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1.
Cell ; 187(12): 3056-3071.e17, 2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38848678

ABSTRACT

The currently accepted intestinal epithelial cell organization model proposes that Lgr5+ crypt-base columnar (CBC) cells represent the sole intestinal stem cell (ISC) compartment. However, previous studies have indicated that Lgr5+ cells are dispensable for intestinal regeneration, leading to two major hypotheses: one favoring the presence of a quiescent reserve ISC and the other calling for differentiated cell plasticity. To investigate these possibilities, we studied crypt epithelial cells in an unbiased fashion via high-resolution single-cell profiling. These studies, combined with in vivo lineage tracing, show that Lgr5 is not a specific ISC marker and that stemness potential exists beyond the crypt base and resides in the isthmus region, where undifferentiated cells participate in intestinal homeostasis and regeneration following irradiation (IR) injury. Our results provide an alternative model of intestinal epithelial cell organization, suggesting that stemness potential is not restricted to CBC cells, and neither de-differentiation nor reserve ISC are drivers of intestinal regeneration.


Subject(s)
Homeostasis , Intestinal Mucosa , Receptors, G-Protein-Coupled , Regeneration , Stem Cells , Animals , Stem Cells/metabolism , Stem Cells/cytology , Mice , Intestinal Mucosa/metabolism , Receptors, G-Protein-Coupled/metabolism , Intestines/cytology , Cell Differentiation , Mice, Inbred C57BL , Epithelial Cells/metabolism , Single-Cell Analysis , Male
2.
STAR Protoc ; 5(1): 102836, 2024 Mar 15.
Article in English | MEDLINE | ID: mdl-38219150

ABSTRACT

Here, we present a protocol for rapidly isolating single cells from the mouse pancreas, minimizing damage caused by digestive enzymes in exocrine cells. We guide you through steps to optimize the dissection sequence, enzyme composition, and operational procedures, resulting in high yields of viable pancreatic single cells. This protocol can be applied across a wide range of research areas, including single-cell sequencing, gene expression profiling, primary cell culture, and even the development of spheroids or organoids. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2023).1.


Subject(s)
Pancreas , Pancreatic Hormones , Animals , Mice , Dissection , Epithelial Cells , Gene Expression Profiling
3.
Am J Physiol Gastrointest Liver Physiol ; 322(6): G583-G597, 2022 06 01.
Article in English | MEDLINE | ID: mdl-35319286

ABSTRACT

Intestinal ganglionic cells in the adult enteric nervous system (ENS) are continually exposed to stimuli from the surrounding microenvironment and need at times to respond to disturbed homeostasis following acute intestinal injury. The kinase DCLK1 and intestinal Dclk1-positive cells have been reported to contribute to intestinal regeneration. Although Dclk1-positive cells are present in adult enteric ganglia, their cellular identity and response to acute injury have not been investigated in detail. Here, we reveal the presence of distinct Dclk1-tdTom+/CD49b+ glial-like and Dclk1-tdTom+/CD49b- neuronal cell types in adult myenteric ganglia. These ganglionic cells demonstrate distinct patterns of tracing over time yet show a similar expansion in response to elevated serotonergic signaling. Interestingly, Dclk1-tdTom+ glial-like and neuronal cell types appear resistant to acute irradiation injury-mediated cell death. Moreover, Dclk1-tdTom+/CD49b+ glial-like cells show prominent changes in gene expression profiles induced by injury, in contrast to Dclk1-tdTom+/CD49b- neuronal cell types. Finally, subsets of Dclk1-tdTom+/CD49b+ glial-like cells demonstrate prominent overlap with Nestin and p75NTR and strong responses to elevated serotonergic signaling or acute injury. These findings, together with their role in early development and their neural crest-like gene expression signature, suggest the presence of reserve progenitor cells in the adult Dclk1 glial cell lineage.NEW & NOTEWORTHY The kinase DCLK1 identifies glial-like and neuronal cell types in adult murine enteric ganglia, which resist acute injury-mediated cell death yet differ in their cellular response to injury. Interestingly, Dclk1-labeled glial-like cells show prominent transcriptional changes in response to injury and harbor features reminiscent of previously described enteric neural precursor cells. Our data thus add to recently emerging evidence of reserve cellular plasticity in the adult enteric nervous system.


Subject(s)
Enteric Nervous System , Neural Stem Cells , Animals , Enteric Nervous System/physiology , Integrin alpha2/metabolism , Mice , Mice, Transgenic , Neuroglia/metabolism , Neurons/metabolism
4.
Cell Mol Gastroenterol Hepatol ; 11(4): 1119-1138, 2021.
Article in English | MEDLINE | ID: mdl-33249238

ABSTRACT

BACKGROUND & AIMS: Histidine decarboxylase (HDC), the histamine-synthesizing enzyme, is expressed in a subset of myeloid cells but also marks quiescent myeloid-biased hematopoietic stem cells (MB-HSCs) that are activated upon myeloid demand injury. However, the role of MB-HSCs in dextran sulfate sodium (DSS)-induced acute colitis has not been addressed. METHODS: We investigated HDC+ MB-HSCs and myeloid cells by flow cytometry in acute intestinal inflammation by treating HDC-green fluorescent protein (GFP) male mice with 5% DSS at various time points. HDC+ myeloid cells in the colon also were analyzed by flow cytometry and immunofluorescence staining. Knockout of the HDC gene by using HDC-/-; HDC-GFP and ablation of HDC+ myeloid cells by using HDC-GFP; HDC-tamoxifen-inducible recombinase Cre system; diphtheria toxin receptor (DTR) mice was performed. The role of H2-receptor signaling in acute colitis was addressed by treatment of DSS-treated mice with the H2 agonist dimaprit dihydrochloride. Kaplan-Meier survival analysis was performed to assess the effect on survival. RESULTS: In acute colitis, rapid activation and expansion of MB-HSC from bone marrow was evident early on, followed by a gradual depletion, resulting in profound HSC exhaustion, accompanied by infiltration of the colon by increased HDC+ myeloid cells. Knockout of the HDC gene and ablation of HDC+ myeloid cells enhance the early depletion of HDC+ MB-HSC, and treatment with H2-receptor agonist ameliorates the depletion of MB-HSCs and resulted in significantly increased survival of HDC-GFP mice with acute colitis. CONCLUSIONS: Exhaustion of bone marrow MB-HSCs contributes to the progression of DSS-induced acute colitis, and preservation of quiescence of MB-HSCs by the H2-receptor agonist significantly enhances survival, suggesting the potential for therapeutic utility.


Subject(s)
Bone Marrow/pathology , Colitis/pathology , Hematopoietic Stem Cells/pathology , Histamine/metabolism , Histidine Decarboxylase/physiology , Inflammation/pathology , Intestines/pathology , Myeloid Cells/pathology , Animals , Bone Marrow/immunology , Bone Marrow/metabolism , Colitis/etiology , Colitis/metabolism , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Inflammation/etiology , Inflammation/metabolism , Intestines/immunology , Intestines/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Cells/immunology , Myeloid Cells/metabolism , Signal Transduction
5.
Cancer Discov ; 10(7): 1038-1057, 2020 07.
Article in English | MEDLINE | ID: mdl-32376773

ABSTRACT

To study genetic factors influencing the progression and therapeutic responses of advanced prostate cancer, we developed a fast and flexible system that introduces genetic alterations relevant to human disease directly into the prostate glands of mice using tissue electroporation. These electroporation-based genetically engineered mouse models (EPO-GEMM) recapitulate features of traditional germline models and, by modeling genetic factors linked to late-stage human disease, can produce tumors that are metastatic and castration-resistant. A subset of tumors with Trp53 alterations acquired spontaneous WNT pathway alterations, which are also associated with metastatic prostate cancer in humans. Using the EPO-GEMM approach and an orthogonal organoid-based model, we show that WNT pathway activation drives metastatic disease that is sensitive to pharmacologic WNT pathway inhibition. Thus, by leveraging EPO-GEMMs, we reveal a functional role for WNT signaling in driving prostate cancer metastasis and validate the WNT pathway as therapeutic target in metastatic prostate cancer. SIGNIFICANCE: Our understanding of the factors driving metastatic prostate cancer is limited by the paucity of models of late-stage disease. Here, we develop EPO-GEMMs of prostate cancer and use them to identify and validate the WNT pathway as an actionable driver of aggressive metastatic disease.This article is highlighted in the In This Issue feature, p. 890.


Subject(s)
Prostatic Neoplasms/genetics , Tissue Engineering/methods , Wnt Signaling Pathway/genetics , Animals , Disease Models, Animal , Female , Humans , Male , Mice , Neoplasm Metastasis
6.
Cell Mol Gastroenterol Hepatol ; 10(2): 434-449.e1, 2020.
Article in English | MEDLINE | ID: mdl-32330731

ABSTRACT

BACKGROUND & AIMS: Enterochromaffin-like (ECL) cells in the stomach express gastrin/cholecystokinin 2 receptor CCK2R and are known to expand under hypergastrinemia, but whether this results from expansion of existing ECL cells or increased production from progenitors has not been clarified. METHODS: We used mice with green fluorescent protein fluorescent reporter expression in ECL cells (histidine decarboxylase [Hdc]-green fluorescent protein), as well as Cck2r- and Hdc-driven Tamoxifen inducible recombinase Cre (Cck2r-CreERT2, Hdc-CreERT2) mice combined with Rosa26Sor-tdTomato (R26-tdTomato) mice, and studied their expression and cell fate in the gastric corpus by using models of hypergastrinemia (gastrin infusion, omeprazole treatment). RESULTS: Hdc-GFP marked the majority of ECL cells, located in the lower third of the gastric glands. Hypergastrinemia led to expansion of ECL cells that was not restricted to the gland base, and promoted cellular proliferation (Ki67) in the gastric isthmus but not in basal ECL cells. Cck2r-CreERT2 mice marked most ECL cells, as well as scattered cell types located higher up in the glands, whose number was increased during hypergastrinemia. Cck2r-CreERT2+ isthmus progenitors, but not Hdc+ mature ECL cells, were the source of ECL cell hyperplasia during hypergastrinemia and could grow as 3-dimensional spheroids in vitro. Moreover, gastrin treatment in vitro promoted sphere formation from sorted Cck2r+Hdc- cells, and increased chromogranin A and phosphorylated- extracellular signal-regulated kinase expression in CCK2R-derived organoids. Gastrin activates extracellular signal-regulated kinase pathways in vivo and in vitro, and treatment with the Mitogen-activated protein kinase kinase 1 inhibitor U0126 blocked hypergastrinemia-mediated changes, including CCK2R-derived ECL cell hyperplasia in vivo as well as sphere formation and chromogranin A expression in vitro. CONCLUSIONS: We show here that hypergastrinemia induces ECL cell hyperplasia that is derived primarily from CCK2R+ progenitors in the corpus. Gastrin-dependent function of CCK2R+ progenitors is regulated by the extracellular signal-regulated kinase pathway.


Subject(s)
Enterochromaffin-like Cells/pathology , Gastric Mucosa/pathology , Gastrins/blood , Animals , Disease Models, Animal , Enterochromaffin-like Cells/metabolism , Gastric Mucosa/cytology , Gastric Mucosa/metabolism , Gastrins/metabolism , Humans , Hyperplasia/blood , Hyperplasia/pathology , MAP Kinase Signaling System , Mice , Receptor, Cholecystokinin B/metabolism , Stem Cells/pathology
7.
Nat Commun ; 11(1): 111, 2020 01 08.
Article in English | MEDLINE | ID: mdl-31913277

ABSTRACT

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.


Subject(s)
Acetylcholine/metabolism , Homeodomain Proteins/metabolism , Intestinal Mucosa/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Doublecortin-Like Kinases , Enteroendocrine Cells/metabolism , Female , Homeodomain Proteins/genetics , Intestinal Mucosa/cytology , Male , Mice , Mice, Inbred C57BL , Neurotransmitter Agents/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Tumor Suppressor Proteins/genetics
8.
Blood Cells Mol Dis ; 80: 102372, 2020 02.
Article in English | MEDLINE | ID: mdl-31710879

ABSTRACT

The marked pro-thrombotic tendency in PNH is likely to be at least partly due to the population of platelets derived from the abnormal stem cell clone. However, identification of GPI (-) platelets by flow cytometry can be technically difficult. Here we describe a technique that involves the addition of aspirin immediately after the separation of platelet rich plasma and the use of gel filtration to isolate platelets away from plasma proteins and other blood cells. In a study of 92 analyses of samples from 50 patients, we have demonstrated that the percentage of PNH platelets correlates well with the percentage of PNH granulocytes. We also provide data on several cases where there was an extreme discrepancy between the proportion of PNH granulocytes and red cells; in these cases, the demonstration of abnormal platelets suggests that the patient is likely to be at risk of thrombosis. We believe this test will be potentially useful in the evaluation of samples from such patients and may serve as a tool to investigate the causes of hypercoagulability in PNH.


Subject(s)
Blood Platelets/metabolism , Hemoglobinuria, Paroxysmal/blood , Hemoglobinuria, Paroxysmal/diagnosis , Biomarkers , Blood Coagulation , Cell Fractionation/methods , Erythrocytes/metabolism , Flow Cytometry/methods , Granulocytes/metabolism , Hemoglobinuria, Paroxysmal/complications , Humans , Platelet Function Tests , ROC Curve , Thrombosis/etiology
9.
Exp Hematol Oncol ; 8: 17, 2019.
Article in English | MEDLINE | ID: mdl-31453016

ABSTRACT

BACKGROUND: Large clonal populations of cells bearing PIG-A mutations are the sine qua non of PNH, but the PIG-A mutation itself is insufficient for clonal expansion. The association between PNH and aplastic anemia supports the immune escape model, but not all PNH patients demonstrate a history of aplasia; therefore, second genetic hits driving clonal expansion have been postulated. Based on the previous identification of JAK2 mutations in patients with a myeloproliferative/PNH overlap syndrome, we considered TET2 as a candidate gene in which mutations might be contributing to clonal expansion. METHODS: Here we sequenced the TET2 and JAK2 genes in 19 patients with large PNH clones. RESULTS: We found one patient with a novel somatic nonsense mutation in TET2 in multiple hematopoietic lineages, which was detectable upon repeat testing. This patient has had severe thromboses and has relatively higher peripheral blood counts compared with the other patients-but does not have other features of a myeloproliferative neoplasm. CONCLUSIONS: We conclude that mutations in TET2 may contribute to clonal expansion in exceptional cases of PNH.

11.
Mutat Res ; 749(1-2): 49-57, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23748046

ABSTRACT

The mutation rate (µ) is likely to be a key parameter in leukemogenesis, but historically, it has been difficult to measure in humans. The PIG-A gene has some advantages for the detection of spontaneous mutations because it is X-linked, and therefore only one mutation is required to disrupt its function. Furthermore, the PIG-A-null phenotype is readily detected by flow cytometry. Using PIG-A, we have now provided the first in vitro measurement of µ in myeloid cells, using cultures of CD34+ cells that are transduced with either the AML-ETO or the MLL-AF9 fusion genes and expanded with cytokines. For the AML-ETO cultures, the median µ value was ∼9.4×10(-7) (range ∼3.6-23×10(-7)) per cell division. In contrast, few spontaneous mutations were observed in the MLL-AF9 cultures. Knockdown of p53 or introduction of mutant NRAS or FLT3 alleles did not have much of an effect on µ. Based on these data, we provide a model to predict whether hypermutability must occur in the process of leukemogenesis.


Subject(s)
Mutation Rate , Myeloid Cells/metabolism , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Core Binding Factor Alpha 2 Subunit/physiology , Fetal Blood/cytology , Flow Cytometry , Gene Knockdown Techniques , Genes, p53 , Glycosylphosphatidylinositols/genetics , Glycosylphosphatidylinositols/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation/physiology , Myeloid Cells/cytology , Myeloid-Lymphoid Leukemia Protein/physiology , Oncogene Proteins, Fusion/physiology , RUNX1 Translocation Partner 1 Protein
12.
Am J Pathol ; 181(5): 1862-9, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22940070

ABSTRACT

It has been proposed that genomic instability is essential to account for the multiplicity of mutations often seen in malignancies. Using the X-linked PIG-A gene as a sentinel gene for spontaneous inactivating somatic mutations, we previously showed that healthy individuals harbor granulocytes with the PIG-A mutant (paroxysmal nocturnal hemoglobinuria) phenotype at a median frequency (f) of ∼12 × 10(-6). Herein, we used a similar approach to determine f in blast cells derived from 19 individuals with acute lymphoblastic leukemia (ALL) and in immortalized Epstein-Barr virus-transformed B-cell cultures (human B-lymphoblastoid cell lines) from 19 healthy donors. The B-lymphoblastoid cell lines exhibited a unimodal distribution, with a median f value of 11 × 10(-6). In contrast, analysis of the f values for the ALL samples revealed at least two distinct populations: one population, representing approximately half of the samples (n = 10), had a median f value of 13 × 10(-6), and the remaining samples (n = 9) had a median f value of 566 × 10(-6). We conclude that in ALL, there are two distinct phenotypes with respect to hypermutability, which we hypothesize will correlate with the number of pathogenic mutations required to produce the leukemia.


Subject(s)
Blast Crisis/complications , Blast Crisis/pathology , Hemoglobinuria, Paroxysmal/complications , Hemoglobinuria, Paroxysmal/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/complications , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Adolescent , Adult , Aged , Aged, 80 and over , B-Lymphocytes/pathology , Case-Control Studies , Cell Line , Child , Child, Preschool , Female , Flow Cytometry , Humans , Male , Middle Aged , Phenotype , Tissue Donors , Young Adult
13.
Mutat Res ; 686(1-2): 1-8, 2010 Apr 01.
Article in English | MEDLINE | ID: mdl-20060400

ABSTRACT

It has been proposed that hypermutability is necessary to account for the high frequency of mutations in cancer. However, historically, the mutation rate (mu) has been difficult to measure directly, and increased cell turnover or selection could provide an alternative explanation. We recently developed an assay for mu using PIG-A as a sentinel gene and estimated that its average value is 10.6 x 10(-7) mutations per cell division in B-lymphoblastoid cell lines (BLCLs) from normal donors. Here we have measured mu in human malignancies and found that it was elevated in cell lines derived from T cell acute lymphoblastic leukemia, mantle cell lymphoma, follicular lymphoma in transformed phase, and 2 plasma cell neoplasms. In contrast, mu was much lower in a marginal zone lymphoma cell line and 5 other plasma cell neoplasms. The highest mu value that we measured, 3286 x 10(-7), is 2 orders of magnitude above the range we have observed in non-malignant human cells. We conclude that the type of genomic instability detected in this assay is a common but not universal feature of hematologic malignancies.


Subject(s)
Genomic Instability , Leukemia, T-Cell/genetics , Lymphoma/genetics , Membrane Proteins/genetics , Neoplasms, Plasma Cell/genetics , Cell Line, Tumor , Clone Cells , Flow Cytometry , Humans , Mutation
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