The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5+) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and to allow for a response to tissue injury. Here, we identify a subpopulation of slowly cycling ISCs marked by mouse telomerase reverse transcriptase (mTert) expression that can give rise to Lgr5+ cells. mTert-expressing cells distribute in a pattern along the crypt–villus axis similar to long-term label-retaining cells (LRCs) and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert+ cells give rise to all differentiated intestinal cell types, persist long term, and contribute to the regenerative response following injury. Consistent with other highly regenerative tissues, our results demonstrate that a slowly cycling stem cell population exists within the intestine.

The peptide hormone, glucagon-like peptide 1 (GLP-1), has been shown to increase glucose-dependent insulin secretion, enhance insulin gene transcription, expand islet cell mass, and inhibit beta-cell apoptosis in animal models of diabetes. The aim of the present study was to evaluate whether GLP-1 could improve function and inhibit apoptosis in freshly isolated human islets. Human islets were cultured for 5 d in the presence, or absence, of GLP-1 (10 nm, added every 12 h) and studied for viability and expression of proapoptotic (caspase-3) and antiapoptotic factors (bcl-2) as well as glucose-dependent insulin production. We observed better-preserved three-dimensional islet morphology in the GLP-1-treated islets, compared with controls. Nuclear condensation, a feature of cell apoptosis, was inhibited by GLP-1. The reduction in the number of apoptotic cells in GLP-1-treated islets was particularly evident at d 3 (6.1% apoptotic nuclei in treated cultures vs. 15.5% in controls; P < 0.01) and at d 5 (8.9 vs. 18.9%; P < 0.01). The antiapoptotic effect of GLP-1 was associated with the down-regulation of active caspase-3 (P < 0.001) and the up-regulation of bcl-2 (P < 0.01). The effect of GLP-1 on the intracellular levels of bcl-2 and caspase-3 was observed at the mRNA and protein levels. Intracellular insulin content was markedly enhanced in islets cultured with GLP-1 vs. control (P < 0.001, at d 5), and there was a parallel GLP-1-dependent potentiation of glucose-dependent insulin secretion (P < 0.01 at d 3; P < 0.05 at d 5). Our findings provide evidence that GLP-1 added to freshly isolated human islets preserves morphology and function and inhibits cell apoptosis.

A constant remodeling of islet cell mass mediated by proliferative and apoptotic stimuli ensures a dynamic response to a changing demand for insulin. In this study, we investigated the effect of glucagon-like peptide-1 (GLP-1) in Zucker diabetic rats, an animal model in which the onset of diabetes occurs when the proliferative potential and the rate of beta-cell apoptosis no longer compensate for the increased demand for insulin. We subjected diabetic rats to a 2-d infusion of GLP-1 and tested their response to an ip glucose tolerance test. GLP-1 produced a significant increase of insulin secretion, which was paralleled by a decrease in plasma glucose levels (P < 0.001 and P < 0.01, respectively). Four days after the removal of the infusion pumps, rats were killed and the pancreas harvested to study the mechanism by which GLP-1 ameliorated glucose tolerance. Ex vivo immunostaining with the marker of cell proliferation, Ki-67, showed that the metabolic changes observed in rats treated with GLP-1 were associated with an increase in cell proliferation of the endocrine and exocrine component of the pancreas. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining, a marker of cellular apoptosis, indicated a reduction of apoptotic cells within the islet as well in the exocrine pancreas in GLP-1-treated rats. Double immunostaining for the apoptotic marker caspase-3 and for insulin showed a significant reduction of caspase-3 expression and an increase in insulin content in GLP-1-treated animals. Finally, staining of pancreatic sections with the nuclear dye 4,6-Diaminidino-2-phenyl-dihydrochloride demonstrated a marked reduction of fragmented nuclei in the islet cells of rats treated with GLP-1. Our findings provide evidence that the beneficial effects of GLP-1 in Zucker diabetic rats is mediated by an increase in islet cell proliferation and a decrease of cellular apoptosis.

Stem cells hold great promise for regenerative medicine, but remain elusive in many tissues in part because universal markers of ''stemness'' have not been identified. The ribonucleoprotein complex telomerase catalyzes the extension of chromosome ends, and its expression is associated with failure of cells to undergo cellular senescence. Because such resistance to senescence is a common characteristic of many stem cells, we hypothesized that telomerase expression may provide a selective biomarker for stem cells in multiple tissues. In fact, telomerase expression has been demonstrated within hematopoietic stem cells. We therefore generated mouse telomerase reverse transcriptase (mTert)-GFP-transgenic mice and assayed the ability of mTert-driven GFP to mark tissue stem cells in testis, bone marrow (BM), and intestine. mTert-GFP mice were generated by using a two-step embryonic stem cell-based strategy, which enabled primary and secondary screening of stably transfected clones before blastocyst injection, greatly increasing the probability of obtaining mTert reporter mice with physiologically appropriate regulation of GFP expression. Analysis of adult mice showed that GFP is expressed in differentiating male germ cells, is enriched among BM-derived hematopoietic stem cells, and specifically marks long-term BrdU-retaining intestinal crypt cells. In addition, telomerase-expressing GFP ؉ BM cells showed longterm, serial, multilineage BM reconstitution, fulfilling the functional definition of hematopoietic stem cells. Together, these data provide direct evidence that mTert-GFP expression marks progenitor cells in blood and small intestine, validating these mice as a useful tool for the prospective identification, isolation, and functional characterization of progenitor/stem cells from multiple tissues.intestinal stem cell ͉ telomerase ͉ tissue stem cells