Prolonged oral antimicrobial administration prevents doxorubicin-induced loss of active intestinal stem cells.

Acute intestinal mucositis is a common off-target effect of chemotherapy, leading to co-morbidities such as vomiting, diarrhea, sepsis, and death. We previously demonstrated that the presence of enteric bacteria modulates the extent of jejunal epithelial damage induced by doxorubicin (DXR) in mice. Despite conventional thinking of the crypt as a sterile environment, recent evidence suggests that bacterial signaling influences aISC function. In this study, we labeled aISCs using transgenic Lgr5-driven fluorescence or with immunostaining for OLFM4. We examined the effect of DXR in both germ free (GF) mice and mice depleted of microbiota using an established antimicrobial treatment protocol (AMBx). We found differences in DXR-induced loss of aISCs between GF mice and mice treated with AMBx. aISCs were decreased after DXR in GF mice, whereas AMBx mice retained aISC expression after DXR. Neither group of mice exhibited an inflammatory response to DXR, suggesting the difference in aISC retention was not due to differences in local tissue inflammation. Therefore, we suspected that there was a protective microbial signal present in the AMBx mice that was not present in the GF mice. 16S rRNA sequencing of jejunal luminal contents demonstrated that AMBx altered the fecal and jejunal microbiota. In the jejunal contents, AMBx mice had increased abundance of Ureaplasma and Burkholderia. These results suggest pro-survival signaling from microbiota in AMBx-treated mice to the aISCs, and that this signaling maintains aISCs in the face of chemotherapeutic injury. Manipulation of the enteric microbiota presents a therapeutic target for reducing the severity of chemotherapy-associated mucositis.

Multiomic analysis defines the first microRNA atlas across all small intestinal epithelial lineages and reveals novel markers of almost all major cell types.

MicroRNA-mediated regulation is critical for the proper development and function of the small intestinal (SI) epithelium. However, it is not known which microRNAs are expressed in each of the cell types of the SI epithelium. To bridge this important knowledge gap, we performed comprehensive microRNA profiling in all major cell types of the mouse SI epithelium. We used flow cytometry and fluorescence-activated cell sorting with multiple reporter mouse models to isolate intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, tuft cells, and secretory progenitors. We then subjected these cell populations to small RNA-sequencing. The resulting atlas revealed highly enriched microRNA markers for almost every major cell type (https://sethupathy-lab.shinyapps.io/SI_miRNA/). Several of these lineage-enriched microRNAs (LEMs) were observed to be embedded in annotated host genes. We used chromatin-run-on sequencing to determine which of these LEMs are likely cotranscribed with their host genes. We then performed single-cell RNA-sequencing to define the cell type specificity of the host genes and embedded LEMs. We observed that the two most enriched microRNAs in secretory progenitors are miR-1224 and miR-672, the latter of which we found is deleted in hominin species. Finally, using several in vivo models, we established that miR-152 is a Paneth cell-specific microRNA.NEW & NOTEWORTHY In this study, first, microRNA atlas (and searchable web server) across all major small intestinal epithelial cell types is presented. We have demonstrated microRNAs that uniquely mark several lineages, including enteroendocrine and tuft. Identification of a key marker of mouse secretory progenitor cells, miR-672, which we show is deleted in humans. We have used several in vivo models to establish miR-152 as a specific marker of Paneth cells, which are highly understudied in terms of microRNAs.

Secretory Sorcery: Paneth Cell Control of Intestinal Repair and Homeostasis.

Paneth cells are professional secretory cells that classically play a role in the innate immune system by secreting antimicrobial factors into the lumen to control enteric bacteria. In this role, Paneth cells are able to sense cues from luminal bacteria and respond by changing production of these factors to protect the epithelial barrier. Paneth cells rely on autophagy to regulate their secretory capability and capacity. Disruption of this pathway through mutation of genes, such as Atg16L1, results in decreased Paneth cell function, dysregulated enteric microbiota, decreased barrier integrity, and increased risk of diseases such as Crohn's disease in humans. Upon differentiation Paneth cells migrate downward and intercalate among active intestinal stem cells at the base of small intestinal crypts. This localization puts them in a unique position to interact with active intestinal stem cells, and recent work shows that Paneth cells play a critical role in influencing the intestinal stem cell niche. This review discusses the numerous ways Paneth cells can influence intestinal stem cells and their niche. We also highlight the ways in which Paneth cells can alter cells and other organ systems.

Epithelial Regeneration After Doxorubicin Arises Primarily From Early Progeny of Active Intestinal Stem Cells.

BACKGROUND & AIMS: aISCs (aISCs) are sensitive to acute insults including chemotherapy and irradiation. Regeneration after aISC depletion has primarily been explored in irradiation (IR). However, the cellular origin of epithelial regeneration after doxorubicin (DXR), a common chemotherapeutic, is poorly understood. METHODS: We monitored DXR's effect on aISCs by enumerating Lgr5-eGFP+ and Olfm4+ crypts, cleaved caspase-3 (CASP3+) immunofluorescence, and time-lapse organoid imaging. Lineage tracing from previously identified regenerative cell populations (Bmi1+, Hopx+, Dll1+, and Defa6+) was performed with DXR damage. Lineage tracing from aISCs was compared with lineage tracing from early progeny cells (transit-amplifying cells arising from aISCs 1 day predamage) in the context of DXR and IR. We compared stem cell and DNA damage response (DDR) transcripts in isolated aISCs and early progeny cells 6 and 24 hours after DXR. RESULTS: Epithelial regeneration after DXR primarily arose from early progeny cells generated by aISCs. Early progeny cells upregulated stem cell gene expression and lacked apoptosis induction (6 hours DXR: 2.5% of CASP3+ cells, p<0.0001). aISCs downregulated stem cell gene expression and underwent rapid apoptosis (6 hours DXR: 63.4% of CASP3+ cells). There was minimal regenerative contribution from Bmi1+, Hopx+, Dll1+, and Defa6+-expressing populations. In homeostasis, 48.4% of early progeny cells were BrdU+, and expressed low levels of DDR transcripts. CONCLUSIONS: We show that DXR effectively depleted aISCs in the small intestine and subsequent epithelial regeneration depended on nonquiescent early progeny cells of aISCs. The chemoresistant phenotype of the early progeny cells may rely on a dampened DDR in contrast to aISCs' robust DDR, which facilitates expeditious apoptosis.

Doxorubicin increases permeability of murine small intestinal epithelium and cultured T84 monolayers.

Enteric bacteria and/or their products are necessary for doxorubicin (DXR)-induced small intestine mucosal damage. While DXR does not induce gross loss of epithelium, others have shown elevated serum endotoxin after DXR administration. However, the mechanism of movement is unknown. We hypothesized that DXR treatment resulted in increased paracellular translocation of bacteria or bacterial products through the small intestinal epithelium. We measured permeability after DXR administration using transepithelial resistance and macromolecular flux and assessed tight junctional gene expression and protein localization both in vitro using T84 cells and ex vivo using murine jejunum. DXR treatment increased flux of 4 kDa dextrans in mouse jejenum, but increased flux of 4, 10 and 20 kDa dextrans in T84 cells. Following DXR, we observed increased permeability, both in vitro and ex vivo, independent of bacteria. DXR induced increased expression of Cldn2 and Cldn4 in murine small intestine but increased only CLDN2 expression in T84 cells. DXR treatment induced disorganization of tight junctional proteins. We conclude that DXR increases paracellular transit of small macromolecules, including bacterial products, through the epithelium, by altering expression of tight junctional components and dynamic loosening of cellular tight junctions.

Enteroendocrine Progenitor Cell-Enriched miR-7 Regulates Intestinal Epithelial Proliferation in an Xiap-Dependent Manner.

BACKGROUND & AIMS: The enteroendocrine cell (EEC) lineage is important for intestinal homeostasis. It was recently shown that EEC progenitors contribute to intestinal epithelial growth and renewal, but the underlying mechanisms remain poorly understood. MicroRNAs are under-explored along the entire EEC lineage trajectory, and comparatively little is known about their contributions to intestinal homeostasis. METHODS: We leverage unbiased sequencing and eight different mouse models and sorting methods to identify microRNAs enriched along the EEC lineage trajectory. We further characterize the functional role of EEC progenitor-enriched miRNA, miR-7, by in vivo dietary study as well as ex vivo enteroid in mice. RESULTS: First, we demonstrate that miR-7 is highly enriched across the entire EEC lineage trajectory and is the most enriched miRNA in EEC progenitors relative to Lgr5+ intestinal stem cells. Next, we show in vivo that in EEC progenitors miR-7 is dramatically suppressed under dietary conditions that favor crypt division and suppress EEC abundance. We then demonstrate by functional assays in mouse enteroids that miR-7 exerts robust control of growth, as determined by budding (proxy for crypt division), EdU and PH3 staining, and likely regulates EEC abundance also. Finally, we show by single-cell RNA sequencing analysis that miR-7 regulates Xiap in progenitor/stem cells and we demonstrate in enteroids that the effects of miR-7 on mouse enteroid growth depend in part on Xiap and Egfr signaling. CONCLUSIONS: This study demonstrates for the first time that EEC progenitor cell-enriched miR-7 is altered by dietary perturbations and that it regulates growth in enteroids via intact Xiap and Egfr signaling.

Mist1 Expression Is Required for Paneth Cell Maturation.

BACKGROUND: Paneth cells are professional secretory cells found within the small intestinal crypt epithelium. Although their role as part of the innate immune complex providing antimicrobial secretory products is well-known, the mechanisms that control secretory capacity are not well-understood. MIST1 is a scaling factor that is thought to control secretory capacity of exocrine cells. METHODS: Mist1+/+ and Mist1-/- mice were used to evaluate the function of MIST1 in small intestinal Paneth cells. We used histologic and immunofluorescence staining to evaluate small intestinal tissue for proliferation and lineage allocation. Total RNA was isolated to evaluate gene expression. Enteroid culture was used to evaluate the impact of the absence of MIST1 expression on intestinal stem cell function. RESULTS: Absence of MIST1 resulted in increased numbers of Paneth cells exhibiting an intermediate cell phenotype but otherwise did not alter overall epithelial cell lineage allocation. Muc2 and lysozyme staining confirmed the presence of intermediate cells at the crypt base of Mist1-/- mice. These changes were not associated with changes in mRNA expression of transcription factors associated with lineage allocation, and they were not abrogated by inhibition of Notch signaling. However, the absence of MIST1 expression was associated with alterations in Paneth cell morphology including decreased granule size and distended rough endoplasmic reticulum. Absence of MIST1 was associated with increased budding of enteroid cultures; however, there was no evidence of increased intestinal stem cell numbers in vivo. CONCLUSIONS: MIST1 plays an important role in organization of the Paneth cell secretory apparatus and managing endoplasmic reticulum stress. This role occurs downstream of Paneth cell lineage allocation.

Cellular Plasticity of Defa4Cre-Expressing Paneth Cells in Response to Notch Activation and Intestinal Injury.

BACKGROUND & AIMS: Loss of leucine-rich repeat-containing G-protein-coupled receptor 5-positive crypt base columnar cells provides permissive conditions for different facultative stem cell populations to dedifferentiate and repopulate the stem cell compartment. In this study, we used a defensin α4-Cre recombinase (Defa4Cre) line to define the potential of Paneth cells to dedifferentiate and contribute to intestinal stem cell (ISC) maintenance during normal homeostasis and after intestinal injury. METHODS: Small intestine and enteroids from Defa4Cre;Rosa26 tandem dimer Tomato (tdTomato), a red fluoresent protein, (or Rosa26 Enhanced Yellow Fluorescent Protein (EYFP)) reporter, Notch gain-of-function (Defa4Cre;Rosa26 Notch Intracellular Domain (NICD)-ires-nuclear Green Fluorescent Protein (nGFP) and Defa4Cre;Rosa26reverse tetracycline transactivator-ires Enhanced Green Fluorescent Protein (EGFP);TetONICD), A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) loss-of-function (Defa4Cre;ADAM10flox/flox), and Adenomatous polyposis coli (APC) inactivation (Defa4Cre;APCflox/flox) mice were analyzed. Doxorubicin treatment was used as an acute intestinal injury model. Lineage tracing, proliferation, and differentiation were assessed in vitro and in vivo. RESULTS: Defa4Cre-expressing cells are fated to become mature Paneth cells and do not contribute to ISC maintenance during normal homeostasis in vivo. However, spontaneous lineage tracing was observed in enteroids, and fluorescent-activated cell sorter-sorted Defa4Cre-marked cells showed clonogenic enteroid growth. Notch activation in Defa4Cre-expressing cells caused dedifferentiation to multipotent ISCs in vivo and was required for adenoma formation. ADAM10 deletion had no significant effect on crypt homeostasis. However, after acute doxorubicin-induced injury, Defa4Cre-expressing cells contributed to regeneration in an ADAM10-Notch-dependent manner. CONCLUSIONS: Our studies have shown that Defa4Cre-expressing Paneth cells possess cellular plasticity, can dedifferentiate into multipotent stem cells upon Notch activation, and can contribute to intestinal regeneration in an acute injury model.

Preservation of reserve intestinal epithelial stem cells following severe ischemic injury.

Intestinal ischemia is an abdominal emergency with a mortality rate >50%, leading to epithelial barrier loss and subsequent sepsis. Epithelial renewal and repair after injury depend on intestinal epithelial stem cells (ISC) that reside within the crypts of Lieberkühn. Two ISC populations critical to epithelial repair have been described: 1) active ISC (aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 positive, sex determining region Y-box 9 positive) and 2) reserve ISC [rISC; less proliferative; homeodomain only protein X (Hopx)+]. Yorkshire crossbred pigs (8-10 wk old) were subjected to 1-4 h of ischemia and 1 h of reperfusion or recovery by reversible mesenteric vascular occlusion. This study was designed to evaluate whether ISC-expressing biomarkers of aISCs or rISCs show differential resistance to ischemic injury and different contributions to the subsequent repair and regenerative responses. Our data demonstrate that, following 3-4 h ischemic injury, aISC undergo apoptosis, whereas rISC are preserved. Furthermore, these rISC are retained ex vivo in spheroids in which cell populations are enriched in the rISC biomarker Hopx. These cells appear to go on to provide a proliferative pool of cells during the recovery period. Taken together, these data indicate that Hopx+ cells are resistant to injury and are the likely source of epithelial renewal following prolonged ischemic injury. It is therefore possible that targeting reserve stem cells will lead to new therapies for patients with severe intestinal injury. NEW & NOTEWORTHY The population of reserve less-proliferative intestinal epithelial stem cells appears resistant to injury despite severe epithelial cell loss, including that of the active stem cell population, which results from prolonged mesenteric ischemia. These cells can change to an activated state and are likely indispensable to regenerative processes. Reserve stem cell targeted therapies may improve treatment and outcome of patients with ischemic disease.

Depletion of enteric bacteria diminishes leukocyte infiltration following doxorubicin-induced small intestinal damage in mice.

BACKGROUND & AIMS: While enteric bacteria have been shown to play a critical role in other forms of intestinal damage, their role in mediating the response to the chemotherapeutic drug Doxorubicin (Doxo) is unclear. In this study, we used a mouse model of intestinal bacterial depletion to evaluate the role enteric bacteria play in mediating Doxo-induced small intestinal damage and, more specifically, in mediating chemokine expression and leukocyte infiltration following Doxo treatment. An understanding of this pathway may allow for development of intervention strategies to reduce chemotherapy-induced small intestinal damage. METHODS: Mice were treated with (Abx) or without (NoAbx) oral antibiotics in drinking water for four weeks and then with Doxo. Jejunal tissues were collected at various time points following Doxo treatment and stained and analyzed for apoptosis, crypt damage and restitution, and macrophage and neutrophil number. In addition, RNA expression of inflammatory markers (TNFα, IL1-ß, IL-10) and cytokines (CCL2, CC7, KC) was assessed by qRT-PCR. RESULTS: In NoAbx mice Doxo-induced damage was associated with rapid induction of apoptosis in jejunal crypt epithelium and an increase weight loss and crypt loss. In addition, we observed an increase in immune-modulating chemokines CCL2, CCL7 and KC and infiltration of macrophages and neutrophils. In contrast, while still positive for induction of apoptosis following Doxo treatment, Abx mice showed neither the overall weight loss nor crypt loss seen in NoAbx mice nor the increased chemokine expression and leukocyte infiltration. CONCLUSION: Enteric bacteria play a critical role in Doxo-induced small intestinal damage and are associated with an increase in immune-modulating chemokines and cells. Manipulation of enteric bacteria or the damage pathway may allow for prevention or treatment of chemotherapy-induced small intestinal damage.

Intestinal bacteria are necessary for doxorubicin-induced intestinal damage but not for doxorubicin-induced apoptosis.

Doxorubicin (DOXO) induces significant, but transient, increases in apoptosis in the stem cell zone of the jejunum, followed by mucosal damage involving a decrease in crypt proliferation, crypt number, and villus height. The gastrointestinal tract is home to a vast population of commensal bacteria and numerous studies have demonstrated a symbiotic relationship between intestinal bacteria and intestinal epithelial cells (IEC) in maintaining homeostatic functions of the intestine. However, whether enteric bacteria play a role in DOXO-induced damage is not well understood. We hypothesized that enteric bacteria are necessary for induction of apoptosis and damage associated with DOXO treatment. Conventionally raised (CONV) and germ free (GF) mice were given a single injection of DOXO, and intestinal tissue was collected at 6, 72, and 120 h after treatment and from no treatment (0 h) controls. Histology and morphometric analyses quantified apoptosis, mitosis, crypt depth, villus height, and crypt density. Immunostaining for muc2 and lysozyme evaluated Paneth cells, goblet cells or dual stained intermediate cells. DOXO administration induced significant increases in apoptosis in jejunal epithelium regardless of the presence of enteric bacteria; however, the resulting injury, as demonstrated by statistically significant changes in crypt depth, crypt number, and proliferative cell number, was dependent upon the presence of enteric bacteria. Furthermore, we observed expansion of Paneth and goblet cells and presence of intermediate cells only in CONV and not GF mice. These findings provide evidence that manipulation and/or depletion of the enteric microbiota may have clinical significance in limiting chemotherapy-induced mucositis.

Tissue underlying the intestinal epithelium elicits proliferation of intestinal stem cells following cytotoxic damage.

The goals of this study were to document the proliferative response of intestinal stem cells (ISCs) during regeneration after damage from doxorubicin (DXR), and to characterize the signals responsible for ISC activation. To this end, jejuni from DXR-treated mice were harvested for histology, assessment of ISC numbers and proliferation by flow cytometry, crypt culture, and RNA analyses. Histology showed that crypt depth and width were increased 4 days after DXR. At this time point, flow cytometry on tissue collected 1 h after EdU administration revealed increased numbers of CD24(lo)UEA(-) ISCs and increased percentage of ISCs cycling. In culture, crypts harvested from DXR-treated mice were equally proliferative as those of control mice. Addition of subepithelial intestinal tissue (SET) collected 4 days after DXR elicited increased budding (1.4 ± 0.3 vs. 5.1 ± 1.0 buds per enteroid). Microarray analysis of SET collected 4 days after DXR revealed 1030 differentially expressed transcripts. Cross-comparison of Gene Ontology terms considered relevant to ISC activation pointed to 10 candidate genes. Of these, the epidermal growth factor (EGF) family member amphiregulin and the BMP antagonist chordin-like 2 were chosen for further study. In crypt culture, amphiregulin alone did not elicit significant budding, but amphiregulin in combination with BMP antagonism showed marked synergism (yielding 6.3 ± 0.5 buds per enteroid). These data suggest a critical role for underlying tissue in regulating ISC behavior after damage, and point to synergism between amphiregulin and chordin-like 2 as factors which may account for activation of ISCs in the regenerative phase.

Paneth cells expand from newly created and preexisting cells during repair after doxorubicin-induced damage.

Paneth cell numbers increase following intestinal damage, but mechanisms driving this process are not understood. We hypothesized that the increase in Paneth cell numbers is due to recruitment of cells from a preexisting pool of secretory progenitors. Mice were given a single injection of doxorubicin (Dox), and intestinal tissue was collected 0-168 h after treatment. Paneth, goblet, and intermediate cells were counted and evaluated for cell morphology. Quantitative RT-PCR was used to measure expression of various genes associated with Paneth cell allocation and maturation. Paneth cells were birth dated using incorporation of thymidine analogs given before or after Dox. Staining revealed "intermediate" cells, which were rarely observed in control crypts but increased significantly in number 96 and 120 h after Dox treatment. Birth dating of intermediate cells 5 days after Dox treatment revealed that 24% of these cells took up thymidine analog given prior to Dox treatment and 36% took up thymidine analog given after Dox treatment. Quantitative RT-PCR demonstrated a significant increase in Spdef, Atoh1, Sox9, EphB3, Mist, Wnt5a, FGF-9, and FGF-18 mRNAs and a significant decrease in Indian hedgehog mRNA. Expansion of the Paneth cell compartment after Dox treatment is due to generation of new cells and recruitment of cells from an existing pool. These cells express Paneth and goblet biomarkers and are found only during repair. Expansion of these cells correlates temporally with reduced Indian hedgehog and increased FGF and Wnt mRNA. These findings are significant, as they provide a first step in understanding mechanisms of Paneth cell expansion during mucosal repair.

Small intestinal stem cells.

PURPOSE OF REVIEW: To summarize our current understanding of small intestinal stem cell biology and the current tools available for studying intestinal stem cells (ISCs). RECENT FINDINGS: Recent reviews and original reports point toward the presence of two distinct populations of stem cells (ISCs) within the intestinal crypts. Until recently, the study of these two populations has been hindered by the lack of biomarkers available for isolation and characterization of ISCs and the absence of suitable culture conditions for expansion of ISCs in vitro. With the accumulation of various surface markers and transgenic mouse models, we have been able to gain a better understanding of the genetic signature of ISCs. In addition, these tools have provided opportunities to begin to study how ISCs are influenced by the various components of the ISC niche, including fibroblasts, bacteria, lymphoid cells, and Paneth cells. Advances in culture conditions now allow for the establishment of in-vitro studies of ISC function and dynamics. SUMMARY: This brief review provides a general historical perspective of our understanding of the delineation of the two ISC populations. Furthermore, it discusses the known ISC markers and how these markers have been used to isolate and characterize ISC populations.

Expansion of intestinal epithelial stem cells during murine development.

Murine small intestinal crypt development is initiated during the first postnatal week. Soon after formation, overall increases in the number of crypts occurs through a bifurcating process called crypt fission, which is believed to be driven by developmental increases in the number of intestinal stem cells (ISCs). Recent evidence suggests that a heterogeneous population of ISCs exists within the adult intestine. Actively cycling ISCs are labeled by Lgr5, Ascl2 and Olfm4; whereas slowly cycling or quiescent ISC are marked by Bmi1 and mTert. The goal of this study was to correlate the expression of these markers with indirect measures of ISC expansion during development, including quantification of crypt fission and side population (SP) sorting. Significant changes were observed in the percent of crypt fission and SP cells consistent with ISC expansion between postnatal day 14 and 21. Quantitative real-time polymerase chain reaction (RT-PCR) for the various ISC marker mRNAs demonstrated divergent patterns of expression. mTert surged earliest, during the first week of life as crypts are initially being formed, whereas Lgr5 and Bmi1 peaked on day 14. Olfm4 and Ascl2 had variable expression patterns. To assess the number and location of Lgr5-expressing cells during this period, histologic sections from intestines of Lgr5-EGFP mice were subjected to quantitative analysis. There was attenuated Lgr5-EGFP expression at birth and through the first week of life. Once crypts were formed, the overall number and percent of Lgr5-EGFP positive cells per crypt remain stable throughout development and into adulthood. These data were supported by Lgr5 in situ hybridization in wild-type mice. We conclude that heterogeneous populations of ISCs are expanding as measured by SP sorting and mRNA expression at distinct developmental time points.

Regeneration of intestinal stem/progenitor cells following doxorubicin treatment of mice.

The intestinal epithelium is in a constant state of renewal. The rapid turnover of cells is fed by a hierarchy of transit amplifying and stem/progenitor cells destined to give rise to the four differentiated epithelial lineages of the small intestine. Doxorubicin (Dox) is a commonly used chemotherapeutic agent that preferentially induces apoptosis in the intestinal stem cell zone (SCZ). We hypothesized that Dox treatment would initially decrease "+4" intestinal stem cell numbers with a subsequent expansion during mucosal repair. Temporal assessment following Dox treatment demonstrated rapid induction of apoptosis in the SCZ leading to a decrease in the number of intestinal stem/progenitor cells as determined by flow cytometry for CD45(-) SP cells, and immunohistochemistry of cells positive for putative +4 stem cell markers beta-cat(Ser552) and DCAMKL1. Between 96 and 168 h postinjection, overall proliferation in the crypts increased concomitant with increases in both absolute and relative numbers of goblet, Paneth, and enteroendocrine cells. This regeneration phase was also associated with increases of CD45(-) SP cells, beta-cat(Ser552)-positive cells, crypt fission, and crypt number. We used Lgr5-lacZ mice to assess behavior of Lgr5-positive stem cells following Dox and found no change in this cell population. Lgr5 mRNA level was also measured and showed no change immediately after Dox but decreased during the regeneration phase. Together these data suggest that, following Dox-induced injury, expansion of intestinal stem cells occurs during mucosal repair. On the basis of available markers this expansion appears to be predominantly the +4 stem cell population rather than those of the crypt base.

Intestinal stem cells.

The epithelial cell lining of the gastrointestinal tract is the most rapidly proliferating tissue in the body. The constant state of renewal of differentiated epithelial cells is sustained by a continual supply of progeny from multipotent progenitors that originate from stem cells located within the intestinal crypts. In addition to supporting normal epithelial homeostasis, intestinal stem cells (ISC) are thought to play an important role in the rapid expansion of the gut during development, tissue regeneration following injury or surgical loss, and malignancy. Because of the lack of specific ISC markers required to isolate and characterize these cells, our current knowledge of the biology of ISC results largely from indirect measures of their behavior published during the past 40 years. The recent description of several potential ISC markers and the use of transgenic mice, both as a tool to lineage trace and to isolate specific cells expressing these markers, have provided a tremendous advancement to our current understanding of these cells. This brief review provides a general historical overview of our understanding of ISC and the tools available to study their behavior in the context of normal and pathological conditions, as well as potential future clinical applications that may result from this exciting area of research.

Early but not late administration of glucagon-like peptide-2 following ileo-cecal resection augments putative intestinal stem cell expansion.

Expansion of intestinal progenitors and putative stem cells (pISC) occurs early and transiently following ileo-cecal resection (ICR). The mechanism controlling this process is not defined. We hypothesized that glucagon-like peptide-2 (GLP-2) would augment jejunal pISC expansion only when administered to mice immediately after ICR. Since recent reports demonstrated increases in intestinal insulin-like growth factor (IGF)-I following GLP-2 administration, we further hypothesized that increased intestinal IGF-I expression would correlate with pISC expansion following ICR. To assess this, GLP-2 or vehicle was administered to mice either immediately after resection (early) or before tissue harvest 6 wk following ICR (late). Histological analysis quantified proliferation and intestinal morphometrics. Serum levels of GLP-2 were measured by ELISA and jejunal IGF-I mRNA by qRT-PCR. Expansion of jejunal pISC was assessed by fluorescent-activated cell sorting of side population cells, immunohistochemistry for phosphorylated beta-catenin at serine 552 (a pISC marker), percent of crypt fission, and total numbers of crypts per jejunal circumference. We found that early but not late GLP-2 treatment after ICR significantly augmented pISC expansion. Increases in jejunal IGF-I mRNA correlated temporally with early pISC expansion and effects of GLP-2. Early GLP-2 increased crypt fission and accelerated adaptive increases in crypt number and intestinal caliber. GLP-2 increased proliferation and intestinal morphometrics in all groups. This study shows that, in mice, GLP-2 promotes jejunal pISC expansion only in the period immediately following ICR. This is associated with increased IGF-I and accelerated adaptive increases in mucosal mass. These data provide clinical rationale relevant to the optimal timing of GLP-2 in patients with intestinal failure.