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1.
Nat Protoc ; 13(1): 59-78, 2018 Jan.
Article in English | MEDLINE | ID: mdl-29215633

ABSTRACT

Characterization of mutational processes in adult stem cells (ASCs) will improve our understanding of aging-related diseases, such as cancer and organ failure, and may ultimately help prevent the development of these diseases. Here, we present a method for cataloging mutations in individual human ASCs without the necessity of using error-prone whole-genome amplification. Single ASCs are expanded in vitro into clonal organoid cultures to generate sufficient DNA for accurate whole-genome sequencing (WGS) analysis. We developed a data-analysis pipeline that identifies with high confidence somatic variants that accumulated in vivo in the original ASC. These genome-wide mutation catalogs are valuable resources for the characterization of the underlying mutational mechanisms. In addition, this protocol can be used to determine the effects of culture conditions or mutagen exposure on mutation accumulation in ASCs in vitro. Here, we describe a protocol for human liver ASCs that can be completed over a period of 3-4 months with hands-on time of ∼5 d.


Subject(s)
Adult Stem Cells/cytology , Mutation Accumulation , Mutation/genetics , Organoids/cytology , Whole Genome Sequencing/methods , Cells, Cultured , DNA/analysis , DNA/genetics , High-Throughput Nucleotide Sequencing , Humans , Liver/cytology
2.
Science ; 358(6360): 234-238, 2017 10 13.
Article in English | MEDLINE | ID: mdl-28912133

ABSTRACT

Mutational processes underlie cancer initiation and progression. Signatures of these processes in cancer genomes may explain cancer etiology and could hold diagnostic and prognostic value. We developed a strategy that can be used to explore the origin of cancer-associated mutational signatures. We used CRISPR-Cas9 technology to delete key DNA repair genes in human colon organoids, followed by delayed subcloning and whole-genome sequencing. We found that mutation accumulation in organoids deficient in the mismatch repair gene MLH1 is driven by replication errors and accurately models the mutation profiles observed in mismatch repair-deficient colorectal cancers. Application of this strategy to the cancer predisposition gene NTHL1, which encodes a base excision repair protein, revealed a mutational footprint (signature 30) previously observed in a breast cancer cohort. We show that signature 30 can arise from germline NTHL1 mutations.


Subject(s)
CRISPR-Cas Systems , Colon , Deoxyribonuclease (Pyrimidine Dimer)/genetics , MutL Protein Homolog 1/genetics , Neoplasms/genetics , Organoids , Breast Neoplasms/genetics , Colorectal Neoplasms/genetics , DNA Mismatch Repair/genetics , DNA Repair/genetics , DNA Replication , Female , Germ-Line Mutation , Humans , INDEL Mutation , Mutagenesis , Stem Cells
3.
Nature ; 538(7624): 260-264, 2016 Oct 13.
Article in English | MEDLINE | ID: mdl-27698416

ABSTRACT

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.


Subject(s)
Adult Stem Cells/metabolism , Aging/genetics , Mutation Accumulation , Mutation Rate , Organ Specificity , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Child , Child, Preschool , Colon/metabolism , DNA Mutational Analysis , Female , Genes, Neoplasm/genetics , Humans , Incidence , Intestine, Small/metabolism , Liver/metabolism , Male , Mice , Middle Aged , Multipotent Stem Cells/metabolism , Neoplasms/epidemiology , Neoplasms/genetics , Organoids/metabolism , Point Mutation/genetics , Young Adult
4.
Cell Stem Cell ; 16(2): 158-70, 2015 Feb 05.
Article in English | MEDLINE | ID: mdl-25620640

ABSTRACT

The Wnt signaling pathway controls stem cell identity in the intestinal epithelium and in many other adult organs. The transcription factor Ascl2 (a Wnt target gene) is a master regulator of intestinal stem cell identity. It is unclear how the continuous Wnt gradient along the crypt axis is translated into discrete expression of Ascl2 and discrete specification of stem cells at crypt bottoms. We show that (1) Ascl2 is regulated in a direct autoactivatory loop, leading to a distinct on/off expression pattern, and (2) Wnt/R-spondin can activate this regulatory loop. This mechanism interprets the Wnt levels in the intestinal crypt and translates the continuous Wnt signal into a discrete Ascl2 "on" or "off" decision. In turn, Ascl2, together with ß-catenin/Tcf, activates the genes fundamental to the stem cell state. In this manner, Ascl2 forms a transcriptional switch that is both Wnt responsive and Wnt dependent to define stem cell identity.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Intestinal Mucosa/cytology , Intestinal Mucosa/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Thrombospondins/metabolism , Wnt Signaling Pathway , Animals , Mice , Thrombospondins/genetics
5.
Cell Stem Cell ; 13(6): 653-8, 2013 Dec 05.
Article in English | MEDLINE | ID: mdl-24315439

ABSTRACT

Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect.


Subject(s)
CRISPR-Associated Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis/pathology , Intestines/pathology , Organoids/metabolism , Stem Cells/metabolism , Adult , Adult Stem Cells/metabolism , Animals , Base Sequence , Genetic Therapy , Humans , Mice , Molecular Sequence Data , RNA Editing/genetics
6.
J Clin Invest ; 123(4): 1763-72, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23478408

ABSTRACT

A highly complex network of intrinsic enteric neurons is required for the digestive and homeostatic functions of the gut. Nevertheless, the genetic and molecular mechanisms that regulate their assembly into functional neuronal circuits are currently unknown. Here we report that the planar cell polarity (PCP) genes Celsr3 and Fzd3 are required during murine embryogenesis to specifically control the guidance and growth of enteric neuronal projections relative to the longitudinal and radial gut axes. Ablation of these genes disrupts the normal organization of nascent neuronal projections, leading to subtle changes of axonal tract configuration in the mature enteric nervous system (ENS), but profound abnormalities in gastrointestinal motility. Our data argue that PCP-dependent modules of connectivity established at early stages of enteric neurogenesis control gastrointestinal function in adult animals and provide the first evidence that developmental deficits in ENS wiring may contribute to the pathogenesis of idiopathic bowel disorders.


Subject(s)
Cadherins/genetics , Embryonic Development/genetics , Enteric Nervous System/embryology , Frizzled Receptors/genetics , Neurogenesis/genetics , Receptors, Cell Surface/genetics , Animals , Axons/physiology , Cadherins/metabolism , Cells, Cultured , Colon/physiology , Enteric Nervous System/metabolism , Enteric Nervous System/physiology , Frizzled Receptors/metabolism , Gastrointestinal Motility , Gastrointestinal Tract/embryology , Gastrointestinal Tract/innervation , Gastrointestinal Tract/physiology , Gene Expression Regulation, Developmental , Mice , Neurons/physiology , Receptors, Cell Surface/metabolism
7.
Curr Protoc Stem Cell Biol ; 27: 5A.6.1-5A.6.8, 2013 Nov 13.
Article in English | MEDLINE | ID: mdl-24510288

ABSTRACT

In this unit, we describe a protocol for regulated gene expression in primary endodermal organoid culture using retroviral vectors. The study of gene function in endodermal epithelia such as those lining the stomach, small intestine, and colon has so far mainly relied on the generation of transgenic mouse lines. Establishing such animal models is laborious, expensive, and time-consuming. Ever-expanding endodermal organoids, grown in an in vitro 3-D epithelial culture system, faithfully recapitulate the in vivo counterpart and represent a sustainable alternative. Gene overexpression and knockdown can be achieved in organoids by retroviral transduction. The technique can also be applied to organoids derived from pre-established mutant mouse lines or used in combination with chemical and biological inhibitors or activators. This method provides a novel, versatile tool for phenotypic analysis of endodermal epithelium in vitro.


Subject(s)
Gene Expression , Organ Culture Techniques/methods , Organoids/metabolism , Retroviridae/genetics , Animals , Genetic Vectors/genetics , Humans , Mice
8.
BMC Gastroenterol ; 12: 81, 2012 Jun 26.
Article in English | MEDLINE | ID: mdl-22735038

ABSTRACT

BACKGROUND: Stem cell-based therapy has recently been explored for the treatment of disorders of the enteric nervous system (ENS). Pluripotent embryonic stem (ES) cells represent an attractive cell source; however, little or no information is currently available on how ES cells will respond to the gut environment. In this study, we investigated the ability of ES cells to respond to environmental cues derived from the ENS and related tissues, both in vitro and in vivo. METHODS: Neurospheres were generated from mouse ES cells (ES-NS) and co-cultured with organotypic preparations of gut tissue consisting of the longitudinal muscle layers with the adherent myenteric plexus (LM-MP). RESULTS: LM-MP co-culture led to a significant increase in the expression of pan-neuronal markers (ßIII-tubulin, PGP 9.5) as well as more specialized markers (peripherin, nNOS) in ES-NS, both at the transcriptional and protein level. The increased expression was not associated with increased proliferation, thus confirming a true neurogenic effect. LM-MP preparations exerted also a myogenic effect on ES-NS, although to a lesser extent. After transplantation in vivo into the mouse pylorus, grafted ES-NS failed to acquire a distinct phenotype al least 1 week following transplantation. CONCLUSIONS: This is the first study reporting that the gut explants can induce neuronal differentiation of ES cells in vitro and induce the expression of nNOS, a key molecule in gastrointestinal motility regulation. The inability of ES-NS to adopt a neuronal phenotype after transplantation in the gastrointestinal tract is suggestive of the presence of local inhibitory influences that prevent ES-NS differentiation in vivo.


Subject(s)
Cell- and Tissue-Based Therapy/methods , Embryonic Stem Cells/transplantation , Enteric Nervous System/embryology , Gastrointestinal Tract/cytology , Muscle, Smooth/cytology , Myenteric Plexus/cytology , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Coculture Techniques , Embryonic Stem Cells/cytology , Gastrointestinal Tract/metabolism , Intestine, Small/cytology , Intestine, Small/metabolism , Mice , Mice, Inbred Strains , Muscle, Smooth/metabolism , Nitric Oxide Synthase Type I/metabolism , Phenotype , Pylorus/cytology , Pylorus/metabolism , Stem Cell Transplantation/methods
9.
Dev Biol ; 366(1): 64-73, 2012 Jun 01.
Article in English | MEDLINE | ID: mdl-22290331

ABSTRACT

The enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, consists of numerous types of neurons, and glial cells, that are distributed in two intramuscular plexuses that extend along the entire length of the gut and control co-ordinated smooth muscle contractile activity and other gut functions. All enteric neurons and glia are derived from neural crest cells (NCC). Vagal (hindbrain) level NCC provide the majority of enteric precursors along the entire length of the gut, while a lesser contribution, that is restricted to the hindgut, arises from the sacral region of the neuraxis. After leaving the dorsal neural tube NCC undergo extensive migration, proliferation, survival and differentiation in order to form a functional ENS. This article reviews the molecular mechanisms underlying these key developmental processes and highlights the major groups of molecules that affect enteric NCC proliferation and survival (Ret/Gdnf and EdnrB/Et-3 pathways, Sox10 and Phox2b transcription factors), cell migration (Ret and EdnrB signalling, semaphorin 3A, cell adhesion molecules, Rho GTPases), and the development of enteric neuronal subtypes and morphologies (Mash1, Gdnf/neurturin, BMPs, Hand2, retinoic acid). Finally, looking to the future, we discuss the need to translate the wealth of data gleaned from animal studies to the clinical area and thus better understand, and develop treatments for, congenital human diseases affecting the ENS.


Subject(s)
Enteric Nervous System , Gastrointestinal Tract/innervation , Neural Crest , Neuroglia , Neurons , Animals , Biomarkers , Cell Differentiation , Cell Movement , Cell Proliferation , Enteric Nervous System/cytology , Enteric Nervous System/physiology , Humans , Neural Crest/cytology , Neural Crest/physiology , Neuroglia/cytology , Neuroglia/physiology , Neurons/cytology , Neurons/physiology , Signal Transduction
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