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1.
Zhonghua Wei Chang Wai Ke Za Zhi ; 22(11): 1095-1100, 2019 Nov 25.
Artigo em Chinês | MEDLINE | ID: mdl-31770844

RESUMO

Colonic organoids are three-dimensional organotypic cultures of the colonic stem cells or pluripotent stem cells. Its essence is the culture of colonic stem cells or pluripotent stem cells, and their derived intestinal epithelial cells, intestinal endocrine cells and goblet cells in basement membrane extract with specific growth factors. Colonic organoids are comprised of all major types of colonic epithelial cells and represent the architecture and function remarkably similar to those of the colonic epithelium, faithfully recapitulating the functional colonic epithelium ex vivo. As a superior basic experimental model, colonic organoids are representing advantages over conventional cell models and animal models in many aspects, such as high successful rate, short productive cycle, and high consistency with source tissue. Since first reported in 2011, colonic organoids have soon become an important topic in the field of colonic diseases. It has now been applied in the field of physiology of colonic epithelium, infectious diarrhea, ulcerative colitis, regeneration of intestinal injury, and colon tumors. In this review, we summarize the research advances of establishment and application of colonic organoids.


Assuntos
Colo/fisiologia , Mucosa Intestinal/fisiologia , Organoides/fisiologia , Células-Tronco/fisiologia , Animais , Colo/citologia , Humanos , Intestinos
2.
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
3.
Mol Cells ; 42(9): 617-627, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31564073

RESUMO

Brain organoids are an exciting new technology with the potential to significantly change our understanding of the development and disorders of the human brain. With step-by-step differentiation protocols, three-dimensional neural tissues are self-organized from pluripotent stem cells, and recapitulate the major millstones of human brain development in vitro. Recent studies have shown that brain organoids can mimic the spatiotemporal dynamicity of neurogenesis, the formation of regional neural circuitry, and the integration of glial cells into a neural network. This suggests that brain organoids could serve as a representative model system to study the human brain. In this review, we will overview the development of brain organoid technology, its current progress and applications, and future prospects of this technology.


Assuntos
Encéfalo/fisiologia , Organoides/fisiologia , Engenharia Tecidual/tendências , Encefalopatias/patologia , Humanos
4.
Biol Sex Differ ; 10(1): 47, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492202

RESUMO

BACKGROUND: Sexual dimorphism in biological responses is a critical knowledge for therapeutic proposals. However, gender differences in intestinal stem cell physiology have been poorly studied. Given the important role of the protease-activated receptor PAR2 in the control of colon epithelial primitive cells and cell cycle genes, we have performed a sex-based comparison of its expression and of the effects of PAR2 activation or knockout on cell proliferation and survival functions. METHODS: Epithelial primitive cells isolated from colons from male and female mice were cultured as colonoids, and their number and size were measured. PAR2 activation was triggered by the addition of SLIGRL agonist peptide in the culture medium. PAR2-deficient mice were used to study the impact of PAR2 expression on colon epithelial cell culture and gene expression. RESULTS: Colonoids from female mice were more abundant and larger compared to males, and these differences were further increased after PAR2 activation by specific PAR2 agonist peptide. The proliferation of male epithelial cells was lower compared to females but was specifically increased in PAR2 knockout male cells. PAR2 expression was higher in male colon cells compared to females and controlled the gene expression and activation of key negative signals of the primitive cell proliferation. This PAR2-dependent brake on the proliferation of male colon primitive cells was correlated with stress resistance. CONCLUSIONS: Altogether, these data demonstrate that there is a sexual dimorphism in the PAR2-dependent regulation of primitive cells of the colon crypt.


Assuntos
Colo/citologia , Receptor PAR-2/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Feminino , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organoides/fisiologia , Receptor PAR-2/genética , Caracteres Sexuais
5.
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
6.
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
7.
Nat Protoc ; 14(6): 1884-1925, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110298

RESUMO

Pediatric liver transplantation is often required as a consequence of biliary disorders because of the lack of alternative treatments for repairing or replacing damaged bile ducts. To address the lack of availability of pediatric livers suitable for transplantation, we developed a protocol for generating bioengineered biliary tissue suitable for biliary reconstruction. Our platform allows the derivation of cholangiocyte organoids (COs) expressing key biliary markers and retaining functions of primary extra- or intrahepatic duct cholangiocytes within 2 weeks of isolation. COs are subsequently seeded on polyglycolic acid (PGA) scaffolds or densified collagen constructs for 4 weeks to generate bioengineered tissue retaining biliary characteristics. Expertise in organoid culture and tissue engineering is desirable for optimal results. COs correspond to mature functional cholangiocytes, differentiating our method from alternative organoid systems currently available that propagate adult stem cells. Consequently, COs provide a unique platform for studies in biliary physiology and pathophysiology, and the resulting bioengineered tissue has broad applications for regenerative medicine and cholangiopathies.


Assuntos
Ductos Biliares/citologia , Ductos Biliares/fisiologia , Organoides/citologia , Organoides/fisiologia , Regeneração , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Separação Celular/métodos , Células Cultivadas , Desenho de Equipamento , Humanos , Camundongos , Engenharia Tecidual/instrumentação , Tecidos Suporte/química
8.
Dev Cell ; 49(3): 317-324, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31063751

RESUMO

Cancer has joined heart disease as the leading source of mortality in the US. In an era of organoids, patient-derived xenografts, and organs on a chip, model organisms continue to thrive with a combination of powerful genetic tools, rapid pace of discovery, and affordability. Model organisms enable the analysis of both the tumor and its associated microenvironment, aspects that are particularly relevant to our understanding of metastasis and drug resistance. In this Perspective, we explore some of the strengths of fruit flies and zebrafish for addressing fundamental cancer questions and how these two organisms can contribute to identifying promising therapeutic candidates.


Assuntos
Modelos Animais de Doenças , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Drosophila melanogaster , Humanos , Organoides/metabolismo , Organoides/fisiologia , Microambiente Tumoral/fisiologia , Peixe-Zebra
11.
Med Sci (Paris) ; 35(5): 470-477, 2019 May.
Artigo em Francês | MEDLINE | ID: mdl-31115331

RESUMO

This review focus on kidney organoids derived from pluripotent stem cells, which become a real alternative to the use of in vitro cellular models or in vivo animals models. The comprehension of the key steps involved during kidney embryonic development led to the establishment of protocols enabling the differentiation of pluripotent stem cells into kidney organoids that are highly complex and organized structures, composed of various renal cell types. These mini-organs are endowed with major applications: the possibility to control iPSC genome (by selecting patients with specific disease or by genome editing) allows the generation of kidney organoïds which recapitulate important physiopathological mechanisms such as cyste formation in renal polycystic disease. Kidney organoids can also be used in high-throughput screening to fasten the screening of nephrotoxic/therapeutic compounds. Finally, kidney organoids have a huge interest in the context of tissue repair, which remains for now a challenging goal linked with technological barriers that need still to be overcome.


Assuntos
Rim , Organoides , Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Avaliação Pré-Clínica de Medicamentos , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas , Rim/embriologia , Rim/fisiologia , Organoides/fisiologia
12.
Mol Biol Cell ; 30(10): 1129-1137, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31034354

RESUMO

Organoids derived from stem cells or tissues in culture can develop into structures that resemble the in vivo anatomy and physiology of intact organs. Human organoid cultures provide the potential to study human development and model disease processes with the same scrutiny and depth of analysis customary for research with nonhuman model organisms. Resembling the complexity of the actual tissue or organ, patient-derived human organoid studies may accelerate medical research, creating new opportunities for tissue engineering and regenerative medicine, generating knowledge and tools for preclinical studies, including drug development and testing. Biologists are drawn to this system as a new "model organism" to study complex disease phenotypes and genetic variability among individuals using patient-derived tissues. The American Society for Cell Biology convened a task force to report on the potential, challenges, and limitations for human organoid research. The task force suggests ways to ease the entry for new researchers into the field and how to facilitate broader use of this new model organism within the research community. This includes guidelines for reproducibility, culturing, sharing of patient materials, patient consent, training, and communication with the public.


Assuntos
Organoides/metabolismo , Organoides/fisiologia , Animais , Pesquisa Biomédica , Técnicas de Cultura de Células/métodos , Humanos , Modelos Biológicos , Organoides/citologia , Medicina Regenerativa , Reprodutibilidade dos Testes , Células-Tronco , Engenharia Tecidual/métodos
13.
BMC Biol ; 17(1): 33, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975131

RESUMO

BACKGROUND: Large animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures. RESULTS: Organoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research. CONCLUSIONS: We establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications.


Assuntos
Intestinos/fisiologia , Organoides/fisiologia , Animais , Doenças do Cão/fisiopatologia , Cães , Gastroenterologia , Intestinos/fisiopatologia , Organoides/fisiopatologia , Células-Tronco/citologia , Pesquisa Médica Translacional
14.
Dig Liver Dis ; 51(6): 753-760, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30948332

RESUMO

Most of the research behind new medical advances is carried out using either animal models or cancer cells, which both have their disadvantage in particular with regard to medical applications such as personalized medicine and novel therapeutic approaches. However, recent advances in stem cell biology have enabled long-term culturing of organotypic intestinal or hepatic tissues derived from tissue resident or pluripotent stem cells. These 3D structures, denoted as organoids, represent a substantial advance in structural and functional complexity over traditional in vitro cell culture models that are often non-physiological and transformed. They can recapitulate the in vivo architecture, functionality and genetic signature of the corresponding tissue. The opportunity to model epithelial cell biology, epithelial turnover, barrier dynamics, immune-epithelial communication and host-microbe interaction more efficiently than previous culture systems, greatly enhance the translational potential of organotypic hepato-gastrointestinal culture systems. Thus there is increasing interest in using such cultured cells as a source for tissue engineering, regenerative medicine and personalized medicine. This review will highlight some of the established and also some exciting novel perspectives on organoids in the fields of gastroenterology and hepatology.


Assuntos
Doença , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Organoides/citologia , Organoides/fisiologia , Animais , Gastroenterologia/tendências , Humanos , Especificidade de Órgãos , Medicina de Precisão/métodos , Medicina Regenerativa/métodos , Pesquisa Médica Translacional
15.
PLoS Genet ; 15(3): e1008076, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30925167

RESUMO

Organoid cultures derived from colorectal cancer (CRC) samples are increasingly used as preclinical models for studying tumor biology and the effects of targeted therapies under conditions capturing in vitro the genetic make-up of heterogeneous and even individual neoplasms. While 3D cultures are initiated from surgical specimens comprising multiple cell populations, the impact of tumor heterogeneity on drug effects in organoid cultures has not been addressed systematically. Here we have used a cohort of well-characterized CRC organoids to study the influence of tumor heterogeneity on the activity of the KRAS/MAPK-signaling pathway and the consequences of treatment by inhibitors targeting EGFR and downstream effectors. MAPK signaling, analyzed by targeted proteomics, shows unexpected heterogeneity irrespective of RAS mutations and is associated with variable responses to EGFR inhibition. In addition, we obtained evidence for intratumoral heterogeneity in drug response among parallel "sibling" 3D cultures established from a single KRAS-mutant CRC. Our results imply that separate testing of drug effects in multiple subpopulations may help to elucidate molecular correlates of tumor heterogeneity and to improve therapy response prediction in patients.


Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias Colorretais/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Neoplasias Colorretais/fisiopatologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Genes erbB-1 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Mutação , Organoides/metabolismo , Organoides/fisiologia , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Transdução de Sinais , Proteínas ras/genética
17.
J Vis Exp ; (143)2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30735176

RESUMO

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.


Assuntos
Organoides/fisiologia , Próstata/fisiologia , Técnicas de Cultura de Tecidos/métodos , Células Epiteliais/citologia , Humanos , Masculino
19.
Neurosci Res ; 138: 33-42, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30326251

RESUMO

Humans have a large and gyrencephalic brain. The higher intellectual ability of humans is dependent on the proper development of the brain. Brain malformation is often associated with cognitive dysfunction. It is thus important to know how our brain grows during development. Several animal species have been used as models to understand the mechanisms of brain development, and have provided us with basic information in this regard. It has been revealed that mammalian brain development basically proceeds through a similar process by common mechanisms, including neural stem cell proliferation and neurogenesis. However, humans also display species-specific features in these processes. These differences seem to be important for building the proper human brain structure. Analysis of these human-specific features requires human brain samples, which are difficult to obtain due to both ethical and practical reasons. Nevertheless, brain organoids derived from human pluripotent stem cells can be used as models to study human brain development and pathology because such organoids can partly recapitulate human fetal developmental processes. In this review, we will review some human-specific features during brain development and discuss brain organoid technology as a model system. We will especially focusing on neocortical development.


Assuntos
Modelos Biológicos , Neocórtex/crescimento & desenvolvimento , Organoides/fisiologia , Animais , Humanos , Especificidade da Espécie , Técnicas de Cultura de Tecidos/métodos
20.
Clin Pharmacol Ther ; 105(1): 79-85, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30415499

RESUMO

Human cardiac drug discovery and disease modeling face challenges in recapitulating cellular complexity and animal-to-human translation due to the limitations of conventional 2D cell culture and animal models. The development of human cardiac organoid technologies could help in stimulating and maintaining differentiated cell functions for extended periods of time. By closely mimicking in vivo organ functions in vitro they could thereby help in overcoming the obstacles mentioned above. Through the construction of human cardiac organoids from pluripotent stem cell-derived cells, derived from patients with specific known genotypes and phenotypes, more complex and robust in vitro tools have recently become available for disease modeling. In this review, we will describe the relevance and importance of evolving organoid platforms in disease biology. We further provide examples of cardiac organoid platforms, which may lead the way toward future personalized medicine and drug discovery.


Assuntos
Cardiopatias/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Miócitos Cardíacos/patologia , Organoides/patologia , Animais , Células Cultivadas , Cardiopatias/fisiopatologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Técnicas de Cultura de Órgãos , Organoides/fisiologia
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