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.
With ageing, normal human tissues experience an expansion of somatic clones that carry cancer mutations
. However, whether such clonal expansion exists in the non-neoplastic intestine remains ...unknown. Here, using whole-exome sequencing data from 76 clonal human colon organoids, we identify a unique pattern of somatic mutagenesis in the inflamed epithelium of patients with ulcerative colitis. The affected epithelium accumulates somatic mutations in multiple genes that are related to IL-17 signalling-including NFKBIZ, ZC3H12A and PIGR, which are genes that are rarely affected in colon cancer. Targeted sequencing validates the pervasive spread of mutations that are related to IL-17 signalling. Unbiased CRISPR-based knockout screening in colon organoids reveals that the mutations confer resistance to the pro-apoptotic response that is induced by IL-17A. Some of these genetic mutations are known to exacerbate experimental colitis in mice
, and somatic mutagenesis in human colon epithelium may be causally linked to the inflammatory process. Our findings highlight a genetic landscape that adapts to a hostile microenvironment, and demonstrate its potential contribution to the pathogenesis of ulcerative colitis.
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.
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•The CRISPR/Cas9 system enables genome editing in intestinal stem cell organoids•cAMP-induced swelling is lost in CFTR mutant organoids of cystic fibrosis patients•CRISPR/Cas9-mediated repair of the CFTR locus restores organoid swelling
Correction of a disease-causing CFTR mutation in cultured intestinal stem cells from cystic fibrosis patients is demonstrated using the CRISPR/Cas9 system.
Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously ...that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.
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•Establishment of a long-term human liver organoid culture•Human liver stem cells retain genetic stability after long-term expansion•Liver organoid cultures differentiate to functional hepatocytes in vitro and in vivo•Organoids derived from patients with genetic disorders model liver disease in vitro
A culture system that allows long-term expansion of human liver can be used to model and study various human diseases.
The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, ...stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.
Continuous-wave laser crystallization of amorphous Si (a-Si) thin films on a polyimide (PI)-coated glass substrate is studied by using a single scan of a highly uniform top-flat line-beam with a 123 ...nm SiO
2
cap layer and a thin buffer layer at room temperature in air. The total buffer layer thickness is reduced to as thin as 1.55
μ
m including the heatsink a-Si layer and two SiO
2
layers. Damage to the polyimide during the crystallization is successfully suppressed by the heatsink layer. An 88.3% {100} surface fraction is obtained for a 60-nm-thick Si thin film within 15° on polyimide even with a low scan velocity of 15 mm/s.
•Stable and huge grain growth of Si thin films.•Orientations of {100} in the surface normal and scan directions.•Length of more than 3000 μm.•Width of 120-150 μm.
A steady-state crystal growth of a ...{100}-oriented grain-boundary free 60 nm-thick Si thin-film is realized so long as the laser scan continues by the continuous-wave laser lateral crystallization of amorphous-Si on quartz, resulting in a huge grain of 3000 μm long and 120-150 μm wide. At an increased power by 0.1 W from the optimum power, a stable grain growth is realized as well, but it takes a longer scan travel to get the steady-state crystal growth than that at the optimum. With a disturbance on the way of the scan at the optimum power, the steady-state crystal growth continues to the vicinity of the disturbance, and it tends to recover with further scan after the disturbance even with the nano-crystal region formed at the disturbance region.
Spheroid and organoid cultures are powerful in vitro models for biology, but size and shape diversity within the culture is largely ignored. To streamline morphometric profiling, we developed ...OrganoSeg, an open-source software that integrates segmentation, filtering, and analysis for archived brightfield images of 3D culture. OrganoSeg is more accurate and flexible than existing platforms, and we illustrate its potential by stratifying 5167 breast-cancer spheroid and 5743 colon and colorectal-cancer organoid morphologies. Organoid transcripts grouped by morphometric signature heterogeneity were enriched for biological processes not prominent in the original RNA sequencing data. OrganoSeg enables complete, objective quantification of brightfield phenotypes, which may give insight into the molecular and multicellular mechanisms of organoid regulation.
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