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1.
PLoS Genet ; 17(11): e1009868, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34752469

RESUMO

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.


Assuntos
Neoplasias Encefálicas/genética , Replicação do DNA/genética , Instabilidade Genômica , Glioma/genética , Histonas/fisiologia , Neoplasias Encefálicas/patologia , Criança , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Humanos , Mitose/genética
2.
EMBO J ; 32(20): 2708-21, 2013 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-24045232

RESUMO

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.


Assuntos
Células-Tronco Adultas/fisiologia , Proliferação de Células , Pâncreas/citologia , Receptores Acoplados a Proteínas G/fisiologia , Trombospondinas/fisiologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos SCID , Camundongos Transgênicos , Modelos Biológicos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/fisiologia , Pâncreas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Trombospondinas/genética , Trombospondinas/metabolismo
3.
Diabetologia ; 59(11): 2387-2392, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27443307

RESUMO

AIMS/HYPOTHESIS: Dynamic processes in pancreatic tissue are difficult to study. We aimed to develop an intravital imaging method to longitudinally examine engraftment, vascularisation, expansion and differentiation in mature islets or embryonic pancreases transplanted under the kidney capsule. METHODS: Isolated pancreatic islets from adult mice and murine embryonic day (E)12.5 pancreases containing fluorescent biomarkers were transplanted under the kidney capsule of immunodeficient recipient mice. Human islet cells were dispersed, transduced with a lentivirus expressing a fluorescent label and reaggregated before transplantation. Graft-containing kidneys were positioned subcutaneously and an imaging window was fitted into the skin on top of the kidney. Intravital imaging using multiphoton microscopy was performed for up to 2 weeks. Volumes of fluorescently labelled cells were determined as a measure of development and survival. RESULTS: Transplanted islets and embryonic pancreases showed good engraftment and remained viable. Engraftment and vascularisation could be longitudinally examined in murine and human islet cells. Murine islet beta cell volume was unchanged over time. Transplanted embryonic pancreases increased to up to 6.1 times of their original volume and beta cell volume increased 90 times during 2 weeks. CONCLUSIONS/INTERPRETATION: This method allows for repeated intravital imaging of grafts containing various sources of pancreatic tissue transplanted under the kidney capsule. Using fluorescent markers, dynamic information concerning engraftment or differentiation can be visualised and measured.


Assuntos
Ilhotas Pancreáticas/citologia , Pâncreas/citologia , Pâncreas/embriologia , Animais , Feminino , Humanos , Técnicas In Vitro , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas , Rim/citologia , Rim/metabolismo , Lentivirus/genética , Camundongos , Pâncreas/metabolismo , Gravidez
4.
Oncogene ; 42(20): 1661-1671, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37020038

RESUMO

Atypical teratoid/rhabdoid tumors (ATRTs) represent a rare, but aggressive pediatric brain tumor entity. They are genetically defined by alterations in the SWI/SNF chromatin remodeling complex members SMARCB1 or SMARCA4. ATRTs can be further classified in different molecular subgroups based on their epigenetic profiles. Although recent studies suggest that the different subgroups have distinct clinical features, subgroup-specific treatment regimens have not been developed thus far. This is hampered by the lack of pre-clinical in vitro models representative of the different molecular subgroups. Here, we describe the establishment of ATRT tumoroid models from the ATRT-MYC and ATRT-SHH subgroups. We demonstrate that ATRT tumoroids retain subgroup-specific epigenetic and gene expression profiles. High throughput drug screens on our ATRT tumoroids revealed distinct drug sensitivities between and within ATRT-MYC and ATRT-SHH subgroups. Whereas ATRT-MYC universally displayed high sensitivity to multi-targeted tyrosine kinase inhibitors, ATRT-SHH showed a more heterogeneous response with a subset showing high sensitivity to NOTCH inhibitors, which corresponded to high expression of NOTCH receptors. Our ATRT tumoroids represent the first pediatric brain tumor organoid model, providing a representative pre-clinical model which enables the development of subgroup-specific therapies.


Assuntos
Neoplasias Encefálicas , Tumor Rabdoide , Teratoma , Criança , Humanos , Teratoma/tratamento farmacológico , Teratoma/genética , Proteína SMARCB1/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Tumor Rabdoide/tratamento farmacológico , Tumor Rabdoide/genética , Tumor Rabdoide/metabolismo , Receptores Notch , Epigenômica , DNA Helicases , Proteínas Nucleares , Fatores de Transcrição/genética
5.
Cell Genom ; 2(2): 100095, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35187519

RESUMO

Pancreatic cancer (PDAC) is a highly aggressive malignancy for which the identification of novel therapies is urgently needed. Here, we establish a human PDAC organoid biobank from 31 genetically distinct lines, covering a representative range of tumor subtypes, and demonstrate that these reflect the molecular and phenotypic heterogeneity of primary PDAC tissue. We use CRISPR-Cas9 genome editing and drug screening to characterize drug-gene interactions with ARID1A and BRCA2. We find that missense- but not frameshift mutations in the PDAC driver gene ARID1A are associated with increased sensitivity to the kinase inhibitors dasatinib (p < 0.0001) and VE-821 (p < 0.0001). We conduct an automated drug-repurposing screen with 1,172 FDA-approved compounds, identifying 26 compounds that effectively kill PDAC organoids, including 19 chemotherapy drugs currently approved for other cancer types. We validate the activity of these compounds in vitro and in vivo. The in vivo validated hits include emetine and ouabain, compounds which are approved for non-cancer indications and which perturb the ability of PDAC organoids to respond to hypoxia. Our study provides proof-of-concept for advancing precision oncology and identifying candidates for drug repurposing via genome editing and drug screening in tumor organoid biobanks.

6.
Cancers (Basel) ; 13(23)2021 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-34885181

RESUMO

Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive soft tissue sarcoma with a lack of effective treatment options and a poor prognosis. DSRCT is characterized by a chromosomal translocation, resulting in the EWSR1-WT1 gene fusion. The molecular mechanisms driving DSRCT are poorly understood, and a paucity of preclinical models hampers DSRCT research. Here, we establish a novel primary patient-derived DSRCT in vitro model, recapitulating the original tumor. We find that EWSR1-WT1 expression affects cell shape and cell survival, and we identify downstream target genes of the EWSR1-WT1 fusion. Additionally, this preclinical in vitro model allows for medium-throughput drug screening. We discover sensitivity to several drugs, including compounds targeting RTKs. MERTK, which has been described as a therapeutic target for several malignancies, correlates with EWSR1-WT1 expression. Inhibition of MERTK with the small-molecule inhibitor UNC2025 results in reduced proliferation of DSRCT cells in vitro, suggesting MERTK as a therapeutic target in DSRCT. This study underscores the usefulness of preclinical in vitro models for studying molecular mechanisms and potential therapeutic options.

7.
Nat Biomed Eng ; 5(2): 179-189, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33495639

RESUMO

Base editors are RNA-programmable deaminases that enable precise single-base conversions in genomic DNA. However, off-target activity is a concern in the potential use of base editors to treat genetic diseases. Here, we report unbiased analyses of transcriptome-wide and genome-wide off-target modifications effected by cytidine base editors in the liver of mice with phenylketonuria. The intravenous delivery of intein-split cytidine base editors by dual adeno-associated viruses led to the repair of the disease-causing mutation without generating off-target mutations in the RNA and DNA of the hepatocytes. Moreover, the transient expression of a cytidine base editor mRNA and a relevant single-guide RNA intravenously delivered by lipid nanoparticles led to ~21% on-target editing and to the reversal of the disease phenotype; there were also no detectable transcriptome-wide and genome-wide off-target edits. Our findings support the feasibility of therapeutic cytidine base editing to treat genetic liver diseases.


Assuntos
Citidina/genética , DNA/genética , Edição de Genes/métodos , Hepatócitos/metabolismo , RNA/genética , Adenoviridae/fisiologia , Animais , Vetores Genéticos/fisiologia , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL
8.
Cell Stem Cell ; 26(3): 431-440.e8, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142663

RESUMO

Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-ß-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-ß resistance and attenuate TGF-ß target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Organoides , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Testes Genéticos , Humanos , Intestinos , Fator de Crescimento Transformador beta
9.
Bio Protoc ; 9(1)2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30675496

RESUMO

The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating small insertion/deletion (indel) mutations, or by homology-directed repair (HDR). If ectopic donor templates are provided, the latter mechanism allows editing with single-nucleotide precision. The preference of mammalian cells to repair DSBs by NHEJ rather than HDR, however, limits the potential of CRISPR-Cas9 for applications where precise editing is needed. To enhance the efficiency of DSB repair by HDR from donor templates, we recently engineered a CRISPR-Cas9 system where the template DNA is bound to the Cas9 enzyme. In short, single-stranded oligonucleotides were labeled with O6-benzylguanine (BG), and covalently linked to a Cas9-SNAP-tag fusion protein to form a ribonucleoprotein-DNA (RNPD) complex consisting of the Cas9 nuclease, the sgRNA, and the repair template. Here, we provide a detailed protocol how to generate O6-benzylguanine (BG)-linked DNA repair templates, produce recombinant Cas9-SNAP-tag fusion proteins, in vitro transcribe single guide RNAs, and transfect RNPDs into various mammalian cells.

10.
Nat Med ; 24(10): 1519-1525, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30297904

RESUMO

CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1, using recently developed CRISPR-Cas-associated base editors2-4. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pahenu2 gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pahenu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.


Assuntos
Sistemas CRISPR-Cas/genética , Hepatopatias/terapia , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/terapia , Animais , DNA/genética , DNA/uso terapêutico , Dependovirus/genética , Modelos Animais de Doenças , Edição de Genes , Terapia Genética/métodos , Humanos , Fígado/metabolismo , Fígado/patologia , Hepatopatias/genética , Hepatopatias/metabolismo , Hepatopatias/patologia , Camundongos , Fenilalanina/sangue , Fenilalanina Hidroxilase/uso terapêutico , Fenilcetonúrias/genética , Fenilcetonúrias/metabolismo , Fenilcetonúrias/patologia , Reparo de DNA por Recombinação/genética
11.
Elife ; 72018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29809142

RESUMO

The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Reparo do DNA por Junção de Extremidades , DNA/metabolismo , Edição de Genes/métodos , Reparo de DNA por Recombinação , Proteína 9 Associada à CRISPR/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/química , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Loci Gênicos , Guanidinas/química , Células HEK293 , Humanos , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem/métodos
12.
Cell Rep ; 24(5): 1363-1376, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30067989

RESUMO

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the second most frequent cause of cancer-related mortality worldwide. The multikinase inhibitor sorafenib is the only treatment option for advanced HCC. Due to tumor heterogeneity, its efficacy greatly varies between patients and is limited due to adverse effects and drug resistance. Current in vitro models fail to recapitulate key features of HCCs. We report the generation of long-term organoid cultures from tumor needle biopsies of HCC patients with various etiologies and tumor stages. HCC organoids retain the morphology as well as the expression pattern of HCC tumor markers and preserve the genetic heterogeneity of the originating tumors. In a proof-of-principle study, we show that liver cancer organoids can be used to test sensitivity to sorafenib. In conclusion, organoid models can be derived from needle biopsies of liver cancers and provide a tool for developing tailored therapies.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Organoides/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Técnicas de Cultura de Tecidos/métodos
13.
Adv Mater ; 30(43): e1801621, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30203567

RESUMO

Epithelial organoids are simplified models of organs grown in vitro from embryonic and adult stem cells. They are widely used to study organ development and disease, and enable drug screening in patient-derived primary tissues. Current protocols, however, rely on animal- and tumor-derived basement membrane extract (BME) as a 3D scaffold, which limits possible applications in regenerative medicine. This prompted us to study how organoids interact with their matrix, and to develop a well-defined hydrogel that supports organoid generation and growth. It is found that soft fibrin matrices provide suitable physical support, and that naturally occurring Arg-Gly-Asp (RGD) adhesion domains on the scaffold, as well as supplementation with laminin-111, are key parameters required for robust organoid formation and expansion. The possibility to functionalize fibrin via factor XIII-mediated anchoring also allows to covalently link fluorescent nanoparticles to the matrix for 3D traction force microscopy. These measurements suggest that the morphogenesis of budding intestinal organoids results from internal pressure combined with higher cell contractility in the regions containing differentiated cells compared to the regions containing stem cells. Since the fibrin/laminin matrix supports long-term expansion of all tested murine and human epithelial organoids, this hydrogel can be widely used as a defined equivalent to BME.


Assuntos
Epitélio/crescimento & desenvolvimento , Fibrina , Hidrogéis , Laminina , Organoides/crescimento & desenvolvimento , Alicerces Teciduais , Animais , Adesão Celular , Linhagem Celular , Humanos , Intestino Delgado/crescimento & desenvolvimento , Fígado/crescimento & desenvolvimento , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pâncreas/crescimento & desenvolvimento , Células-Tronco/fisiologia , Propriedades de Superfície , Técnicas de Cultura de Tecidos
14.
Stem Cell Reports ; 10(3): 712-724, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29539434

RESUMO

Generating an unlimited source of human insulin-producing cells is a prerequisite to advance ß cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDHhi), express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC), and can form new organoids in contrast to ALDHlo cells. Interestingly, gene expression profiling revealed that ALDHhi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS+) cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDHhi cells formed INS+ cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential.


Assuntos
Diferenciação Celular/fisiologia , Organoides/fisiologia , Células-Tronco/patologia , Adulto , Animais , Proliferação de Células/fisiologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Camundongos , Organoides/metabolismo , Células-Tronco/metabolismo
15.
Diabetes ; 63(2): 410-20, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24130334

RESUMO

Upon a nutrient challenge, L cells produce glucagon-like peptide 1 (GLP-1), a powerful stimulant of insulin release. Strategies to augment endogenous GLP-1 production include promoting L-cell differentiation and increasing L-cell number. Here we present a novel in vitro platform to generate functional L cells from three-dimensional cultures of mouse and human intestinal crypts. We show that short-chain fatty acids selectively increase the number of L cells, resulting in an elevation of GLP-1 release. This is accompanied by the upregulation of transcription factors associated with the endocrine lineage of intestinal stem cell development. Thus, our platform allows us to study and modulate the development of L cells in mouse and human crypts as a potential basis for novel therapeutic strategies in patients with type 2 diabetes.


Assuntos
Células Enteroendócrinas/fisiologia , Intestino Delgado/citologia , Intestino Delgado/fisiologia , Organoides/citologia , Animais , Técnicas de Cultura de Células , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Tecidos
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