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1.
Biomater Biosyst ; 8: 100068, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36824378

RESUMO

Current in vivo disease models and analysis methods for cardiac drug development have been insufficient in providing accurate and reliable predictions of drug efficacy and safety. Here, we propose a custom optical flow-based analysis method to quantitatively measure recordings of contracting cardiomyocytes on polydimethylsiloxane (PDMS), compatible with medium-throughput systems. Movement of the PDMS was examined by covalently bound fluorescent beads on the PDMS surface, differences caused by increased substrate stiffness were compared, and cells were stimulated with ß-agonist. We further validated the system using cardiomyocytes treated with endothelin-1 and compared their contractions against control and cells incubated with receptor antagonist bosentan. After validation we examined two MYBPC3-mutant patient-derived cell lines. Recordings showed that higher substrate stiffness resulted in higher contractile pressure, while beating frequency remained similar to control. ß-agonist stimulation resulted in both higher beating frequency as well as higher pressure values during contraction and relaxation. Cells treated with endothelin-1 showed an increased beating frequency, but a lower contraction pressure. Cells treated with both endothelin-1 and bosentan remained at control level of beating frequency and pressure. Lastly, both MYBPC3-mutant lines showed a higher beating frequency and lower contraction pressure. Our validated method is capable of automatically quantifying contraction of hiPSC-derived cardiomyocytes on a PDMS substrate of known shear modulus, returning an absolute value. Our method could have major benefits in a medium-throughput setting.

2.
PLoS Comput Biol ; 16(9): e1008203, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32976482

RESUMO

Novel studies conducting cardiac safety assessment using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are promising but might be limited by their specificity and predictivity. It is often challenging to correctly classify ion channel blockers or to sufficiently predict the risk for Torsade de Pointes (TdP). In this study, we developed a method combining in vitro and in silico experiments to improve machine learning approaches in delivering fast and reliable prediction of drug-induced ion-channel blockade and proarrhythmic behaviour. The algorithm is based on the construction of a dictionary and a greedy optimization, leading to the definition of optimal classifiers. Finally, we present a numerical tool that can accurately predict compound-induced pro-arrhythmic risk and involvement of sodium, calcium and potassium channels, based on hiPSC-CM field potential data.


Assuntos
Algoritmos , Arritmias Cardíacas , Canais Iônicos , Modelos Cardiovasculares , Miócitos Cardíacos , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/fisiopatologia , Fármacos Cardiovasculares/farmacologia , Biologia Computacional , Bases de Dados Factuais , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Torsades de Pointes/fisiopatologia
3.
Europace ; 22(9): 1431-1441, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32770183

RESUMO

AIMS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have proven valuable for studies in drug discovery and safety, although limitations regarding their structural and electrophysiological characteristics persist. In this study, we investigated the electrophysiological properties of Pluricyte® CMs, a commercially available hiPSC-CMs line with a ventricular phenotype, and assessed arrhythmia incidence by IKr block at the single-cell and 2D monolayer level. METHODS AND RESULTS: Action potentials were measured at different pacing frequencies, using dynamic clamp. Through voltage-clamp experiments, we determined the properties of INa, IKr, and ICaL. Intracellular Ca2+ measurements included Ca2+-transients at baseline and during caffeine perfusion. Effects of IKr block were assessed in single hiPSC-CMs and 2D monolayers (multi-electrode arrays). Action-potential duration (APD) and its rate dependence in Pluricyte® CMs were comparable to those reported for native human CMs. INa, IKr, and ICaL revealed amplitudes, kinetics, and voltage dependence of activation/inactivation similar to other hiPSC-CM lines and, to some extent, to native CMs. Near-physiological Ca2+-induced Ca2+ release, response to caffeine and excitation-contraction coupling gain characterized the cellular Ca2+-handling. Dofetilide prolonged the APD and field-potential duration, and induced early afterdepolarizations. Beat-to-beat variability of repolarization duration increased significantly before the first arrhythmic events in single Pluricyte® CMs and 2D monolayers, and predicted pending arrhythmias better than action-potential prolongation. CONCLUSION: Taking their ion-current characteristics and Ca2+ handling into account, Pluricyte® CMs are suitable for in vitro studies on action potentials and field potentials. Beat-to-beat variability of repolarization duration proved useful to evaluate the dynamics of repolarization instability and demonstrated its significance as proarrhythmic marker in hiPSC-CMs during IKr block.


Assuntos
Células-Tronco Pluripotentes Induzidas , Potenciais de Ação , Arritmias Cardíacas , Fenômenos Eletrofisiológicos , Humanos , Miócitos Cardíacos
4.
J Pharmacol Toxicol Methods ; 105: 106889, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32565326

RESUMO

Safety pharmacology is an essential part of drug development aiming to identify, evaluate and investigate undesirable pharmacodynamic properties of a drug primarily prior to clinical trials. In particular, cardiovascular adverse drug reactions (ADR) have halted many drug development programs. Safety pharmacology has successfully implemented a screening strategy to detect cardiovascular liabilities, but there is room for further refinement. In this setting, we present the INSPIRE project, a European Training Network in safety pharmacology for Early Stage Researchers (ESRs), funded by the European Commission's H2020-MSCA-ITN programme. INSPIRE has recruited 15 ESR fellows that will conduct an individual PhD-research project for a period of 36 months. INSPIRE aims to be complementary to ongoing research initiatives. With this as a goal, an inventory of collaborative research initiatives in safety pharmacology was created and the ESR projects have been designed to be complementary to this roadmap. Overall, INSPIRE aims to improve cardiovascular safety evaluation, either by investigating technological innovations or by adding mechanistic insight in emerging safety concerns, as observed in the field of cardio-oncology. Finally, in addition to its hands-on research pillar, INSPIRE will organize a number of summer schools and workshops that will be open to the wider community as well. In summary, INSPIRE aims to foster both research and training in safety pharmacology and hopes to inspire the future generation of safety scientists.


Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Desenvolvimento de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Farmacologia/métodos , Humanos , Segurança
5.
Artigo em Inglês | MEDLINE | ID: mdl-29355722

RESUMO

Safety pharmacology studies that evaluate drug candidates for potential cardiovascular liabilities remain a critical component of drug development. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have recently emerged as a new and promising tool for preclinical hazard identification and risk assessment of drugs. Recently, Pluriomics organized its first User Meeting entitled 'Combining Pluricyte® Cardiomyocytes & MEA for Safety Pharmacology applications', consisting of scientific sessions and live demonstrations, which provided the opportunity to discuss the application of hiPSC-CMs (Pluricyte® Cardiomyocytes) in cardiac safety assessment to support early decision making in safety pharmacology. This report summarizes the outline and outcome of this Pluriomics User Meeting, which took place on November 24-25, 2016 in Leiden (The Netherlands). To reflect the content of the communications presented at this meeting we have cited key scientific articles and reviews.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Miócitos Cardíacos/efeitos dos fármacos , Cardiotoxicidade/prevenção & controle , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/normas , Eletrodos , Guias como Assunto , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp/instrumentação , Técnicas de Patch-Clamp/métodos
6.
Methods Mol Biol ; 1353: 163-80, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-25626427

RESUMO

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can differentiate to cardiomyocytes in vitro, offering unique opportunities to investigate cardiac development and disease as well as providing a platform to perform drug and toxicity tests. Initial cardiac differentiation methods were based on either inductive co-culture or aggregation as embryoid bodies, often in the presence of fetal calf serum. More recently, monolayer differentiation protocols have evolved as feasible alternatives and are often performed in completely defined culture medium and substrates. Thus, our ability to efficiently and reproducibly generate cardiomyocytes from multiple different hESC and hiPSC lines has improved significantly.We have developed a directed differentiation monolayer protocol that can be used to generate cultures comprising ~50% cardiomyocytes, in which both the culture of the undifferentiated human pluripotent stem cells (hPSCs) and the differentiation procedure itself are defined and serum-free. The differentiation method is also effective for hPSCs maintained in other culture systems. In this chapter, we outline the differentiation protocol and describe methods to assess cardiac differentiation efficiency as well as to identify and quantify the yield of cardiomyocytes.


Assuntos
Técnicas de Cultura de Células/métodos , Reprogramação Celular , Células-Tronco Embrionárias/citologia , Inibidores Enzimáticos/farmacologia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Ativinas/farmacologia , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular , Colágeno/química , Combinação de Medicamentos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Laminina/química , Imagem Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Cultura Primária de Células , Proteoglicanas/química , Piridinas/farmacologia , Pirimidinas/farmacologia , Troponina T/genética , Troponina T/metabolismo
7.
Development ; 142(18): 3231-8, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26209647

RESUMO

Differentiated derivatives of human pluripotent stem cells (hPSCs) are often considered immature because they resemble foetal cells more than adult, with hPSC-derived cardiomyocytes (hPSC-CMs) being no exception. Many functional features of these cardiomyocytes, such as their cell morphology, electrophysiological characteristics, sarcomere organization and contraction force, are underdeveloped compared with adult cardiomyocytes. However, relatively little is known about how their gene expression profiles compare with the human foetal heart, in part because of the paucity of data on the human foetal heart at different stages of development. Here, we collected samples of matched ventricles and atria from human foetuses during the first and second trimester of development. This presented a rare opportunity to perform gene expression analysis on the individual chambers of the heart at various stages of development, allowing us to identify not only genes involved in the formation of the heart, but also specific genes upregulated in each of the four chambers and at different stages of development. The data showed that hPSC-CMs had a gene expression profile similar to first trimester foetal heart, but after culture in conditions shown previously to induce maturation, they cluster closer to the second trimester foetal heart samples. In summary, we demonstrate how the gene expression profiles of human foetal heart samples can be used for benchmarking hPSC-CMs and also contribute to determining their equivalent stage of development.


Assuntos
Diferenciação Celular/fisiologia , Feto/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/citologia , Transcriptoma , Feto/metabolismo , Perfilação da Expressão Gênica , Humanos
8.
J Cell Mol Med ; 18(8): 1509-18, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24981391

RESUMO

It has been known for over 20 years that foetal calf serum can induce hypertrophy in cultured cardiomyocytes but this is rarely considered when examining cardiomyocytes derived from pluripotent stem cells (PSC). Here, we determined how serum affected cardiomyocytes from human embryonic- (hESC) and induced pluripotent stem cells (hiPSC) and hiPSC from patients with hypertrophic cardiomyopathy linked to a mutation in the MYBPC3 gene. We first confirmed previously published hypertrophic effects of serum on cultured neonatal rat cardiomyocytes demonstrated as increased cell surface area and beating frequency. We then found that serum increased the cell surface area of hESC- and hiPSC-derived cardiomyocytes and their spontaneous contraction rate. Phenylephrine, which normally induces cardiac hypertrophy, had no additional effects under serum conditions. Likewise, hiPSC-derived cardiomyocytes from three MYBPC3 patients which had a greater surface area than controls in the absence of serum as predicted by their genotype, did not show this difference in the presence of serum. Serum can thus alter the phenotype of human PSC derived cardiomyocytes under otherwise defined conditions such that the effects of hypertrophic drugs and gene mutations are underestimated. It is therefore pertinent to examine cardiac phenotypes in culture media without or in low concentrations of serum.


Assuntos
Cardiomiopatia Hipertrófica/patologia , Proteínas de Transporte/genética , Meios de Cultura/química , Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Soro/química , Potenciais de Ação , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Proteínas de Transporte/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Derme/citologia , Derme/metabolismo , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Mutação/genética , Miócitos Cardíacos/citologia , Fenótipo , Ratos
9.
Exp Cell Res ; 327(2): 297-306, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24836851

RESUMO

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis.


Assuntos
Diferenciação Celular , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Provírus/genética , Integração Viral/genética , Southern Blotting , Proliferação de Células , Células Cultivadas , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Derme/citologia , Derme/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Imunofluorescência , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo
10.
Expert Opin Drug Saf ; 11(2): 285-98, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22103746

RESUMO

INTRODUCTION: The field of cardiac safety pharmacology has been experiencing exciting changes over the recent years. Drug induced arrhythmia of the torsade des pointes types has been the reason for the denial of approval of novel drug candidates. The aim of cardiac safety pharmacology is to detect undesirable pharmacodynamic drug effects within and above the therapeutic range. A special focus is on the identification of potential arrhythmogenic effects within the drug discovery chain. AREAS COVERED: Here, the authors discuss the relevance of induced pluripotent stem (iPS) cell derived cardiomyocytes for safety pharmacology. The technology of obtaining functional cardiomyocytes from somatic cells of healthy donors and patients with inherited diseases is the basis for diverse disease models in multi-level safety pharmacology screening. The reader will gain an overview of stem cell based technologies in cardiac safety pharmacology in cardiac and disease modeling by iPS cell derived cardiomyocytes from patients with an inherited cardiac syndrome. EXPERT OPINION: iPS cell derived cardiomyocytes - especially from patients with increased risk of cardiac arrhythmia - are on the verge of offering new options for drug testing. More reliable assays can be expected to predict the arrhythmogenic risk of drug candidates in humans. However, this technology is still new and extensive validation studies are due.


Assuntos
Arritmias Cardíacas/induzido quimicamente , Bioensaio , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Testes de Toxicidade/métodos , Potenciais de Ação , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Células Cultivadas , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Risco
11.
Nat Methods ; 8(12): 1037-40, 2011 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-22020065

RESUMO

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.


Assuntos
Separação Celular/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Mioblastos Cardíacos/citologia , Miócitos Cardíacos/citologia , Fatores de Transcrição/metabolismo , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Biomarcadores/análise , Diferenciação Celular , Perfilação da Expressão Gênica , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Humanos , Mioblastos Cardíacos/metabolismo , Miócitos Cardíacos/metabolismo , Reação em Cadeia da Polimerase , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Fatores de Transcrição/genética , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
12.
Trends Mol Med ; 17(9): 475-84, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21703926

RESUMO

Recent advances in pluripotent stem cell biology now make it possible to generate human cardiomyocytes in vitro from both healthy individuals and from patients with cardiac abnormalities. This offers unprecedented opportunities to study cardiac disease development 'in a dish' and establish novel platforms for drug discovery, either to prevent disease progression or to reverse it. In this review paper, we discuss some of the genetic diseases that affect the heart and illustrate how these new paradigms could assist our understanding of cardiac pathogenesis and aid in drug discovery. In particular, we highlight the limitations of other commonly used model systems in predicting the consequences of drug exposure on the human heart.


Assuntos
Cardiomiopatias/genética , Descoberta de Drogas , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Potenciais de Ação , Animais , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Linhagem Celular , Modelos Animais de Doenças , Eletrofisiologia , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais
14.
Ann N Y Acad Sci ; 1188: 52-7, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20201886

RESUMO

In recent years the differentiation efficiency of human embryonic stem cells (hESCs) to cardiomyocytes has improved considerably. In general, hESC-derived cardiomyocytes are formed in aggregates, which require dissociation for follow-up experimental analyses and (clinical) applications. Here, we show that inhibition of the Rho-associated kinase (ROCK) by Y-27632 improved survival of dissociated hESC-derived differentiated cells. A maximum effect on cell survival was already observed within the first 24 hours. Hereafter, no further differences in the percentage of apoptotic and proliferating cells were observed with or without ROCK-inhibitor treatment. Improved survival was observed in both cardiomyocyte as well as non-cardiomyocyte cell populations. Viable cardiomyocytes were indicated by the appearance of beating, sarcomeric organization of cardiac-specific proteins, and fluorescence of a mitochondrion-selective dye. These results facilitate development of applications of hESC-derived cardiomyocytes in multiple research areas. Furthermore, these findings may be applied to other cell types differentiated from hESCs or other stem cells.


Assuntos
Amidas/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Diferenciação Celular , Linhagem Celular , Separação Celular , Sobrevivência Celular , Células-Tronco Embrionárias/enzimologia , Humanos , Miócitos Cardíacos/enzimologia , Quinases Associadas a rho/metabolismo
15.
J Proteome Res ; 9(3): 1610-8, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20088484

RESUMO

The absence of identified cell surface proteins and corresponding antibodies to most differentiated derivatives of human embryonic stem cells (hESCs) has largely limited selection of specific cell types from mixed cell populations to genetic approaches. Here, we describe the use of mass spectrometry (MS)-based proteomics on cell membrane proteins isolated from hESCs that were differentiated into cardiomyocytes to identify candidate proteins for this particular lineage. Quantitative MS distinguished cardiomyocyte-specific plasma membrane proteins that were highly enriched or detected only in cardiomyocytes derived from hESCs and human fetal hearts compared with a heterogeneous pool of hESC-derived differentiated cells. For several candidates, cardiomyocyte-specific expression and cell surface localization were verified by conventional antibody-based methodologies. Using an antibody against elastin microfibril interfacer 2 (EMILIN2), we demonstrate that cardiomyocytes can be sorted from live cell populations. Besides showing that MS-based membrane proteomics is a powerful tool to identify candidate proteins that allow purification of specific cell lineages from heterogeneous populations, this approach generated a plasma membrane proteome profile suggesting signaling pathways that control cell behavior.


Assuntos
Biomarcadores/análise , Células-Tronco Embrionárias/metabolismo , Proteínas de Membrana/análise , Miócitos Cardíacos/metabolismo , Proteômica/métodos , Anticorpos/metabolismo , Biomarcadores/metabolismo , Western Blotting , Diferenciação Celular , Separação Celular , Células-Tronco Embrionárias/citologia , Fibrilinas , Glicoproteínas/metabolismo , Humanos , Marcação por Isótopo , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência , Miócitos Cardíacos/citologia
16.
Methods Mol Biol ; 584: 413-23, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19907990

RESUMO

Generic methods for genetic manipulation of human embryonic stem cells (hESCs) are important for both present research and future commercial applications. To date, differences in cell derivation and culture have required independent optimization of transfection and transduction protocols and some lines have remained refractile to all methods. Here we describe a culture protocol that has been extensively tested in 12 different hESC lines (1, 2) and shown to support efficient gene transfer independent of the method of gene delivery or history of the cell line. The system is based on Matrigel monolayer culture and conditioned medium from mouse embryonic feeder cells (MEFs) and entails transient high-density culture followed by rapid adaptation to low density for gene transfer. Under these conditions, plasmid transfection, virus infection, and siRNA transfection are highly effective. Stable genetically modified hESC lines can be generated with plasmid transfection, viral infection, or electroporation without loss of pluripotency or differentiation potential. The majority of lines generated in this system display a normal karyotype.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Técnicas de Transferência de Genes , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem Celular , Separação Celular , Criopreservação , Meios de Cultura , Meios de Cultivo Condicionados , Eletroporação/métodos , Citometria de Fluxo , Humanos , Camundongos , Plasmídeos/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA Interferente Pequeno/genética , Transdução Genética/métodos , Transfecção/métodos
17.
Stem Cell Res ; 4(2): 107-16, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20034863

RESUMO

Recent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. Block of the human Ether-a-go go Related Gene (hERG) ion channel in particular is associated with life-threatening arrhythmias, such as Torsade de Pointes (TdP). Here we investigated human cardiomyocytes derived from pluripotent (embryonic) stem cells (hESC) as a renewable, scalable, and reproducible system on which to base cardiac safety pharmacology assays. Analyses of extracellular field potentials in hESC-derived cardiomyocytes (hESC-CM) and generation of derivative field potential duration (FPD) values showed dose-dependent responses for 12 cardiac and noncardiac drugs. Serum levels in patients of drugs with known effects on QT interval overlapped with prolonged FPD values derived from hESC-CM, as predicted. We thus propose hESC-CM FPD prolongation as a safety criterion for preclinical evaluation of new drugs in development. This is the first study in which dose responses of such a wide range of compounds on hESC-CM have been generated and shown to be predictive of clinical effects. We propose that assays based on hESC-CM could complement or potentially replace some of the preclinical cardiac toxicity screening tests currently used for lead optimization and further development of new drugs.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Células-Tronco Embrionárias/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Eletrofisiologia , Humanos , Lidocaína/toxicidade , Síndrome do QT Longo/induzido quimicamente , Camundongos , Técnicas de Patch-Clamp , Quinidina/toxicidade , Sotalol/toxicidade
18.
Trends Pharmacol Sci ; 30(10): 536-45, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19762090

RESUMO

Stem cells derived from pre-implantation human embryos or from somatic cells by reprogramming are pluripotent and self-renew indefinitely in culture. Pluripotent stem cells are unique in being able to differentiate to any cell type of the human body. Differentiation towards the cardiac lineage has attracted significant attention, initially with a strong focus on regenerative medicine. Although an important research area, the heart has proven challenging to repair by cardiomyocyte replacement. However, the ability to reprogramme adult cells to pluripotent stem cells and genetically manipulate stem cells presented opportunities to develop models of human disease. The availability of human cardiomyocytes from stem cell sources is expected to accelerate the discovery of cardiac drugs and safety pharmacology by offering more clinically relevant human culture models than presently available. Here we review the state-of-the-art using stem cell-derived human cardiomyocytes in drug discovery, drug safety pharmacology, and regenerative medicine.


Assuntos
Pesquisa Biomédica , Descoberta de Drogas/métodos , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes , Diferenciação Celular , Células Cultivadas , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/transplante , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Medicina Regenerativa/métodos
19.
Cell Stem Cell ; 5(2): 214-26, 2009 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-19664995

RESUMO

Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during differentiation induced by bone morphogenetic protein (BMP) and removal of hESC growth factors. Of 5222 proteins identified, 1399 were phosphorylated on 3067 residues. Approximately 50% of these phosphosites were regulated within 1 hr of differentiation induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways and kinase activities. Among the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated as a result of phosphorylation. Using the data to predict kinase-substrate relationships, we reconstructed the hESC kinome; CDK1/2 emerged as central in controlling self-renewal and lineage specification. The findings provide new insights into how hESCs exit the pluripotent state and present the hESC (phospho)proteome resource as a complement to existing pluripotency network databases.


Assuntos
Células-Tronco Embrionárias/metabolismo , Fosfoproteínas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteoma/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Proteína Quinase CDC2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Quinase 2 Dependente de Ciclina/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Células HeLa , Humanos , Fosfoproteínas/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Fatores de Transcrição SOXB1/antagonistas & inibidores , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
20.
Nat Protoc ; 3(9): 1435-43, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18772870

RESUMO

Realizing the potential of human embryonic stem cells (hESCs) in research and commercial applications requires generic protocols for culture, expansion and genetic modification that function between multiple lines. Here we describe a feeder-free hESC culture protocol that was tested in 13 independent hESC lines derived in five different laboratories. The procedure is based on Matrigel adaptation in mouse embryonic fibroblast conditioned medium (CM) followed by monolayer culture of hESC. When combined, these techniques provide a robust hESC culture platform, suitable for high-efficiency genetic modification via plasmid transfection (using lipofection or electroporation), siRNA knockdown and viral transduction. In contrast to other available protocols, it does not require optimization for individual lines. hESC transiently expressing ectopic genes are obtained within 9 d and stable transgenic lines within 3 weeks.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/fisiologia , Meios de Cultivo Condicionados , Técnicas Genéticas , Humanos
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