trust, Doris Duke Foundation (grant 2014103), Japan Society for the Promotion of Science (KAKENHI grants JP15H04805 and JP15K15284), Crohn's and Colitis Foundation grant 274415, NIH (grants 1R56DK095820, K01DK109081, and UL1 TR000448).

Single cell lineage tracing reveals a role for TgfßR2 in intestinal stem cell dynamics and differentiation.

Intestinal stem cells (ISCs) are maintained by a niche mechanism, in which multiple ISCs undergo differential fates where a single ISC clone ultimately occupies the niche. Importantly, mutations continually accumulate within ISCs creating a potential competitive niche environment. Here we use single cell lineage tracing following stochastic transforming growth factor ß receptor 2 (TgfßR2) mutation to show cell autonomous effects of TgfßR2 loss on ISC clonal dynamics and differentiation. Specifically, TgfßR2 mutation in ISCs increased clone survival while lengthening times to monoclonality, suggesting that Tgfß signaling controls both ISC clone extinction and expansion, independent of proliferation. In addition, TgfßR2 loss in vivo reduced crypt fission, irradiation-induced crypt regeneration, and differentiation toward Paneth cells. Finally, altered Tgfß signaling in cultured mouse and human enteroids supports further the in vivo data and reveals a critical role for Tgfß signaling in generating precursor secretory cells. Overall, our data reveal a key role for Tgfß signaling in regulating ISCs clonal dynamics and differentiation, with implications for cancer, tissue regeneration, and inflammation.

NOD2 up-regulates TLR2-mediated IL-23p19 expression via NF-κB subunit c-Rel in Paneth cell-like cells.

IL-23p19 plays important roles in intestinal antimicrobial immunity, while its over-expression can lead to intestinal inflammation. However, the bacterial compounds and the type of pattern recognition receptor involved in the inducible expression of IL-23p19 in Paneth cells remain unclear. Here we show that the mRNA expression of IL-23p19 was increased in Paneth cell (PC)-like cells stimulated by Toll-like receptor 2 (TLR2) ligands, peptidoglycan (PGN) and Pam3CSK4, and was further increased in the presence of nucleotide-binding oligomerization domain 2 (NOD2)-ligand muramyl dipeptide (MDP). However, its mRNA expression was decreased in NOD2-knockdown PC-like cells. Additionally, the c-Rel activation was increased in Pam3CSK4- or PGN-stimulated PC-like cells, but the PGN-induced c-Rel activation was decreased in NOD2-knockdown PC-like cells and had no significant difference compared with Pam3CSK4-induced c-Rel activation. Our results suggest that NOD2 up-regulates TLR2-mediated IL-23p19 expression via increasing c-Rel activation in PC-like cells. This finding might provide us with a novel therapeutic target for inflammatory bowel disease to inhibit IL-23p19 over-expression via the NOD2-c-Rel pathway.

Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon.

Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5(+) stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5(+) stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4(+) DCS cells promote organoid formation of single Lgr5(+) colon stem cells. DCS cells can be massively produced from Lgr5(+) colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4(+) DCS cells serve as Paneth cell equivalents in the colon crypt niche.