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1.
STAR Protoc ; 5(3): 103189, 2024 Jul 12.
Article in English | MEDLINE | ID: mdl-39003744

ABSTRACT

Isogenic disease models, such as genetically engineered organoids, provide insight into the impact of genetic variants on organ function. Here, we present a protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools. We describe steps for single guide RNA (sgRNA) design and cloning, electroporation, and selecting electroporated cells. We then detail procedures for clonal line generation. Next-generation CRISPR tools do not require double-stranded break (DSB) induction for their function, thus simplifying in vitro disease model generation. For complete details on the use and execution of this protocol, please refer to Geurts et al.1,2.

2.
Proc Natl Acad Sci U S A ; 121(12): e2309902121, 2024 Mar 19.
Article in English | MEDLINE | ID: mdl-38483988

ABSTRACT

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.


Subject(s)
F-Box Proteins , Neoplasms , Humans , F-Box-WD Repeat-Containing Protein 7/genetics , F-Box-WD Repeat-Containing Protein 7/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Epidermal Growth Factor/genetics , Epidermal Growth Factor/pharmacology , Epidermal Growth Factor/metabolism , Proteomics , ErbB Receptors/genetics , ErbB Receptors/metabolism , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , F-Box Proteins/genetics
3.
Mol Cancer ; 23(1): 39, 2024 02 21.
Article in English | MEDLINE | ID: mdl-38378518

ABSTRACT

BACKGROUND: Focal adhesion signaling involving receptor tyrosine kinases (RTK) and integrins co-controls cancer cell survival and therapy resistance. However, co-dependencies between these receptors and therapeutically exploitable vulnerabilities remain largely elusive in HPV-negative head and neck squamous cell carcinoma (HNSCC). METHODS: The cytotoxic and radiochemosensitizing potential of targeting 10 RTK and ß1 integrin was determined in up to 20 3D matrix-grown HNSCC cell models followed by drug screening and patient-derived organoid validation. RNA sequencing and protein-based biochemical assays were performed for molecular characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts. RESULTS: Fibroblast growth factor receptor (FGFR 1-4) targeting exhibited the strongest cytotoxic and radiosensitizing effects as monotherapy and combined with ß1 integrin inhibition, exceeding the efficacy of the other RTK studied. Pharmacological pan-FGFR inhibition elicited responses ranging from cytotoxicity/radiochemosensitization to resistance/radiation protection. RNA sequence analysis revealed a mesenchymal-to-epithelial transition (MET) in sensitive cell models, whereas resistant cell models exhibited a partial epithelial-to-mesenchymal transition (EMT). Accordingly, inhibition of EMT-associated kinases such as EGFR caused reduced adaptive resistance and enhanced (radio)sensitization to FGFR inhibition cell model- and organoid-dependently. Transferring the EMT-associated transcriptomic profiles to HNSCC patient cohorts not only demonstrated their prognostic value but also provided a conclusive validation of the presence of EGFR-related vulnerabilities that can be strategically exploited for therapeutic interventions. CONCLUSIONS: This study demonstrates that pan-FGFR inhibition elicits a beneficial radiochemosensitizing and a detrimental radioprotective potential in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically.


Subject(s)
Antineoplastic Agents , Carcinoma, Squamous Cell , Head and Neck Neoplasms , Humans , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/genetics , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics , Integrin beta1/genetics , Cell Line, Tumor , Receptor Protein-Tyrosine Kinases/genetics , Antineoplastic Agents/therapeutic use , ErbB Receptors/metabolism , Phenotype , Epithelial-Mesenchymal Transition/genetics
4.
Cell Stem Cell ; 31(2): 227-243.e12, 2024 02 01.
Article in English | MEDLINE | ID: mdl-38215738

ABSTRACT

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.


Subject(s)
Conjunctiva , Goblet Cells , Humans , Mice , Animals , Conjunctiva/metabolism , Goblet Cells/metabolism , Epithelium , Interleukin-13 , Homeostasis , Organoids
5.
Br J Cancer ; 130(3): 369-379, 2024 02.
Article in English | MEDLINE | ID: mdl-38102228

ABSTRACT

BACKGROUND: Bladder cancer is one of the most common cancer types worldwide. Generally, research relies on invasive sampling strategies. METHODS: Here, we generate bladder cancer organoids directly from urine (urinoids). In this project, we establish 12 urinoid lines from 22 patients with non-muscle and muscle-invasive bladder tumours, with an efficiency of 55%. RESULTS: The histopathological features of the urinoids accurately resemble those of the original bladder tumours. Genetically, there is a high concordance of single nucleotide polymorphisms (92.56%) and insertions & deletions (91.54%) between urinoids and original tumours from patient 4. Furthermore, these urinoids show sensitivity to bladder cancer drugs, similar to their tissue-derived organoid counterparts. Genetic analysis of longitudinally generated tumoroids and urinoids from one patient receiving systemic immunotherapy, identify alterations that may guide the choice for second-line therapy. Successful treatment adaptation was subsequently demonstrated in the urinoid setting. CONCLUSION: Therefore, urinoids can advance precision medicine in bladder cancer as a non-invasive platform for tumour pathogenesis, longitudinal drug-response monitoring, and therapy adaptation.


Subject(s)
Urinary Bladder Neoplasms , Humans , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology , Urinary Bladder/pathology , Immunotherapy , Precision Medicine , Organoids/pathology
6.
Science ; 382(6669): 451-458, 2023 10 27.
Article in English | MEDLINE | ID: mdl-37883554

ABSTRACT

Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI), and colon. EECs regulate aspects of metabolic activity, including insulin levels, satiety, gastrointestinal secretion, and motility. The generation of different EEC lineages is not completely understood. In this work, we report a CRISPR knockout screen of the entire repertoire of transcription factors (TFs) in adult human SI organoids to identify dominant TFs controlling EEC differentiation. We discovered ZNF800 as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional CRISPR screens for genes that program cell fate.


Subject(s)
CRISPR-Cas Systems , Cell Lineage , Enteroendocrine Cells , Gene Expression Regulation , Repressor Proteins , Zinc Fingers , Humans , Cell Differentiation/genetics , Enteroendocrine Cells/cytology , Enteroendocrine Cells/metabolism , Organoids , Adult , Cell Lineage/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism
7.
Nat Commun ; 14(1): 5411, 2023 09 05.
Article in English | MEDLINE | ID: mdl-37669929

ABSTRACT

Intestinal stem cells (ISCs) maintain the epithelial lining of the intestines, but mechanisms regulating ISCs and their niche after damage remain poorly understood. Utilizing radiation injury to model intestinal pathology, we report here that the Interleukin-33 (IL-33)/ST2 axis, an immunomodulatory pathway monitored clinically as an intestinal injury biomarker, regulates intrinsic epithelial regeneration by inducing production of epidermal growth factor (EGF). Three-dimensional imaging and lineage-specific RiboTag induction within the stem cell compartment indicated that ISCs expressed IL-33 in response to radiation injury. Neighboring Paneth cells responded to IL-33 by augmenting production of EGF, which promoted ISC recovery and epithelial regeneration. These findings reveal an unknown pathway of niche regulation and crypt regeneration whereby the niche responds dynamically upon injury and the stem cells orchestrate regeneration by regulating their niche. This regenerative circuit also highlights the breadth of IL-33 activity beyond immunomodulation and the therapeutic potential of EGF administration for treatment of intestinal injury.


Subject(s)
Interleukin-33 , Radiation Injuries , Humans , Epidermal Growth Factor , Imaging, Three-Dimensional , Immunomodulation
8.
Nat Commun ; 14(1): 4998, 2023 08 17.
Article in English | MEDLINE | ID: mdl-37591832

ABSTRACT

Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.


Subject(s)
Adult Stem Cells , RNA, Guide, CRISPR-Cas Systems , Adult , Humans , Oncogenes , Carcinogenesis/genetics , Cell Transformation, Neoplastic , Organoids
10.
J Cell Biol ; 219(9)2020 09 07.
Article in English | MEDLINE | ID: mdl-32573693

ABSTRACT

In vitro experiments have shown that GRASP65 (GORASP1) and GRASP55 (GORASP2) proteins function in stacking Golgi cisternae. However, in vivo depletion of GORASPs in metazoans has given equivocal results. We have generated a mouse lacking both GORASPs and find that Golgi cisternae remained stacked. However, the stacks are disconnected laterally from each other, and the cisternal cross-sectional diameters are significantly reduced compared with their normal counterparts. These data support earlier findings on the role of GORASPs in linking stacks, and we suggest that unlinking of stacks likely affects dynamic control of COPI budding and vesicle fusion at the rims. The net result is that cisternal cores remain stacked, but cisternal diameter is reduced by rim consumption.


Subject(s)
Golgi Apparatus/metabolism , Golgi Matrix Proteins/metabolism , Animals , COP-Coated Vesicles/metabolism , Female , Intracellular Membranes/metabolism , Membrane Fusion/physiology , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL
11.
Nat Commun ; 11(1): 2660, 2020 05 27.
Article in English | MEDLINE | ID: mdl-32461556

ABSTRACT

High-grade serous ovarian cancer (HG-SOC)-often referred to as a "silent killer"-is the most lethal gynecological malignancy. The fallopian tube (murine oviduct) and ovarian surface epithelium (OSE) are considered the main candidate tissues of origin of this cancer. However, the relative contribution of each tissue to HG-SOC is not yet clear. Here, we establish organoid-based tumor progression models of HG-SOC from murine oviductal and OSE tissues. We use CRISPR-Cas9 genome editing to introduce mutations into genes commonly found mutated in HG-SOC, such as Trp53, Brca1, Nf1 and Pten. Our results support the dual origin hypothesis of HG-SOC, as we demonstrate that both epithelia can give rise to ovarian tumors with high-grade pathology. However, the mutated oviductal organoids expand much faster in vitro and more readily form malignant tumors upon transplantation. Furthermore, in vitro drug testing reveals distinct lineage-dependent sensitivities to the common drugs used to treat HG-SOC in patients.


Subject(s)
CRISPR-Cas Systems/genetics , Organoids , Ovarian Neoplasms/etiology , Animals , Antineoplastic Agents/pharmacology , BRCA1 Protein/genetics , CRISPR-Associated Protein 9 , Epithelium/pathology , Fallopian Tubes/pathology , Female , Gene Editing/methods , Mice , Mutation , Neurofibromatosis 1/genetics , Organ Culture Techniques/methods , Organoids/drug effects , Organoids/physiopathology , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovary/pathology , PTEN Phosphohydrolase/genetics , Tumor Suppressor Protein p53/genetics
12.
Cell Stem Cell ; 26(3): 377-390.e6, 2020 03 05.
Article in English | MEDLINE | ID: mdl-32084390

ABSTRACT

Ablation of LGR5+ intestinal stem cells (ISCs) is associated with rapid restoration of the ISC compartment. Different intestinal crypt populations dedifferentiate to provide new ISCs, but the transcriptional and signaling trajectories that guide this process are unclear, and a large body of work suggests that quiescent "reserve" ISCs contribute to regeneration. By timing the interval between LGR5+ lineage tracing and lethal injury, we show that ISC regeneration is explained nearly completely by dedifferentiation, with contributions from absorptive and secretory progenitors. The ISC-restricted transcription factor ASCL2 confers measurable competitive advantage to resting ISCs and is essential to restore the ISC compartment. Regenerating cells re-express Ascl2 days before Lgr5, and single-cell RNA sequencing (scRNA-seq) analyses reveal transcriptional paths underlying dedifferentiation. ASCL2 target genes include the interleukin-11 (IL-11) receptor Il11ra1, and recombinant IL-11 enhances crypt cell regenerative potential. These findings reveal cell dedifferentiation as the principal means for ISC restoration and highlight an ASCL2-regulated signal that enables this adaptive response.


Subject(s)
Cell Dedifferentiation , Stem Cells , Intestinal Mucosa , Intestines , Signal Transduction
13.
Proc Natl Acad Sci U S A ; 116(52): 26599-26605, 2019 Dec 26.
Article in English | MEDLINE | ID: mdl-31843916

ABSTRACT

Cycling intestinal Lgr5+ stem cells are intermingled with their terminally differentiated Paneth cell daughters at crypt bottoms. Paneth cells provide multiple secreted (e.g., Wnt, EGF) as well as surface-bound (Notch ligand) niche signals. Here we show that ablation of Paneth cells in mice, using a diphtheria toxin receptor gene inserted into the P-lysozyme locus, does not affect the maintenance of Lgr5+ stem cells. Flow cytometry, single-cell sequencing, and histological analysis showed that the ablated Paneth cells are replaced by enteroendocrine and tuft cells. As these cells physically occupy Paneth cell positions between Lgr5 stem cells, they serve as an alternative source of Notch signals, which are essential for Lgr5+ stem cell maintenance. Our combined in vivo results underscore the adaptive flexibility of the intestine in maintaining normal tissue homeostasis.

14.
Proc Natl Acad Sci U S A ; 116(52): 26580-26590, 2019 Dec 26.
Article in English | MEDLINE | ID: mdl-31818951

ABSTRACT

We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to target MTAP (a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs.

15.
Cancer Discov ; 9(7): 852-871, 2019 07.
Article in English | MEDLINE | ID: mdl-31053628

ABSTRACT

Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)-derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro. Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs. SIGNIFICANCE: This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. In vitro drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice.See related commentary by Hill and D'Andrea, p. 828.This article is highlighted in the In This Issue feature, p. 813.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/therapy , Mouth Mucosa/pathology , Organoids/pathology , Precision Medicine/methods , Squamous Cell Carcinoma of Head and Neck/pathology , Squamous Cell Carcinoma of Head and Neck/therapy , Animals , Carboplatin/administration & dosage , Cetuximab/administration & dosage , Chemoradiotherapy , Cisplatin/administration & dosage , Drug Screening Assays, Antitumor/methods , Head and Neck Neoplasms/metabolism , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Mouth Mucosa/drug effects , Mouth Mucosa/radiation effects , Organoids/drug effects , Organoids/radiation effects , Squamous Cell Carcinoma of Head and Neck/metabolism , Xenograft Model Antitumor Assays
16.
Cell Stem Cell ; 24(6): 927-943.e6, 2019 06 06.
Article in English | MEDLINE | ID: mdl-31130514

ABSTRACT

The deubiquitinating enzyme BAP1 is a tumor suppressor, among others involved in cholangiocarcinoma. BAP1 has many proposed molecular targets, while its Drosophila homolog is known to deubiquitinate histone H2AK119. We introduce BAP1 loss-of-function by CRISPR/Cas9 in normal human cholangiocyte organoids. We find that BAP1 controls the expression of junctional and cytoskeleton components by regulating chromatin accessibility. Consequently, we observe loss of multiple epithelial characteristics while motility increases. Importantly, restoring the catalytic activity of BAP1 in the nucleus rescues these cellular and molecular changes. We engineer human liver organoids to combine four common cholangiocarcinoma mutations (TP53, PTEN, SMAD4, and NF1). In this genetic background, BAP1 loss results in acquisition of malignant features upon xenotransplantation. Thus, control of epithelial identity through the regulation of chromatin accessibility appears to be a key aspect of BAP1's tumor suppressor function. Organoid technology combined with CRISPR/Cas9 provides an experimental platform for mechanistic studies of cancer gene function in a human context.


Subject(s)
Cholangiocarcinoma/genetics , Chromatin/metabolism , Epithelial Cells/physiology , Liver Neoplasms/genetics , Liver/physiology , Tumor Suppressor Proteins/metabolism , Ubiquitin Thiolesterase/metabolism , Animals , Bioengineering , Carcinogenesis , Cells, Cultured , Chromatin/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Cytoskeleton/metabolism , Female , Humans , Loss of Function Mutation/genetics , Mice , Mice, SCID , Organoids , Transplantation, Heterologous , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics
17.
Cell ; 176(5): 1158-1173.e16, 2019 02 21.
Article in English | MEDLINE | ID: mdl-30712869

ABSTRACT

Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.


Subject(s)
Cell Lineage/genetics , Enteroendocrine Cells/metabolism , Gene Expression Profiling/methods , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/genetics , Cell Lineage/physiology , Enteroendocrine Cells/physiology , Fluorescent Dyes , Homeodomain Proteins/genetics , Intestinal Mucosa/cytology , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Optical Imaging/methods , Organoids , Phenotype , Single-Cell Analysis/methods , Stem Cells , Transcription Factors/genetics , Transcriptome/genetics
18.
Gastroenterology ; 156(1): 145-159.e19, 2019 01.
Article in English | MEDLINE | ID: mdl-30273559

ABSTRACT

BACKGROUND & AIMS: RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS: We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS: The H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS: In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Epithelial Cells/enzymology , Genomic Instability , Intestinal Neoplasms/prevention & control , Intestine, Small/enzymology , Ribonuclease H/metabolism , Animals , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Colitis/chemically induced , Colitis/enzymology , Colitis/genetics , Colitis/pathology , DNA Damage , Dextran Sulfate , Disease Models, Animal , Epithelial Cells/pathology , Female , Genetic Predisposition to Disease , Humans , Intestinal Neoplasms/enzymology , Intestinal Neoplasms/genetics , Intestinal Neoplasms/pathology , Intestine, Small/pathology , Male , Mice, Knockout , Phenotype , Ribonuclease H/deficiency , Ribonuclease H/genetics , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics
19.
Proc Natl Acad Sci U S A ; 115(52): E12245-E12254, 2018 12 26.
Article in English | MEDLINE | ID: mdl-30530645

ABSTRACT

The significance of cardiac stem cell (CSC) populations for cardiac regeneration remains disputed. Here, we apply the most direct definition of stem cell function (the ability to replace lost tissue through cell division) to interrogate the existence of CSCs. By single-cell mRNA sequencing and genetic lineage tracing using two Ki67 knockin mouse models, we map all proliferating cells and their progeny in homoeostatic and regenerating murine hearts. Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adult myocardium represented various noncardiomyocyte cell types. Proliferative postdamage fibroblasts expressing follistatin-like protein 1 (FSTL1) closely resemble neonatal cardiac fibroblasts and form the fibrotic scar. Genetic deletion of Fstl1 in cardiac fibroblasts results in postdamage cardiac rupture. We find no evidence for the existence of a quiescent CSC population, for transdifferentiation of other cell types toward cardiomyocytes, or for proliferation of significant numbers of cardiomyocytes in response to cardiac injury.


Subject(s)
Cell Proliferation , Heart Injuries/physiopathology , Animals , Cells, Cultured , Disease Models, Animal , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Follistatin-Related Proteins/genetics , Follistatin-Related Proteins/metabolism , Heart Injuries/genetics , Heart Injuries/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardium/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Pregnancy , Stem Cells/cytology , Stem Cells/metabolism
20.
Cell ; 175(6): 1591-1606.e19, 2018 11 29.
Article in English | MEDLINE | ID: mdl-30500538

ABSTRACT

The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The "oval cell" response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.


Subject(s)
Cell Proliferation , Hepatocytes/metabolism , Organoids/metabolism , Animals , Cell Culture Techniques , Cells, Cultured , Hepatocytes/cytology , Humans , Mice , Mice, Inbred BALB C , Mice, Knockout , Organoids/cytology , Stem Cells/cytology , Stem Cells/metabolism , Time Factors
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