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1.
Nat Cell Biol ; 22(3): 321-331, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32123335

RESUMO

CRISPR-Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR-Cas9-mediated homology-independent organoid transgenesis (CRISPR-HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR-HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter-in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin-uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Introdução de Genes/métodos , Organoides/citologia , Hepatócitos/citologia , Hepatócitos/ultraestrutura , Humanos , Intestinos/citologia , Fígado/citologia , Organoides/ultraestrutura , Fuso Acromático/ultraestrutura , Proteína Supressora de Tumor p53/fisiologia
2.
Nat Commun ; 11(1): 997, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081850

RESUMO

Prostate development depends on balanced cell proliferation and differentiation, and acetylated KLF5 is known to alter epithelial proliferation. It remains elusive whether post-translational modifications of transcription factors can differentially determine adult stem/progenitor cell fate. Here we report that, in human and mouse prostates, Klf5 is expressed in both basal and luminal cells, with basal cells preferentially expressing acetylated Klf5. Functionally, Klf5 is indispensable for maintaining basal progenitors, their luminal differentiation, and the proliferation of their basal and luminal progenies. Acetylated Klf5 is also essential for basal progenitors' maintenance and proper luminal differentiation, as deacetylation of Klf5 causes excess basal-to-luminal differentiation; attenuates androgen-mediated organoid organization; and retards postnatal prostate development. In basal progenitor-derived luminal cells, Klf5 deacetylation increases their proliferation and attenuates their survival and regeneration following castration and subsequent androgen restoration. Mechanistically, Klf5 deacetylation activates Notch signaling. Klf5 and its acetylation thus contribute to postnatal prostate development and regeneration by controlling basal progenitor cell fate.


Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Próstata/crescimento & desenvolvimento , Próstata/metabolismo , Acetilação , Androgênios/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Humanos , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Orquiectomia , Organoides/citologia , Organoides/metabolismo , Próstata/citologia , Regeneração , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo
3.
Nat Cell Biol ; 22(1): 60-73, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31907413

RESUMO

Defining the ontogeny of the human adaptive immune system during embryogenesis has implications for understanding childhood diseases including leukaemias and autoimmune conditions. Using RAG1:GFP human pluripotent stem cell reporter lines, we examined human T-cell genesis from pluripotent-stem-cell-derived haematopoietic organoids. Under conditions favouring T-cell development, RAG1+ cells progressively upregulated a cohort of recognized T-cell-associated genes, arresting development at the CD4+CD8+ stage. Sort and re-culture experiments showed that early RAG1+ cells also possessed B-cell, myeloid and erythroid potential. Flow cytometry and single-cell-RNA-sequencing data showed that early RAG1+ cells co-expressed the endothelial/haematopoietic progenitor markers CD34, VECAD and CD90, whereas imaging studies identified RAG1+ cells within CD31+ endothelial structures that co-expressed SOX17+ or the endothelial marker CAV1. Collectively, these observations provide evidence for a wave of human T-cell development that originates directly from haemogenic endothelium via a RAG1+ intermediate with multilineage potential.


Assuntos
Endotélio/citologia , Hemangioblastos/citologia , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Linhagem Celular , Desenvolvimento Embrionário/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Organoides/citologia
4.
Nat Commun ; 11(1): 215, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924806

RESUMO

Efficient generation of human induced pluripotent stem cell (hiPSC)-derived human intestinal organoids (HIOs) would facilitate the development of in vitro models for a variety of diseases that affect the gastrointestinal tract, such as inflammatory bowel disease or Cystic Fibrosis. Here, we report a directed differentiation protocol for the generation of mesenchyme-free HIOs that can be primed towards more colonic or proximal intestinal lineages in serum-free defined conditions. Using a CDX2eGFP iPSC knock-in reporter line to track the emergence of hindgut progenitors, we follow the kinetics of CDX2 expression throughout directed differentiation, enabling the purification of intestinal progenitors and robust generation of mesenchyme-free organoids expressing characteristic markers of small intestinal or colonic epithelium. We employ HIOs generated in this way to measure CFTR function using cystic fibrosis patient-derived iPSC lines before and after correction of the CFTR mutation, demonstrating their future potential for disease modeling and therapeutic screening applications.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Intestinos/fisiologia , Mesoderma/metabolismo , Organoides/metabolismo , Fator de Transcrição CDX2/metabolismo , Diferenciação Celular , Fibrose Cística , Células Epiteliais , Técnicas de Introdução de Genes , Vetores Genéticos , Humanos , Intestino Delgado , Organoides/citologia , Fator Nuclear 1 de Tireoide/genética
5.
Adv Exp Med Biol ; 1212: 179-220, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31025308

RESUMO

Insulin-dependent diabetes mellitus or type 1 diabetes mellitus (T1DM) is an auto-immune condition characterized by the loss of pancreatic ß-cells. The curative approach for highly selected patients is the pancreas or the pancreatic islet transplantation. Nevertheless, these options are limited by a growing shortage of donor organs and by the requirement of immunosuppression.Xenotransplantation of porcine islets has been extensively investigated. Nevertheless, the strong xenoimmunity and the risk of transmission of porcine endogenous retroviruses, have limited their application in clinic. Generation of ß-like cells from stem cells is one of the most promising strategies in regenerative medicine. Embryonic, and more recently, adult stem cells are currently the most promising cell sources exploited to generate functional ß-cells in vitro. A number of studies demonstrated that stem cells could generate functional pancreatic organoids (POs), able to restore normoglycemia when implanted in different preclinical diabetic models. Nevertheless, a gradual loss of function and cell dead are commonly detected when POs are transplanted in immunocompetent animals. So far, the main issue to be solved is the post-transplanted islet loss, due to the host immune attack. To avoid this hurdle, nanotechnology has provided a number of polymers currently under investigation for islet micro and macro-encapsulation. These new approaches, besides conferring PO immune protection, are able to supply oxygen and nutrients and to preserve PO morphology and long-term viability.Herein, we summarize the current knowledge on bioengineered POs and the stem cell differentiation platforms. We also discuss the in vitro strategies used to generate functional POs, and the protocols currently used to confer immune-protection against the host immune attack (micro- and macro-encapsulation). In addition, the most relevant ongoing clinical trials, and the most relevant hurdles met to move towards clinical application are revised.


Assuntos
Diabetes Mellitus Tipo 1/terapia , Ilhotas Pancreáticas/citologia , Organoides/citologia , Medicina Regenerativa/métodos , Células-Tronco/citologia , Animais , Diferenciação Celular , Diabetes Mellitus Tipo 1/patologia , Humanos , Transplante das Ilhotas Pancreáticas/métodos
6.
Nat Cell Biol ; 21(11): 1321-1333, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31685987

RESUMO

Following severe or chronic liver injury, adult ductal cells (cholangiocytes) contribute to regeneration by restoring both hepatocytes and cholangiocytes. We recently showed that ductal cells clonally expand as self-renewing liver organoids that retain their differentiation capacity into both hepatocytes and ductal cells. However, the molecular mechanisms by which adult ductal-committed cells acquire cellular plasticity, initiate organoids and regenerate the damaged tissue remain largely unknown. Here, we describe that ductal cells undergo a transient, genome-wide, remodelling of their transcriptome and epigenome during organoid initiation and in vivo following tissue damage. TET1-mediated hydroxymethylation licences differentiated ductal cells to initiate organoids and activate the regenerative programme through the transcriptional regulation of stem-cell genes and regenerative pathways including the YAP-Hippo signalling. Our results argue in favour of the remodelling of genomic methylome/hydroxymethylome landscapes as a general mechanism by which differentiated cells exit a committed state in response to tissue damage.


Assuntos
Proteínas de Ligação a DNA/genética , Epigênese Genética , Regeneração Hepática/genética , Fígado/metabolismo , Organoides/metabolismo , Proteínas Proto-Oncogênicas/genética , Transcriptoma , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ductos Biliares/citologia , Ductos Biliares/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Fígado/citologia , Masculino , Camundongos Transgênicos , Organoides/citologia , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais
7.
PLoS Biol ; 17(10): e3000498, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31613879

RESUMO

During gastrulation, the pluripotent epiblast self-organizes into the 3 germ layers-endoderm, mesoderm and ectoderm, which eventually form the entire embryo. Decades of research in the mouse embryo have revealed that a signaling cascade involving the Bone Morphogenic Protein (BMP), WNT, and NODAL pathways is necessary for gastrulation. In vivo, WNT and NODAL ligands are expressed near the site of gastrulation in the posterior of the embryo, and knockout of these ligands leads to a failure to gastrulate. These data have led to the prevailing view that a signaling gradient in WNT and NODAL underlies patterning during gastrulation; however, the activities of these pathways in space and time have never been directly observed. In this study, we quantify BMP, WNT, and NODAL signaling dynamics in an in vitro model of human gastrulation. Our data suggest that BMP signaling initiates waves of WNT and NODAL signaling activity that move toward the colony center at a constant rate. Using a simple mathematical model, we show that this wave-like behavior is inconsistent with a reaction-diffusion-based Turing system, indicating that there is no stable signaling gradient of WNT/NODAL. Instead, the final signaling state is homogeneous, and spatial differences arise only from boundary effects. We further show that the durations of WNT and NODAL signaling control mesoderm differentiation, while the duration of BMP signaling controls differentiation of CDX2-positive extra-embryonic cells. The identity of these extra-embryonic cells has been controversial, and we use RNA sequencing (RNA-seq) to obtain their transcriptomes and show that they closely resemble human trophoblast cells in vivo. The domain of BMP signaling is identical to the domain of differentiation of these trophoblast-like cells; however, neither WNT nor NODAL forms a spatial pattern that maps directly to the mesodermal region, suggesting that mesoderm differentiation is controlled dynamically by the combinatorial effect of multiple signals. We synthesize our data into a mathematical model that accurately recapitulates signaling dynamics and predicts cell fate patterning upon chemical and physical perturbations. Taken together, our study shows that the dynamics of signaling events in the BMP, WNT, and NODAL cascade in the absence of a stable signaling gradient control fate patterning of human gastruloids.


Assuntos
Proteína Morfogenética Óssea 4/genética , Gastrulação/genética , Mesoderma/metabolismo , Proteína Nodal/genética , Transdução de Sinais , Proteínas Wnt/genética , Benzotiazóis/farmacologia , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Gástrula/citologia , Gástrula/efeitos dos fármacos , Gástrula/metabolismo , Gastrulação/efeitos dos fármacos , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Modelos Biológicos , Modelos Estatísticos , Proteína Nodal/deficiência , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Proteínas Wnt/metabolismo
8.
Nat Commun ; 10(1): 4647, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31604927

RESUMO

Human embryonic stem cell-derived beta cells offer a promising cell-based therapy for diabetes. However, efficient stem cell to beta cell differentiation has proven difficult, possibly due to the lack of cross-talk with the appropriate mesenchymal niche. To define organ-specific niche signals, we isolated pancreatic and gastrointestinal stromal cells, and analyzed their gene expression during development. Our genetic studies reveal the importance of tightly regulated Hedgehog signaling in the pancreatic mesenchyme: inactivation of mesenchymal signaling leads to annular pancreas, whereas stroma-specific activation of signaling via loss of Hedgehog regulators, Sufu and Spop, impairs pancreatic growth and beta cell genesis. Genetic rescue and transcriptome analyses show that these Sufu and Spop knockout defects occur through Gli2-mediated activation of gastrointestinal stromal signals such as Wnt ligands. Importantly, inhibition of Wnt signaling in organoid and human stem cell cultures significantly promotes insulin-producing cell generation, altogether revealing the requirement for organ-specific regulation of stromal niche signals.


Assuntos
Células-Tronco Embrionárias/citologia , Proteínas Hedgehog/metabolismo , Células Secretoras de Insulina/citologia , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Diabetes Mellitus/terapia , Regulação para Baixo , Humanos , Células Secretoras de Insulina/transplante , Proteínas Nucleares/metabolismo , Organoides/citologia , Proteínas Repressoras/metabolismo , Proteínas Wnt/metabolismo
9.
Genes Dev ; 33(19-20): 1319-1345, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575677

RESUMO

There are now many reports of human kidney organoids generated via the directed differentiation of human pluripotent stem cells (PSCs) based on an existing understanding of mammalian kidney organogenesis. Such kidney organoids potentially represent tractable tools for the study of normal human development and disease with improvements in scale, structure, and functional maturation potentially providing future options for renal regeneration. The utility of such organotypic models, however, will ultimately be determined by their developmental accuracy. While initially inferred from mouse models, recent transcriptional analyses of human fetal kidney have provided greater insight into nephrogenesis. In this review, we discuss how well human kidney organoids model the human fetal kidney and how the remaining differences challenge their utility.


Assuntos
Rim/fisiologia , Modelos Biológicos , Organoides/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Rim/citologia , Rim/embriologia , Rim/crescimento & desenvolvimento , Organoides/citologia
10.
Nat Protoc ; 14(11): 3082-3100, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31554955

RESUMO

Blood vessels are fundamental to animal life and have critical roles in many diseases, such as stroke, myocardial infarction and diabetes. The vasculature is formed by endothelial cells that line the vessel and are covered with mural cells, specifically pericytes in smaller vessels and vascular smooth muscle cells (vSMCs) in larger-diameter vessels. Both endothelial cells and mural cells are essential for proper blood vessel function and can be derived from human pluripotent stem cells (hPSCs). Here, we describe a protocol to generate self-organizing 3D human blood vessel organoids from hPSCs that exhibit morphological, functional and molecular features of human microvasculature. These organoids are differentiated via mesoderm induction of hPSC aggregates and subsequent differentiation into endothelial networks and pericytes in a 3D collagen I-Matrigel matrix. Blood vessels form within 2-3 weeks and can be further grown in scalable suspension culture. Importantly, in vitro-differentiated human blood vessel organoids transplanted into immunocompromised mice gain access to the mouse circulation and specify into functional arteries, arterioles and veins.


Assuntos
Vasos Sanguíneos/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Engenharia Tecidual/métodos , Diferenciação Celular , Linhagem Celular , Colágeno/química , Combinação de Medicamentos , Endotélio Vascular/citologia , Humanos , Laminina/química , Microvasos/citologia , Neovascularização Fisiológica , Pericitos/citologia , Proteoglicanas/química , Tecidos Suporte/química
11.
Nature ; 574(7776): 112-116, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554966

RESUMO

Organogenesis is a complex and interconnected process that is orchestrated by multiple boundary tissue interactions1-7. However, it remains unclear how individual, neighbouring components coordinate to establish an integral multi-organ structure. Here we report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cells. The boundary interactions between anterior and posterior gut spheroids differentiated from human pluripotent stem cells enables retinoic acid-dependent emergence of hepato-biliary-pancreatic organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factors. Whereas transplant-derived tissues are dominated by midgut derivatives, long-term-cultured microdissected hepato-biliary-pancreatic organoids develop into segregated multi-organ anlages, which then recapitulate early morphogenetic events including the invagination and branching of three different and interconnected organ structures, reminiscent of tissues derived from mouse explanted foregut-midgut culture. Mis-segregation of multi-organ domains caused by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo8,9. In sum, we demonstrate that the experimental multi-organ integrated model can be established by the juxtapositioning of foregut and midgut tissues, and potentially serves as a tractable, manipulatable and easily accessible model for the study of complex human endoderm organogenesis.


Assuntos
Sistema Biliar/embriologia , Intestinos/embriologia , Fígado/embriologia , Modelos Biológicos , Morfogênese , Pâncreas/embriologia , Animais , Sistema Biliar/citologia , Biomarcadores/análise , Biomarcadores/metabolismo , Padronização Corporal , Endoderma/citologia , Endoderma/embriologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Intestinos/citologia , Fígado/citologia , Masculino , Camundongos , Organoides/citologia , Organoides/embriologia , Pâncreas/citologia , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Esferoides Celulares/transplante , Fatores de Transcrição HES-1/análise , Fatores de Transcrição HES-1/metabolismo
12.
Nat Commun ; 10(1): 4407, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562298

RESUMO

Understanding urothelial stem cell biology and differentiation has been limited by the lack of methods for their unlimited propagation. Here, we establish mouse urothelial organoids that can be maintained uninterruptedly for >1 year. Organoid growth is dependent on EGF and Wnt activators. High CD49f/ITGA6 expression features a subpopulation of organoid-forming cells expressing basal markers. Upon differentiation, multilayered organoids undergo reduced proliferation, decreased cell layer number, urothelial program activation, and acquisition of barrier function. Pharmacological modulation of PPARγ and EGFR promotes differentiation. RNA sequencing highlighted genesets enriched in proliferative organoids (i.e. ribosome) and transcriptional networks involved in differentiation, including expression of Wnt ligands and Notch components. Single-cell RNA sequencing (scRNA-Seq) analysis of the organoids revealed five clusters with distinct gene expression profiles. Together, with the use of γ-secretase inhibitors and scRNA-Seq, confirms that Notch signaling is required for differentiation. Urothelial organoids provide a powerful tool to study cell regeneration and differentiation.


Assuntos
Diferenciação Celular/genética , Integrina alfa6/genética , Organoides/metabolismo , Receptores Notch/metabolismo , Células-Tronco/metabolismo , Urotélio/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/farmacologia , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Integrina alfa6/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Organoides/citologia , Organoides/efeitos dos fármacos , Receptores Notch/genética , Análise de Célula Única/métodos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Urotélio/citologia
13.
Nat Cell Biol ; 21(8): 1015-1026, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31332348

RESUMO

Human liver cancers, including hepatocellular carcinomas and intra-hepatic cholangiocarcinomas, are often diagnosed late with poor prognosis. A better understanding of cancer initiation could provide potential preventive therapies and increase survival. Models for studying human liver cancer initiation are largely missing. Here, using directly reprogrammed human hepatocytes (hiHeps) and inactivation of p53 and RB, we established organoids possessing liver architecture and function. HiHep organoids were genetically engineered to model the initial alterations in human liver cancers. Bona fide hepatocellular carcinomas were developed by overexpressing c-Myc. Excessive mitochondrion-endoplasmic reticulum coupling induced by c-Myc facilitated hepatocellular carcinoma initiation and seemed to be a target of preventive treatment. Furthermore, through the analysis of human intra-hepatic cholangiocarcinoma-enriched mutations, we demonstrate that the RAS-induced lineage conversion from hepatocytes to intra-hepatic cholangiocarcinoma cells can be prevented by the combined inhibition of Notch and JAK-STAT. Together, hiHep organoids represent a system that can be genetically manipulated to model cancer initiation and identify potential preventive therapies.


Assuntos
Hepatócitos/citologia , Neoplasias Hepáticas/patologia , Fígado/patologia , Organoides/citologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Colangiocarcinoma/patologia , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Hepáticas/genética , Camundongos , Proteína Supressora de Tumor p53/genética
14.
EMBO J ; 38(14): e99299, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31304629

RESUMO

The metastatic progression of cancer is a multi-step process initiated by the local invasion of the peritumoral stroma. To identify the mechanisms underlying colorectal carcinoma (CRC) invasion, we collected live human primary cancer specimens at the time of surgery and monitored them ex vivo. This revealed that conventional adenocarcinomas undergo collective invasion while retaining their epithelial glandular architecture with an inward apical pole delineating a luminal cavity. To identify the underlying mechanisms, we used microscopy-based assays on 3D organotypic cultures of Caco-2 cysts as a model system. We performed two siRNA screens targeting Rho-GTPases effectors and guanine nucleotide exchange factors. These screens revealed that ROCK2 inhibition triggers the initial leader/follower polarization of the CRC cell cohorts and induces collective invasion. We further identified FARP2 as the Rac1 GEF necessary for CRC collective invasion. However, FARP2 activation is not sufficient to trigger leader cell formation and the concomitant inhibition of Myosin-II is required to induce invasion downstream of ROCK2 inhibition. Our results contrast with ROCK pro-invasive function in other cancers, stressing that the molecular mechanism of metastatic spread likely depends on tumour types and invasion mode.


Assuntos
Adenocarcinoma/metabolismo , Técnicas de Cultura de Células/métodos , Neoplasias Colorretais/metabolismo , Quinases Associadas a rho/metabolismo , Adenocarcinoma/genética , Animais , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Organoides/citologia , Organoides/metabolismo , RNA Interferente Pequeno/farmacologia , Quinases Associadas a rho/genética
16.
EMBO J ; 38(15): e101654, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31282586

RESUMO

Patient-derived tumour xenografts and tumour organoids have become important preclinical model systems for cancer research. Both models maintain key features from their parental tumours, such as genetic and phenotypic heterogeneity, which allows them to be used for a wide spectrum of applications. In contrast to patient-derived xenografts, organoids can be established and expanded with high efficiency from primary patient material. On the other hand, xenografts retain tumour-stroma interactions, which are known to contribute to tumorigenesis. In this review, we discuss recent advances in patient-derived tumour xenograft and tumour organoid model systems and compare their promises and challenges as preclinical models in cancer research.


Assuntos
Neoplasias/patologia , Organoides/patologia , Animais , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Humanos , Modelos Biológicos , Organoides/citologia , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Gut ; 68(12): 2228-2237, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300517

RESUMO

Organoid cultures have emerged as an alternative in vitro system to recapitulate tissues in a dish. While mouse models and cell lines have furthered our understanding of liver biology and associated diseases, they suffer in replicating key aspects of human liver tissue, in particular its complex architecture and metabolic functions. Liver organoids have now been established for multiple species from induced pluripotent stem cells, embryonic stem cells, hepatoblasts and adult tissue-derived cells. These represent a promising addition to our toolbox to gain a deeper understanding of this complex organ. In this perspective we will review the advances in the liver organoid field, its limitations and potential for biomedical applications.


Assuntos
Pesquisa Biomédica/métodos , Fígado/citologia , Modelos Biológicos , Organoides/fisiologia , Medicina Regenerativa/métodos , Animais , Técnicas de Cultura de Células , Linhagem Celular , Humanos , Organoides/citologia
18.
PLoS One ; 14(7): e0213114, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31295264

RESUMO

BACKGROUND: 2-Cl-C.OXT-A (COA-Cl) is a novel synthesized adenosine analog that activates Sphingosine-1-phosphate 1 receptor (S1P1R) and combines with the adenosine A1 receptor (A1R) in G proteins and was shown to enhance angiogenesis and improve the brain function in rat stroke models. However, the role of COA-Cl in hearts remains unclear. COA-Cl, which has a similar structure to xanthine derivatives, has the potential to suppress phosphodiesterase (PDE), which is an important factor involved in the beating of heart muscle. METHODS AND RESULTS: Cardiac organoids with fibroblasts, human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs), and hiPSC-derived endothelial cells (hiPSC-ECs) were cultured until they started beating. The beating and contraction of organoids were observed before and after the application of COA-Cl. COA-Cl significantly increased the beating rate and fractional area change in organoids. To elucidate the mechanism underlying these effects of COA-Cl on cardiac myocytes, pure hiPSC-CM spheroids were evaluated in the presence/absence of Suramin (antagonist of A1R). The effects of COA-Cl, SEW2871 (direct stimulator of S1P1R), two positive inotropes (Isoproterenol [ISO] and Forskolin [FSK]), and negative inotrope (Propranolol [PRP]) on spheroids were assessed based on the beating rates and cAMP levels. COA-Cl stimulated the beating rates about 1.5-fold compared with ISO and FSK, while PRP suppressed the beating rate. However, no marked changes were observed with SEW2871. COA-Cl, ISO, and FSK increased the cAMP level. In contrast, the level of cAMP did not change with PRP or SEW2871 treatment. The results were the same in the presence of Suramin as absence. Furthermore, an enzyme analysis showed that COA-Cl suppressed the PDE activity by half. CONCLUSIONS: COA-Cl, which has neovascularization effects, suppressed PDE and increased the contraction of cardiac organoids, independent of S1P1R and A1R. These findings suggest that COA-Cl may be useful as an inotropic agent for promoting angiogenesis in the future.


Assuntos
Adenosina/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Adenosina/análogos & derivados , Linhagem Celular , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/metabolismo
19.
Dev Cell ; 50(1): 102-116.e6, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31265809

RESUMO

The renal corpuscle of the kidney comprises a glomerular vasculature embraced by podocytes and supported by mesangial myofibroblasts, which ensure plasma filtration at the podocyte-generated slit diaphragm. With a spectrum of podocyte-expressed gene mutations causing chronic disease, an enhanced understanding of podocyte development and function to create relevant in vitro podocyte models is a clinical imperative. To characterize podocyte development, scRNA-seq was performed on human fetal kidneys, identifying distinct transcriptional signatures accompanying the differentiation of functional podocytes from progenitors. Interestingly, organoid-generated podocytes exhibited highly similar, progressive transcriptional profiles despite an absence of the vasculature, although abnormal gene expression was pinpointed in late podocytes. On transplantation into mice, organoid-derived podocytes recruited the host vasculature and partially corrected transcriptional profiles. Thus, human podocyte development is mostly intrinsically regulated and vascular interactions refine maturation. These studies support the application of organoid-derived podocytes to model disease and to restore or replace normal kidney functions.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes Induzidas/citologia , Glomérulos Renais/citologia , Organoides/citologia , Podócitos/citologia , Análise de Célula Única/métodos , Células Cultivadas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Glomérulos Renais/metabolismo , Organoides/metabolismo , Podócitos/metabolismo
20.
Med Sci (Paris) ; 35(6-7): 549-555, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31274085

RESUMO

The study of gut diseases is often limited by the access to human biological tissues and animal models that do not faithfully mimic the human pathologies. In this context, the development of intestinal organoids from human pluripotent stem cells is paving the way of gastrointestinal physiology and digestive disease study. In this review, we recall the embryonic development of the digestive tract and its translation to human pluripotent stem cell differentiation. We also present the different types of intestinal organoids that can be generated, as well as their applications in research.


Assuntos
Intestinos/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Gastroenteropatias/patologia , Gastroenteropatias/terapia , Trato Gastrointestinal/citologia , Trato Gastrointestinal/crescimento & desenvolvimento , Trato Gastrointestinal/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Intestinos/fisiologia , Organoides/fisiologia , Células-Tronco Pluripotentes/fisiologia , Regeneração/fisiologia , Técnicas de Cultura de Tecidos
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