Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 154
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Cell ; 176(4): 913-927.e18, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30686581

RESUMO

Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.


Assuntos
Miócitos Cardíacos/citologia , Técnicas de Cultura de Tecidos/instrumentação , Engenharia Tecidual/métodos , Potenciais de Ação , Diferenciação Celular , Células Cultivadas , Fenômenos Eletrofisiológicos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Miocárdio/citologia , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Tecidos/métodos
2.
Nat Rev Mol Cell Biol ; 18(1): 56-67, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27876786

RESUMO

Human pluripotent stem cells (hPSCs) provide an unparalleled opportunity to establish in vitro differentiation models that will transform our approach to the study of human development. In the case of the blood system, these models will enable investigation of the earliest stages of human embryonic haematopoiesis that was previously not possible. In addition, they will provide platforms for studying the origins of human blood cell diseases and for generating de novo haematopoietic stem and progenitor cell populations for cell-based regenerative therapies.


Assuntos
Hematopoese/fisiologia , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Células-Tronco Pluripotentes/fisiologia
3.
Cell ; 155(1): 215-27, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-24074870

RESUMO

Hematopoietic stem cells (HSCs) develop from a specialized subpopulation of endothelial cells known as hemogenic endothelium (HE). Although the HE origin of HSCs is now well established in different species, the signaling pathways that control this transition remain poorly understood. Here, we show that activation of retinoic acid (RA) signaling in aorta-gonad-mesonephros-derived HE ex vivo dramatically enhanced its HSC potential, whereas conditional inactivation of the RA metabolizing enzyme retinal dehydrogenase 2 in VE-cadherin expressing endothelial cells in vivo abrogated HSC development. Wnt signaling completely blocked the HSC inductive effects of RA modulators, whereas inhibition of the pathway promoted the development of HSCs in the absence of RA signaling. Collectively, these findings position RA and Wnt signaling as key regulators of HSC development and in doing so provide molecular insights that will aid in developing strategies for their generation from pluripotent stem cells.


Assuntos
Células-Tronco Hematopoéticas/citologia , Tretinoína/metabolismo , Aldeído Oxirredutases/metabolismo , Animais , Aorta/citologia , Aorta/embriologia , Regulação para Baixo , Embrião de Mamíferos , Gônadas/citologia , Gônadas/embriologia , Células-Tronco Hematopoéticas/metabolismo , Mesonefro/citologia , Camundongos , Receptores do Ácido Retinoico/metabolismo , Via de Sinalização Wnt
4.
Cell ; 151(1): 221-32, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22981225

RESUMO

Directed differentiation of human embryonic stem cells (ESCs) into cardiovascular cells provides a model for studying molecular mechanisms of human cardiovascular development. Although it is known that chromatin modification patterns in ESCs differ markedly from those in lineage-committed progenitors and differentiated cells, the temporal dynamics of chromatin alterations during differentiation along a defined lineage have not been studied. We show that differentiation of human ESCs into cardiovascular cells is accompanied by programmed temporal alterations in chromatin structure that distinguish key regulators of cardiovascular development from other genes. We used this temporal chromatin signature to identify regulators of cardiac development, including the homeobox gene MEIS2. Using the zebrafish model, we demonstrate that MEIS2 is critical for proper heart tube formation and subsequent cardiac looping. Temporal chromatin signatures should be broadly applicable to other models of stem cell differentiation to identify regulators and provide key insights into major developmental decisions.


Assuntos
Diferenciação Celular , Cromatina , Células-Tronco Embrionárias/metabolismo , Coração/embriologia , Miocárdio/citologia , Animais , Epigênese Genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismo
5.
Cell ; 151(1): 206-20, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22981692

RESUMO

Heart development is exquisitely sensitive to the precise temporal regulation of thousands of genes that govern developmental decisions during differentiation. However, we currently lack a detailed understanding of how chromatin and gene expression patterns are coordinated during developmental transitions in the cardiac lineage. Here, we interrogated the transcriptome and several histone modifications across the genome during defined stages of cardiac differentiation. We find distinct chromatin patterns that are coordinated with stage-specific expression of functionally related genes, including many human disease-associated genes. Moreover, we discover a novel preactivation chromatin pattern at the promoters of genes associated with heart development and cardiac function. We further identify stage-specific distal enhancer elements and find enriched DNA binding motifs within these regions that predict sets of transcription factors that orchestrate cardiac differentiation. Together, these findings form a basis for understanding developmentally regulated chromatin transitions during lineage commitment and the molecular etiology of congenital heart disease.


Assuntos
Epigênese Genética , Redes Reguladoras de Genes , Miocárdio/citologia , Animais , Diferenciação Celular , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Coração/embriologia , Humanos , Camundongos , Fatores de Transcrição/metabolismo , Transcriptoma
6.
Eur J Haematol ; 113(4): 530-542, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38967591

RESUMO

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure disorder that often presents at infancy. Progress has been made in revealing causal mutated genes (SBDS and others), ribosome defects, and hematopoietic aberrations in SDS. However, the mechanism underlying the hematopoietic failure remained unknown, and treatment options are limited. Herein, we investigated the onset of SDS embryonic hematopoietic impairments. We generated SDS and control human-derived induced pluripotent stem cells (iPSCs). SDS iPSCs recapitulated the SDS hematological phenotype. Detailed stepwise evaluation of definitive hematopoiesis revealed defects that started at the early emerging hematopoietic progenitor (EHP) stage after mesoderm and hemogenic endothelium were normally induced. Hematopoietic potential of EHPs was markedly reduced, and the introduction of SBDS in SDS iPSCs improved colony formation. Transcriptome analysis revealed reduced expression of ribosome and oxidative phosphorylation-related genes in undifferentiated and differentiated iPSCs. However, certain pathways (e.g., DNA replication) and genes (e.g., CHCHD2) were exclusively or more severely dysregulated in EHPs compared with earlier and later stages. To our knowledge, this study offers for the first time an insight into the embryonic onset of human hematopoietic defects in an inherited bone marrow failure syndrome and reveals cellular and molecular aberrations at critical stages of hematopoietic development toward EHPs.


Assuntos
Diferenciação Celular , Hematopoese , Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes Induzidas , Síndrome de Shwachman-Diamond , Humanos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Perfilação da Expressão Gênica , Fenótipo , Mutação , Lipomatose/genética , Lipomatose/patologia , Lipomatose/metabolismo , Ribossomos/metabolismo , Ribossomos/genética , Biomarcadores , Transcriptoma , Proteínas
7.
Circulation ; 145(18): 1412-1426, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35089805

RESUMO

BACKGROUND: Human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs) have tremendous promise for application in cardiac regeneration, but their translational potential is limited by an immature phenotype. We hypothesized that large-scale manufacturing of mature hPSC-CMs could be achieved through culture on polydimethylsiloxane (PDMS)-lined roller bottles and that the transplantation of these cells would mediate better structural and functional outcomes than with conventional immature hPSC-CM populations. METHODS: We comprehensively phenotyped hPSC-CMs after in vitro maturation for 20 and 40 days on either PDMS or standard tissue culture plastic substrates. All hPSC-CMs were generated from a transgenic hPSC line that stably expressed a voltage-sensitive fluorescent reporter to facilitate in vitro and in vivo electrophysiological studies, and cardiomyocyte populations were also analyzed in vitro by immunocytochemistry, ultrastructure and fluorescent calcium imaging, and bulk and single-cell transcriptomics. We next compared outcomes after the transplantation of these populations into a guinea pig model of myocardial infarction using end points including histology, optical mapping of graft- and host-derived action potentials, echocardiography, and telemetric electrocardiographic monitoring. RESULTS: We demonstrated the economic generation of >1×108 mature hPSC-CMs per PDMS-lined roller bottle. Compared with their counterparts generated on tissue culture plastic substrates, PDMS-matured hPSC-CMs exhibited increased cardiac gene expression and more mature structural and functional properties in vitro. More important, intracardiac grafts formed with PDMS-matured myocytes showed greatly enhanced structure and alignment, better host-graft electromechanical integration, less proarrhythmic behavior, and greater beneficial effects on contractile function. CONCLUSIONS: We describe practical methods for the scaled generation of mature hPSC-CMs and provide the first evidence that the transplantation of more mature cardiomyocytes yields better outcomes in vivo.


Assuntos
Miócitos Cardíacos , Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Linhagem Celular , Cobaias , Humanos , Miócitos Cardíacos/metabolismo , Plásticos/metabolismo , Células-Tronco Pluripotentes/metabolismo
8.
Cell ; 132(4): 661-80, 2008 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-18295582

RESUMO

The potential to generate virtually any differentiated cell type from embryonic stem cells (ESCs) offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. To realize this potential, it is essential to be able to control ESC differentiation and to direct the development of these cells along specific pathways. Embryology has offered important insights into key pathways regulating ESC differentiation, resulting in advances in modeling gastrulation in culture and in the efficient induction of endoderm, mesoderm, and ectoderm and many of their downstream derivatives. This has led to the identification of new multipotential progenitors for the hematopoietic, neural, and cardiovascular lineages and to the development of protocols for the efficient generation of a broad spectrum of cell types including hematopoietic cells, cardiomyocytes, oligodendrocytes, dopamine neurons, and immature pancreatic beta cells. The next challenge will be to demonstrate the functional utility of these cells, both in vitro and in preclinical models of human disease.


Assuntos
Diferenciação Celular , Desenvolvimento Embrionário , Células-Tronco Embrionárias/citologia , Humanos , Transplante de Células-Tronco
9.
Nature ; 545(7655): 432-438, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28514439

RESUMO

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders.


Assuntos
Diferenciação Celular , Linhagem da Célula , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , Animais , Reprogramação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Endotélio/citologia , Feminino , Transplante de Células-Tronco Hematopoéticas , Proteínas Homeobox A10 , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Regulador Transcricional ERG/metabolismo
10.
Circulation ; 135(19): 1832-1847, 2017 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-28167635

RESUMO

BACKGROUND: Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions. METHODS: We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM. RESULTS: EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to ß-adrenergic stimulation mediated via canonical ß1- and ß2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair. CONCLUSIONS: We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions.


Assuntos
Células-Tronco Embrionárias/transplante , Insuficiência Cardíaca/terapia , Células-Tronco Pluripotentes Induzidas/transplante , Miócitos Cardíacos/transplante , Engenharia Tecidual/métodos , Remodelação Ventricular/fisiologia , Animais , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Insuficiência Cardíaca/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Miocárdio/citologia , Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Impressão Tridimensional , Ratos , Ratos Nus
11.
Development ; 142(24): 4253-65, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26493401

RESUMO

The efficient generation of hepatocytes from human pluripotent stem cells (hPSCs) requires the induction of a proper endoderm population, broadly characterized by the expression of the cell surface marker CXCR4. Strategies to identify and isolate endoderm subpopulations predisposed to the liver fate do not exist. In this study, we generated mouse monoclonal antibodies against human embryonic stem cell-derived definitive endoderm with the goal of identifying cell surface markers that can be used to track the development of this germ layer and its specification to a hepatic fate. Through this approach, we identified two endoderm-specific antibodies, HDE1 and HDE2, which stain different stages of endoderm development and distinct derivative cell types. HDE1 marks a definitive endoderm population with high hepatic potential, whereas staining of HDE2 tracks with developing hepatocyte progenitors and hepatocytes. When used in combination, the staining patterns of these antibodies enable one to optimize endoderm induction and hepatic specification from any hPSC line.


Assuntos
Biomarcadores/metabolismo , Diferenciação Celular , Endoderma/citologia , Hepatócitos/citologia , Células-Tronco Pluripotentes/citologia , Animais , Anticorpos/metabolismo , Linhagem Celular , Separação Celular , Ectoderma/citologia , Hepatócitos/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cinética , Mesoderma/citologia , Camundongos Endogâmicos BALB C , Pâncreas/embriologia , Células-Tronco Pluripotentes/metabolismo , Coloração e Rotulagem
12.
Blood ; 128(6): 783-93, 2016 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-27301863

RESUMO

Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro differentiation of human induced pluripotent stem cells (iPSCs) generated from patients with different recombination-activating gene 1 (RAG1) mutations to assess T-cell development and T-cell receptor (TCR) V(D)J recombination. RAG1-mutants from severe combined immunodeficient (SCID) patient cells showed a failure to sustain progression beyond the CD3(--)CD4(-)CD8(-)CD7(+)CD5(+)CD38(-)CD31(-/lo)CD45RA(+) stage of T-cell development to reach the CD3(-/+)CD4(+)CD8(+)CD7(+)CD5(+)CD38(+)CD31(+)CD45RA(-) stage. Despite residual mutant RAG1 recombination activity from an Omenn syndrome (OS) patient, similar impaired T-cell differentiation was observed, due to increased single-strand DNA breaks that likely occur due to heterodimers consisting of both an N-terminal truncated and a catalytically dead RAG1. Furthermore, deep-sequencing analysis of TCR-ß (TRB) and TCR-α (TRA) rearrangements of CD3(-)CD4(+)CD8(-) immature single-positive and CD3(+)CD4(+)CD8(+) double-positive cells showed severe restriction of repertoire diversity with preferential usage of few Variable, Diversity, and Joining genes, and skewed length distribution of the TRB and TRA complementary determining region 3 sequences from SCID and OS iPSC-derived cells, whereas control iPSCs yielded T-cell progenitors with a broadly diversified repertoire. Finally, no TRA/δ excision circles (TRECs), a marker of TRA/δ locus rearrangements, were detected in SCID and OS-derived T-lineage cells, consistent with a pre-TCR block in T-cell development. This study compares human T-cell development of SCID vs OS patients, and elucidates important differences that help to explain the wide range of immunologic phenotypes that result from different mutations within the same gene of various patients.


Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes Induzidas/patologia , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/patologia , Linfócitos T/patologia , Células Cultivadas , Quebras de DNA , Genes RAG-1 , Humanos , Lactente , Mutação , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Recombinação V(D)J
13.
Nat Mater ; 15(6): 669-78, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26950595

RESUMO

We report the fabrication of a scaffold (hereafter referred to as AngioChip) that supports the assembly of parenchymal cells on a mechanically tunable matrix surrounding a perfusable, branched, three-dimensional microchannel network coated with endothelial cells. The design of AngioChip decouples the material choices for the engineered vessel network and for cell seeding in the parenchyma, enabling extensive remodelling while maintaining an open-vessel lumen. The incorporation of nanopores and micro-holes in the vessel walls enhances permeability, and permits intercellular crosstalk and extravasation of monocytes and endothelial cells on biomolecular stimulation. We also show that vascularized hepatic tissues and cardiac tissues engineered by using AngioChips process clinically relevant drugs delivered through the vasculature, and that millimetre-thick cardiac tissues can be engineered in a scalable manner. Moreover, we demonstrate that AngioChip cardiac tissues implanted with direct surgical anastomosis to the femoral vessels of rat hindlimbs establish immediate blood perfusion.


Assuntos
Materiais Biocompatíveis/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Dispositivos Lab-On-A-Chip , Fígado/metabolismo , Monócitos/metabolismo , Miocárdio/citologia , Engenharia Tecidual , Alicerces Teciduais/química , Anastomose Cirúrgica , Animais , Fêmur/irrigação sanguínea , Fêmur/citologia , Fêmur/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Fígado/irrigação sanguínea , Fígado/citologia , Monócitos/citologia , Miocárdio/metabolismo , Porosidade , Ratos , Ratos Endogâmicos Lew , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos
14.
Mol Ther ; 24(3): 582-91, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26444081

RESUMO

Many applications of pluripotent stem cells (PSCs) require efficient editing of silent chromosomal genes. Here, we show that a major limitation in isolating edited clones is silencing of the selectable marker cassette after homologous recombination and that this can be overcome by using a ubiquitous chromatin opening element (UCOE) promoter-driven transgene. We use this strategy to edit the silent IL2RG locus in human PSCs with a recombinant adeno-associated virus (rAAV)-targeting vector in the absence of potentially genotoxic, site-specific nucleases and show that IL2RG is required for natural killer and T-cell differentiation of human PSCs. Insertion of an active UCOE promoter into a silent locus altered the histone modification and cytosine methylation pattern of surrounding chromatin, but these changes resolved when the UCOE promoter was removed. This same approach could be used to correct IL2RG mutations in X-linked severe combined immunodeficiency patient-derived induced PSCs (iPSCs), to prevent graft versus host disease in regenerative medicine applications, or to edit other silent genes.


Assuntos
Edição de Genes , Inativação Gênica , Subunidade gama Comum de Receptores de Interleucina/genética , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Sobrevivência Celular/genética , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Epigênese Genética , Técnicas de Inativação de Genes , Marcação de Genes , Loci Gênicos , Humanos , Células Matadoras Naturais/citologia , Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas , Subpopulações de Linfócitos T/citologia , Transgenes , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
15.
Development ; 140(12): 2597-610, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23715552

RESUMO

Osteoarthritis primarily affects the articular cartilage of synovial joints. Cell and/or cartilage replacement is a promising therapy, provided there is access to appropriate tissue and sufficient numbers of articular chondrocytes. Embryonic stem cells (ESCs) represent a potentially unlimited source of chondrocytes and tissues as they can generate a broad spectrum of cell types under appropriate conditions in vitro. Here, we demonstrate that mouse ESC-derived chondrogenic mesoderm arises from a Flk-1(-)/Pdgfrα(+) (F(-)P(+)) population that emerges in a defined temporal pattern following the development of an early cardiogenic F(-)P(+) population. Specification of the late-arising F(-)P(+) population with BMP4 generated a highly enriched population of chondrocytes expressing genes associated with growth plate hypertrophic chondrocytes. By contrast, specification with Gdf5, together with inhibition of hedgehog and BMP signaling pathways, generated a population of non-hypertrophic chondrocytes that displayed properties of articular chondrocytes. The two chondrocyte populations retained their hypertrophic and non-hypertrophic properties when induced to generate spatially organized proteoglycan-rich cartilage-like tissue in vitro. Transplantation of either type of chondrocyte, or tissue generated from them, into immunodeficient recipients resulted in the development of cartilage tissue and bone within an 8-week period. Significant ossification was not observed when the tissue was transplanted into osteoblast-depleted mice or into diffusion chambers that prevent vascularization. Thus, through stage-specific manipulation of appropriate signaling pathways it is possible to efficiently and reproducibly derive hypertrophic and non-hypertrophic chondrocyte populations from mouse ESCs that are able to generate distinct cartilage-like tissue in vitro and maintain a cartilage tissue phenotype within an avascular and/or osteoblast-free niche in vivo.


Assuntos
Cartilagem Articular/citologia , Condrócitos/citologia , Condrogênese , Células-Tronco Embrionárias/citologia , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Cartilagem Articular/metabolismo , Diferenciação Celular , Linhagem da Célula , Condrócitos/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Feminino , Fator 5 de Diferenciação de Crescimento/genética , Fator 5 de Diferenciação de Crescimento/metabolismo , Hipertrofia/metabolismo , Imuno-Histoquímica , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Osteoblastos/metabolismo , Osteogênese , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Fatores de Tempo
16.
Development ; 140(15): 3285-96, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23861064

RESUMO

Human pluripotent stem cells (hPSCs) represent a novel source of hepatocytes for drug metabolism studies and cell-based therapy for the treatment of liver diseases. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, owing to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood. Here, we show that the combination of three-dimensional cell aggregation and cAMP signaling enhance the maturation of hPSC-derived hepatoblasts to a hepatocyte-like population that displays expression profiles and metabolic enzyme levels comparable to those of primary human hepatocytes. Importantly, we also demonstrate that generation of the hepatoblast population capable of responding to cAMP is dependent on appropriate activin/nodal signaling in the definitive endoderm at early stages of differentiation. Together, these findings provide new insights into the pathways that regulate maturation of hPSC-derived hepatocytes and in doing so provide a simple and reproducible approach for generating metabolically functional cell populations.


Assuntos
AMP Cíclico/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Ativinas/metabolismo , Agregação Celular , Técnicas de Cultura de Células , Diferenciação Celular , Endoderma/citologia , Endoderma/metabolismo , Humanos , Proteína Nodal/metabolismo , Transdução de Sinais , Transcriptoma
17.
Nat Methods ; 10(8): 781-7, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23793239

RESUMO

Directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) and permit engineering of human myocardium in vitro. However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium. Here we describe a platform that combines three-dimensional cell cultivation with electrical stimulation to mature hPSC-derived cardiac tissues. We used quantitative structural, molecular and electrophysiological analyses to explain the responses of immature human myocardium to electrical stimulation and pacing. We demonstrated that the engineered platform allows for the generation of three-dimensional, aligned cardiac tissues (biowires) with frequent striations. Biowires submitted to electrical stimulation had markedly increased myofibril ultrastructural organization, elevated conduction velocity and improved both electrophysiological and Ca(2+) handling properties compared to nonstimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Engenharia Tecidual/métodos , Diferenciação Celular/fisiologia , Estimulação Elétrica , Fenômenos Eletrofisiológicos , Humanos , Microscopia Eletrônica de Transmissão , Miocárdio/ultraestrutura
18.
Stem Cells ; 33(7): 2148-57, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25865043

RESUMO

Recent advances in pluripotent stem cell biology and directed differentiation have identified a population of human cardiovascular progenitors that give rise to cardiomyocytes, smooth muscle, and endothelial cells. Because the heart develops from progenitors in 3D under constant mechanical load, we sought to test the effects of a 3D microenvironment and mechanical stress on differentiation and maturation of human cardiovascular progenitors into myocardial tissue. Progenitors were derived from embryonic stem cells, cast into collagen hydrogels, and left unstressed or subjected to static or cyclic mechanical stress. Compared to 2D culture, the unstressed 3D environment increased cardiomyocyte numbers and decreased smooth muscle numbers. Additionally, 3D culture suppressed smooth muscle α-actin content, suggesting diminished cell maturation. Cyclic stress-conditioning increased expression of several cardiac markers, including ß-myosin heavy chain and cardiac troponin T, and the tissue showed enhanced calcium dynamics and force production. There was no effect of mechanical loading on cardiomyocyte or smooth muscle specification. Thus, 3D growth conditions favor cardiac differentiation from cardiovascular progenitors, whereas 2D conditions promote smooth muscle differentiation. Mechanical loading promotes cardiomyocyte structural and functional maturation. Culture in 3-D facilitates understanding how cues such as mechanical stress affect the differentiation and morphogenesis of distinct cardiovascular cell populations into organized, functional human cardiovascular tissue. Stem Cells 2015;33:2148-2157.


Assuntos
Miocárdio/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Humanos , Miocárdio/citologia , Estresse Mecânico , Engenharia Tecidual
19.
Proc Natl Acad Sci U S A ; 110(49): E4698-707, 2013 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-24255110

RESUMO

Access to robust and information-rich human cardiac tissue models would accelerate drug-based strategies for treating heart disease. Despite significant effort, the generation of high-fidelity adult-like human cardiac tissue analogs remains challenging. We used computational modeling of tissue contraction and assembly mechanics in conjunction with microfabricated constraints to guide the design of aligned and functional 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues that we term cardiac microwires (CMWs). Miniaturization of the platform circumvented the need for tissue vascularization and enabled higher-throughput image-based analysis of CMW drug responsiveness. CMW tissue properties could be tuned using electromechanical stimuli and cell composition. Specifically, controlling self-assembly of 3D tissues in aligned collagen, and pacing with point stimulation electrodes, were found to promote cardiac maturation-associated gene expression and in vivo-like electrical signal propagation. Furthermore, screening a range of hPSC-derived cardiac cell ratios identified that 75% NKX2 Homeobox 5 (NKX2-5)+ cardiomyocytes and 25% Cluster of Differentiation 90 OR (CD90)+ nonmyocytes optimized tissue remodeling dynamics and yielded enhanced structural and functional properties. Finally, we demonstrate the utility of the optimized platform in a tachycardic model of arrhythmogenesis, an aspect of cardiac electrophysiology not previously recapitulated in 3D in vitro hPSC-derived cardiac microtissue models. The design criteria identified with our CMW platform should accelerate the development of predictive in vitro assays of human heart tissue function.


Assuntos
Microambiente Celular/fisiologia , Miocárdio/citologia , Células-Tronco Pluripotentes/citologia , Engenharia Tecidual/métodos , Fenômenos Biomecânicos , Estimulação Elétrica , Análise de Elementos Finitos , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Humanos , Antígenos Thy-1/metabolismo , Fatores de Transcrição/metabolismo
20.
Semin Cell Dev Biol ; 23(6): 701-10, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22750147

RESUMO

The loss of beta cells in Type I diabetes ultimately leads to insulin dependence and major complications that are difficult to manage by insulin injections. Given the complications associated with long-term administration of insulin, cell-replacement therapy is now under consideration as an alternative treatment that may someday provide a cure for this disease. Over the past 10 years, islet transplantation trials have demonstrated that it is possible to replenish beta cell function in Type I diabetes patients and, at least temporarily, eliminate their dependency on insulin. While not yet optimal, the success of these trials has provided proof-of-principle that cell replacement therapy is a viable option for treating diabetes. Limited access to donor islets has launched a search for alternative source of beta cells for cell therapy purposes and focused the efforts of many investigators on the challenge of deriving such cells from human embryonic (hESCs) and induced pluripotent stem cells (hiPSCs). Over the past five years, significant advances have been made in understanding the signaling pathways that control lineage development from human pluripotent stem cells (hPSCs) and as a consequence, it is now possible to routinely generate insulin producing cells from both hESCs and hiPSCs. While these achievements are impressive, significant challenges do still exist, as the majority of insulin producing cells generated under these conditions are polyhormonal and non functional, likely reflecting the emergence of the polyhormonal population that is known to arise in the early embryo during the phase of pancreatic development known as the 'first transition'. Functional beta cells, which arise during the second phase or transition of pancreatic development have been generated from hESCs, however they are detected only following transplantation of progenitor stage cells into immunocompromised mice. With this success, our challenge now is to define the pathways that control the development and maturation of this second transition population from hPSCs, and establish conditions for the generation of functional beta cells in vitro.


Assuntos
Diferenciação Celular , Diabetes Mellitus/terapia , Células Secretoras de Insulina/citologia , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células , Ensaios Clínicos como Assunto , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/transplante , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Células Secretoras de Insulina/metabolismo , Células-Tronco Pluripotentes/transplante
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa