Isthmus progenitor cells contribute to homeostatic cellular turnover and support regeneration following intestinal injury.

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.

An optimized protocol for isolation of murine pancreatic single cells with high yield and purity.

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.

Acute Intestinal Inflammation Depletes/Recruits Histamine-Expressing Myeloid Cells From the Bone Marrow Leading to Exhaustion of MB-HSCs.

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.

Somatic Tissue Engineering in Mouse Models Reveals an Actionable Role for WNT Pathway Alterations in Prostate Cancer Metastasis.

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.

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.

Analysis of TET2 mutations in paroxysmal nocturnal hemoglobinuria (PNH).

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.

The rate of spontaneous mutations in human myeloid cells.

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.

Leukemic blasts with the paroxysmal nocturnal hemoglobinuria phenotype in children with acute lymphoblastic leukemia.

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.

A quantitative analysis of genomic instability in lymphoid and plasma cell neoplasms based on the PIG-A gene.

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.