Prox1-positive cells monitor and sustain the murine intestinal epithelial cholinergic niche

The enteric neurotransmitter acetylcholine governs important intestinal epithelial secretory and immune functions through its actions on epithelial muscarinic Gq-coupled receptors such as M3R. Its role in the regulation of intestinal stem cell function and differentiation, however, has not been clarified. Here, we find that nonselective muscarinic receptor antagonism in mice as well as epithelial-specific ablation of M3R induces a selective expansion of DCLK1-positive tuft cells, suggesting a model of feedback inhibition. Cholinergic blockade reduces Lgr5-positive intestinal stem cell tracing and cell number. In contrast, Prox1-positive endocrine cells appear as primary sensors of cholinergic blockade inducing the expansion of tuft cells, which adopt an enteroendocrine phenotype and contribute to increased mucosal levels of acetylcholine. This compensatory mechanism is lost with acute irradiation injury, resulting in a paucity of tuft cells and acetylcholine production. Thus, enteroendocrine tuft cells appear essential to maintain epithelial homeostasis following modifications of the cholinergic intestinal niche

BHLHA15-Positive Secretory Precursor Cells Can Give Rise to Tumors in Intestine and Colon in Mice

Background & Aims: The intestinal epithelium is maintained by long-lived intestinal stem cells (ISCs) that reside near the crypt base. Above the ISC zone, there are short-lived progenitors that normally give rise to lineage-specific differentiated cell types but can dedifferentiate into ISCs in certain circumstances. However, the role of epithelial dedifferentiation in cancer development has not been fully elucidated. Methods: We performed studies with Bhlha15-CreERT, Lgr5-DTR-GFP, Apc flox/flox , LSL-Notch (IC), and R26-reporter strains of mice. Some mice were given diphtheria toxin to ablate Lgr5-positive cells, were irradiated, or were given 5-fluorouracil, hydroxyurea, doxorubicin, or dextran sodium sulfate to induce intestinal or colonic tissue injury. In intestinal tissues, we analyzed the fate of progeny that expressed Bhlha15. We used microarrays and reverse-transcription PCR to analyze gene expression patterns in healthy and injured intestinal tissues and in tumors. We analyzed gene expression patterns in human colorectal tumors using The Cancer Genome Atlas data set. Results: Bhlha15 identified Paneth cells and short-lived secretory precursors (including pre-Paneth label-retaining cells) located just above the ISC zone in the intestinal epithelium. Bhlha15 + cells had no plasticity after loss of Lgr5-positive cells or irradiation. However, Bhlha15 + secretory precursors started to supply the enterocyte lineage after doxorubicin-induced epithelial injury in a Notch-dependent manner. Sustained activation of Notch converts Bhlha15 + secretory precursors to long-lived enterocyte progenitors. Administration of doxorubicin and expression of an activated form of Notch resulted in a gene expression pattern associated with enterocyte progenitors, whereas only sustained activation of Notch altered gene expression patterns in Bhlha15 + precursors toward those of ISCs. Bhlha15 + enterocyte progenitors with sustained activation of Notch formed intestinal tumors with serrated features in mice with disruption of Apc. In the colon, Bhlha15 marked secretory precursors that became stem-like, cancer-initiating cells after dextran sodium sulfate–induced injury, via activation of Src and YAP signaling. In analyses of human colorectal tumors, we associated activation of Notch with chromosome instability-type tumors with serrated features in the left colon. Conclusions: In mice, we found that short-lived precursors can undergo permanent reprogramming by activation of Notch and YAP signaling. These cells could mediate tumor formation in addition to traditional ISCs

Nerve Growth Factor Promotes Gastric Tumorigenesis through Aberrant Cholinergic Signaling

Within the gastrointestinal stem cell niche, nerves help to regulate both normal and neoplastic stem cell dynamics. Here, we reveal the mechanisms underlying the cancer-nerve partnership. We find that Dclk1+ tuft cells and nerves are the main sources of acetylcholine (ACh) within the gastric mucosa. Cholinergic stimulation of the gastric epithelium induced nerve growth factor (NGF) expression, and in turn NGF overexpression within gastric epithelium expanded enteric nerves and promoted carcinogenesis. Ablation of Dclk1+ cells or blockade of NGF/Trk signaling inhibited epithelial proliferation and tumorigenesis in an ACh muscarinic receptor-3 (M3R)-dependent manner, in part through suppression of yes-associated protein (YAP) function. This feedforward ACh-NGF axis activates the gastric cancer niche and offers a compelling target for tumor treatment and prevention

Bone Marrow Myeloid Cells Regulate Myeloid-Biased Hematopoietic Stem Cells via a Histamine-Dependent Feedback Loop

Myeloid-biased hematopoietic stem cells (MB-HSCs) play critical roles in recovery from injury, but little is known about how they are regulated within the bone marrow niche. Here we describe an auto-/paracrine physiologic circuit that controls quiescence of MB-HSCs and hematopoietic progenitors marked by histidine decarboxylase (Hdc). Committed Hdc+ myeloid cells lie in close anatomical proximity to MB-HSCs and produce histamine, which activates the H2 receptor on MB-HSCs to promote their quiescence and self-renewal. Depleting histamine-producing cells enforces cell cycle entry, induces loss of serial transplant capacity, and sensitizes animals to chemotherapeutic injury. Increasing demand for myeloid cells via lipopolysaccharide (LPS) treatment specifically recruits MB-HSCs and progenitors into the cell cycle; cycling MB-HSCs fail to revert into quiescence in the absence of histamine feedback, leading to their depletion, while an H2 agonist protects MB-HSCs from depletion after sepsis. Thus, histamine couples lineage-specific physiological demands to intrinsically primed MB-HSCs to enforce homeostasis. Chen et al. show that histidine decarboxylase (Hdc) marks quiescent myeloid-biased HSCs (MB-HSCs). Daughter myeloid cells form a spatial cluster with Hdc+ MB-HSCs and secrete histamine to enforce their quiescence and protect them from depletion, following activation by a variety of physiologic insults

Three-Dimensional Gastrointestinal Organoid Culture in Combination with Nerves or Fibroblasts : A Method to Characterize the Gastrointestinal Stem Cell Niche

The gastrointestinal epithelium is characterized by a high turnover of cells and intestinal stem cells predominantly reside at the bottom of crypts and their progeny serve to maintain normal intestinal homeostasis. Accumulating evidence demonstrates the pivotal role of a niche surrounding intestinal stem cells in crypts, which consists of cellular and soluble components and creates an environment constantly influencing the fate of stem cells. Here we describe different 3D culture systems to culture gastrointestinal epithelium that should enable us to study the stem cell niche in vitro in the future: organoid culture and multilayered systems such as organotypic cell culture and culture of intestinal tissue fragments ex vivo. These methods mimic the in vivo situation in vitro by creating 3D culture conditions that reflect the physiological situation of intestinal crypts. Modifications of the composition of the culture media as well as coculturing epithelial organoids with previously described cellular components such as myofibroblasts, collagen, and neurons show the impact of the methods applied to investigate niche interactions in vitro. We further present a novel method to isolate labeled nerves from the enteric nervous system using Dclk1-CreGFP mice

Hemostatic therapy in experimental intracerebral hemorrhage associated with the direct thrombin inhibitor dabigatran

Background and Purpose: Dabigatran-etexilate (DE) recently has been approved for stroke prevention in atrial fibrillation. However, lack of effective antagonists represents a major concern in the event of intracerebral hemorrhage (ICH). The aims of the present study were to establish a murine model of ICH associated with dabigatran, and to test the efficacy of different hemostatic factors in preventing hematoma growth. Methods: In C57BL/6 mice receiving DE (4.5 or 9.0 mg/kg), in vivo and in vitro coagulation assays and dabigatran plasma levels were measured repeatedly. Thirty minutes after inducing ICH by striatal collagenase injection, mice received an intravenous injection of saline, prothrombin complex concentrate (PCC; 100 U/kg), murine fresh-frozen plasma (200 μL), or recombinant human factor VIIa (8.0 mg/kg). ICH volume was quantified on brain cryosections 24 hours later. Results: DE substantially prolonged tail vein bleeding time and ecarin clotting time for 4 hours correspondi