Base Of Intestinal Crypts Research Articles

Mo1863 Characterization of the Gastric Cancer-Associated Human Microbiota

crypt. By 24 hours, tdTomato epifluorescence was observed in Paneth (α-defensin 5+), transit-amplifying cells (Ki67+) and scattered enterocytes (DPP IV+) throughout the villus. Colocalization studies revealed that the tdTomato fluorescent cells were not enteroendocrine (Chromogranin A+), tuft (DCAMKL1+), or goblet cells (Mucin 2+) at the 24h time point in the intestine. Additionally, we analyzed Zfp148 promoter activity in secretory progenitor cells by MATH1 staining and found that these cells also did not express tdTomato fluorescence. By 3 weeks post TX treatment, long tracts of cells were labeled along the length of the villi in addition to persistent and more intense tdTomato fluorescence occurring at the crypt base. In the colon, activated cells gave rise to scattered non-adjacent glands that were completely labeled with tdTomato fluorescence. Conclusion: Zfp148 gene expression (ZBP89) occurred in cells at the intestinal crypt base that morphologically coincide with cells involved in the stem cell niche. Over time these cells were able to propagate and label entire colonic glands and, in the intestine, a majority of the intestinal villus. Although a ubiquitous protein, Zfp148 gene expression is more robust in the stem cell niche implicating a role for this transcription factor in modulating stem cell fate.

Vitamin D is a determinant of mouse intestinal Lgr5 stem cell functions

Lgr5+ intestinal crypt base columnar cells function as stem cells whose progeny populate the villi, and Lgr5+ cells in which Apc is inactivated can give rise to tumors. Surprisingly, these Lgr5+ stem cell properties were abrogated by the lower dietary vitamin D and calcium in a semi-purified diet that promotes both genetically initiated and sporadic intestinal tumors. Inactivation of the vitamin D receptor in Lgr5+ cells established that compromise of Lgr5 stem cell function was a rapid, cell autonomous effect of signaling through the vitamin D receptor. The loss of Lgr5 stem cell function was associated with presence of Ki67 negative Lgr5+ cells at the crypt base. Therefore, vitamin D, a common nutrient and inducer of intestinal cell maturation, is an environmental factor that is a determinant of Lgr5+ stem cell functions in vivo. Since diets used in reports that establish and dissect mouse Lgr5+ stem cell activity likely provided vitamin D levels well above the range documented for human populations, the contribution of Lgr5+ cells to intestinal homeostasis and tumor formation in humans may be significantly more limited, and variable in the population, then suggested by published rodent studies.

Determination using synchrotron radiation-based Fourier transform infrared microspectroscopy of putative stem cells in human adenocarcinoma of the intestine: corresponding benign tissue as a template.

The epithelial-cell layer lining the two morphologically and functionally distinct segments of the mammalian intestinal tract, small intestine, and colon is constantly being renewed. This renewal is necessitated by a harsh lumen environment and is hypothesized to be driven by a small population of stem cells (SCs) that are believed to reside at the base of intestinal crypts. A lack of specific markers has hampered previous attempts to identify their exact location. We obtained tissue sections containing small intestine and colon crypts derived from normal (benign) or adenocarcinoma (AC) human intestine. The samples were floated onto BaF2 windows and analyzed using synchrotron radiation-based Fourier transform infrared microspectroscopy via an aperture size of 10 × 10 μm. Derived infrared (IR) spectral data was then analyzed using principal component analysis and/or linear discriminant analysis. Hypothesized cell types (as a function of aperture location along the length of individual crypts) within benign crypts were classed based on exploratory unsupervised IR spectral point clustering. Scores plots derived from individual small intestine crypts consistently generated one or two distinct spectra that clustered away from the remaining cell categories; these were retrospectively classed as "distinct base region" spectra. In these plots, a clear progression of locations along crypt lengths designated as from putative stem cells (SCs) to transit-amplifying (TA) cells to terminally differentiated (TD) cells was observed in benign small intestine and colon crypts. This progression of spectral points was crypt specific, pointing away from a unifying cell lineage model in human intestinal crypts. On comparison of AC-derived spectra versus corresponding benign, a subpopulation of AC-derived spectra suggested a putative SC-like spectral fingerprint; remaining IR spectra were classed as exhibiting TA cell-like or TD cell-like spectral characteristics. These observations could point to a cancer SC phenotype; an approach capable of identifying their in situ location has enormous therapeutic applications.

Ultrastructural and histochemical study on the Paneth cells in the rat ascending colon.

Paneth cells (PCs) contribute to the host defense against indigenous bacteria in the small intestine. We found Paneth cell-like cells (PLCs) in the rat ascending colon, but the nature of PLCs is never clarified. Therefore, the present study aimed to clarify the cytological characteristics of PLCs and discuss their cellular differentiation. PLCs were localized in the bases of intestinal crypts, especially follicle-associated intestinal crypts in proximal colonic lymphoid tissue, but were very seldom found in the ordinary intestinal crypts of the ascending colon. PLCs possessed specific granules with highly electron-dense cores and haloes, as well as PCs in the small intestine. The secretory granules of PLCs were positive for PAS reaction, lysozyme and soluble phospholipase A2, but negative for Alcian blue staining, β-defensin-1 and -2, as well as the ones of PCs. Furthermore, intermediate cells possessing both the PLC-specific granules and the mucus granules similar to those of goblet cells (GCs) were occasionally found in the vicinity of PLCs. Intermediate cells ranged from goblet cell-like cells rich in mucus granules to PLC-like cells with few mucus granules. The cellular condensation and fragmentation were exclusively found in PLCs but never seen in intermediate cells or GCs. The PLCs, which were identified as PC, were suggested to be transformed from GCs through intermediate cells and finally to die by apoptosis in intestinal crypts of proximal colonic lymphoid tissue in the rat ascending colon.

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin

Paneth cells at the base of small intestinal crypts secrete α-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted α-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal α-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell α-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8±5.2ng/ml in proximal small intestine to 54.3±10.3ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal α-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota.

Covering the Cover

Covering the Cover

Somatostatin receptor haploinsufficiency accelerates KrasG12D-initiated pancreatic carcinogenesis by promoting inflammation

While a number of mouse models have been made to recapituate FAP, they fall short of truly mimicking the hallmarks of the human disorder. In lineage tracing studies (Powell et al., Cell, March 2012), we found that Leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1), a pan-ErbB inhibitor, marks a population of stem cells at the base of small intestinal and colonic crypts. In our current study, we combined our Lrig1-CreERT2/+ mice with Apcflox/+ mice and eliminated one copy of Apc with tamoxifen injection. Over a period of 50-100 days, the mice developmultiple distal colonic tumors as monitored by colonoscopy, as well as by novel non-invasive imaging (tumor formation measured by TSPO ligand binding monitored via PET). These mice also exhibit periampullary and duodenal tumors, as well as gastric lesions, thus mimicking additional features of FAP. Of interest, Lrig1-CreERT2/ +;Apcflox/+ mice also have CHRPE-like lesions in their retinal pigment epithelium. Finally, when induced Lrig1-CreERT2/+;Apcflox/flox mice were treated (i.p.) with a novel Wnt inhibitor, VU-WS113, cytosolic β-catenin was markedly reduced and TSPO uptake was also dramatically decreased, providing in vivo evidence that VU-WS113 may be an effective therapy for Wnt-driven tumors. We are in the process of comparing the transcriptional profiles of Lrig1-CreERT2/+;Apcflox/+ colonic tumors and human FAP tumors to understand their similarities and differences at a molecular level. In summary, in addition to recapitulating a number of features of human FAP the most important of which is distal, colonic tumors our results suggest this model represents a tractable system to test the efficacy of innovative therapeutic interventions whose efficacy can be monitored over time by colonoscopy and non-invasive imaging modalities.

Abstract LB-44: A novel PUMA inhibitor protects against chemotherapy-induced intestinal injury without compromising tumor response.

Abstract PUMA is a BH3-only member of the Bcl2 family that is a critical mediator of apoptosis in response to a variety of stimuli, and plays a particularly strong role in gastrointestinal and hematopoietic stem cell apoptosis following radiation. In this study we found that PUMA-dependent apoptosis mediates gastrointestinal injury induced by chemotherapeutic agents, indicating its critical role in DNA damage induced intestinal injury. PUMA neutralizes all known anti-apoptotic Bcl-2 family members through high-affinity interactions with its BH3 domain, thus facilitating activation of the intrinsic apoptotic pathway. Based on these interactions, we devised a pharmacophore model to mimic them, with the goal being a small molecule that prevents PUMA interaction with Bcl-2 family proteins. In silico screening of the ZINC 8.0 database yielded 142 potential compounds, 13 of which had favorable toxicity profiles. Extensive in vitro testing further narrowed the list to two lead compounds, one of which demonstrated significant in vivo activities. This lead compound significantly protected the intestinal stem cells from apoptosis induced by Irinotecan (CPT-11) and 5-Fluorouracil (5-FU). Utilizing a Lewis Lung Carcinoma (LLC) syngeneic tumor model, we found that treatment of C57/B6 mice with the experimental PUMA inhibitor (I.P.) significantly decreased CPT-11-induced apoptosis in the intestinal crypt base, similarly to that observed in PUMA KO animals. This treatment significantly preserved intestinal morphology and decreased weight loss in tumor bearing mice. Further, we found that administration of the PUMA inhibitor did not alter the tumor response to CPT-11. These findings suggest the feasibility of retaining the efficacy of chemotherapy while mitigating GI adverse effects with a small molecule. Further experiments will determine the inhibitor's pharmacokinetics and pharmacodynamics, as well as its mechanisms of action in vivo. Citation Format: Brian J. Leibowitz, Mei Li, Liang Wei, Lin Zhang, Jian Yu. A novel PUMA inhibitor protects against chemotherapy-induced intestinal injury without compromising tumor response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-44. doi:10.1158/1538-7445.AM2013-LB-44

Proteomic insights into mammalian reproduction biology

Over the past 10 years, insights into the biology of intestinal stem cells have rapidly increased. The adult intestinal epithelium represents one of the most suitable models to study tissue stem cells. It is the rapidly self-renewing tissue and the design of its basic units, the crypt-villus is highly stereotypical. Since the discovery of Lgr5+ stem cells at the base of intestinal crypts, it has become possible to unveil the molecular mechanisms that control homeostasis of the stem cells, including the extrinsic signaling cues from the crypt niche. Based on these insights, in vitro propagation of murine and human intestinal stem cells in the form of ever-expanding organoids has become possible. The three-dimensional culture technology of organoids opens new avenues for studying fundamental questions on the stem cells and their niche, but has also allowed several applications in personalized medicine. Here, we discuss these recent developments and insights.

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.

Deficiency of secreted cryptdin‐4 detected in a mouse model of Crohn fs disease using a new sandwich ELISA

Paneth cells in the base of small intestinal crypts secrete α‐defensins in response to bacteria, contribute to innate immunity, and shape the composition of intestinal microbiota. Among mouse α‐defensins, cryptdin‐4 (Crp4) is the most potent microbicide. However, a lack of sensitive assays to quantify α‐defensins released into the intestinal lumen has limited efforts to establish a relationship between secreted α‐defensin and diseases. Here we establish a highly sensitive sandwich ELISA, and analyze secreted Crp4 in feces from a mouse model of Crohn's disease. SD rats were immunized with native Crp4, and monoclonal antibodies recognizing different epitopes were identified and paired to optimize ELISA conditions. To measure levels of secreted Crp4, feces were collected from BALB/c mice and IL10 null, Crohn's disease model mice, and fecal proteins were extracted. The established ELISA was specific and highly sensitive with a Crp4 detection range of 120~1600 pg/ml. The Crp4 concentration measured in feces of normal mice was 20.4 ± 5.1 ng/ml (n=5). In contrast, feces from IL10 null mice contained 8.7 ± 5.0 ng/ml of Crp4 (n=5). Secreted Crp4 was significantly reduced in IL10 null mice. Quantifying secreted α‐defensin in feces enable us to know luminal α‐defensin levels for the first time, and contributes to a deeper understanding of diverse diseases such as inflammatory bowel disease, infectious disease, and obesity.

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

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.

Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium.

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth.

Paneth cell α-defensins in enteric innate immunity.

Paneth cells at the base of small intestinal crypts of Lieberkühn secrete high levels of α-defensins in response to cholinergic and microbial stimuli. Paneth cell α-defensins are broad spectrum microbicides that function in the extracellular environment of the intestinal lumen, and they are responsible for the majority of secreted bactericidal peptide activity. Paneth cell α-defensins confer immunity to oral infection by Salmonella enterica serovar Typhimurium, and they are major determinants of the composition of the small intestinal microbiome. In addition to host defense molecules such as α-defensins, lysozyme, and Pla2g2a, Paneth cells also produce and release proinflammatory mediators as components of secretory granules. Disruption of Paneth cell homeostasis, with subsequent induction of endoplasmic reticulum stress, autophagy, or apoptosis, contributes to inflammation in diverse genetic and experimental mouse models.

Fibroblast Growth Factor Receptor-3 (FGFR-3) Regulates Expression of Paneth Cell Lineage-specific Genes in Intestinal Epithelial Cells through both TCF4/β-Catenin-dependent and -independent Signaling Pathways

Fibroblast growth factor receptor-3 (FGFR-3) expression in the developing intestine is restricted to the undifferentiated epithelial cells within the lower portion of the crypt. We previously showed that mice lacking functional FGFR-3 have a significant decrease in the number of Paneth cells in the small intestine. Here, we used Caco2 cells to investigate whether FGFR-3 signaling can directly modulate expression of Paneth cell differentiation markers through its effects on TCF4/β-catenin or through other signaling pathways downstream of this receptor. Caco2 cells treated with FGFR-3 ligands or expressing FGFR-3(K650E), a constitutively active mutant, resulted in a significantly increased expression of genes characteristic of mature Paneth cells, including human α-defensins 5 and 6 (HD5 and HD6) and Paneth cell lysozyme, whereas enterocytic differentiation markers were reduced. Activation of FGFR-3 signaling sustained high levels of β-catenin mRNA expression, leading to increased TCF4/β-catenin-regulated transcriptional activity in Caco2 cells. Sustained activity of the TCF4/β-catenin pathway was required for the induction of Paneth cell markers. Activation of the MAPK pathway by FGFR-3 is also required for the induction of Paneth cell markers in addition to and independent of the effect of FGFR-3 on TCF4/β-catenin activity. These studies suggest that coordinate activation of multiple independent signaling pathways downstream of FGFR-3 is involved in regulation of Paneth cell differentiation.

Heterogeneity in expression of antimicrobial proteins in normal and crohn's disease small intestinal paneth cells

IntroductionPaneth cells are normally located at the base of small intestinal crypts and express a number of antimicrobial factors that are believed to be important in host protection against luminal...

Strain-specific polymorphisms in Paneth cell α-defensins of C57BL/6 mice and evidence of vestigial myeloid α-defensin pseudogenes.

Paneth cells at the base of small intestinal crypts secrete microbicidal α-defensins, termed cryptdins (Crps) in mice, as mediators of innate immunity. Proteomic studies show that five abundant Paneth cell α-defensins in C57BL/6 mice are strain specific in that they have not been identified in other inbred strains of mice. Two C57BL/6-specific peptides are coded for by the Defcr20 and -21 genes evident in the NIH C57BL/6 genome but absent from the Celera mixed-strain assembly, which excludes C57BL/6 data and differs from the NIH build with respect to the organization of the α-defensin gene locus. Conversely, C57BL/6 mice lack the Crp1, -2, -4, and -6 peptides and their corresponding Defcr1, -2, -4, and -6 genes, which are common to several mouse strains, including those of the Celera assembly. In C57BL/6 mice, α-defensin gene diversification appears to have occurred by tandem duplication of a multigene cassette that was not found in the mixed-strain assembly. Both mouse genome assemblies contain conserved α-defensin pseudogenes that are closely related to functional myeloid α-defensin genes in the rat, suggesting that the neutrophil α-defensin defect in mice resulted from progressive gene loss. Given the role of α-defensins in shaping the composition of the enteric microflora, such polymorphisms may influence outcomes in mouse models of disease or infection.

Intestinal Crypt Homeostasis Results from Neutral Competition between Symmetrically Dividing Lgr5 Stem Cells

Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5(hi) stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5(hi) cells. These reveal that most Lgr5(hi) cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5(hi) cell division adopt divergent fates (i.e., one Lgr5(hi) cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics.