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1.
Europace ; 22(9): 1431-1441, 2020 09 01.
Article in English | MEDLINE | ID: mdl-32770183

ABSTRACT

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.


Subject(s)
Induced Pluripotent Stem Cells , Action Potentials , Arrhythmias, Cardiac , Electrophysiological Phenomena , Humans , Myocytes, Cardiac
2.
Article in English | MEDLINE | ID: mdl-29355722

ABSTRACT

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.


Subject(s)
Action Potentials/drug effects , Drug Evaluation, Preclinical/methods , Myocytes, Cardiac/drug effects , Cardiotoxicity/prevention & control , Cell Line , Drug Evaluation, Preclinical/instrumentation , Drug Evaluation, Preclinical/standards , Electrodes , Guidelines as Topic , Humans , Induced Pluripotent Stem Cells/physiology , Myocardial Contraction/drug effects , Myocytes, Cardiac/physiology , Patch-Clamp Techniques/instrumentation , Patch-Clamp Techniques/methods
3.
Methods Mol Biol ; 1353: 163-80, 2016.
Article in English | MEDLINE | ID: mdl-25626427

ABSTRACT

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.


Subject(s)
Cell Culture Techniques/methods , Cellular Reprogramming , Embryonic Stem Cells/cytology , Enzyme Inhibitors/pharmacology , Myocytes, Cardiac/cytology , Pluripotent Stem Cells/cytology , Activins/pharmacology , Biomarkers/metabolism , Bone Morphogenetic Protein 4/pharmacology , Cell Differentiation , Collagen/chemistry , Drug Combinations , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Gene Expression , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Intercellular Signaling Peptides and Proteins/pharmacology , Laminin/chemistry , Molecular Imaging , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Pluripotent Stem Cells/drug effects , Pluripotent Stem Cells/metabolism , Primary Cell Culture , Proteoglycans/chemistry , Pyridines/pharmacology , Pyrimidines/pharmacology , Troponin T/genetics , Troponin T/metabolism
4.
Development ; 142(18): 3231-8, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26209647

ABSTRACT

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.


Subject(s)
Cell Differentiation/physiology , Fetus/physiology , Gene Expression Regulation, Developmental/physiology , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Pluripotent Stem Cells/cytology , Transcriptome , Fetus/metabolism , Gene Expression Profiling , Humans
5.
J Cell Mol Med ; 18(8): 1509-18, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24981391

ABSTRACT

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.


Subject(s)
Cardiomyopathy, Hypertrophic/pathology , Carrier Proteins/genetics , Culture Media/chemistry , Embryonic Stem Cells/physiology , Induced Pluripotent Stem Cells/physiology , Myocytes, Cardiac/physiology , Serum/chemistry , Action Potentials , Animals , Animals, Newborn , Calcium/metabolism , Cardiomyopathy, Hypertrophic/genetics , Cardiomyopathy, Hypertrophic/metabolism , Carrier Proteins/metabolism , Case-Control Studies , Cell Differentiation , Cells, Cultured , Dermis/cytology , Dermis/metabolism , Embryonic Stem Cells/cytology , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Image Processing, Computer-Assisted , Induced Pluripotent Stem Cells/cytology , Mice , Mutation/genetics , Myocytes, Cardiac/cytology , Phenotype , Rats
6.
Exp Cell Res ; 327(2): 297-306, 2014 Oct 01.
Article in English | MEDLINE | ID: mdl-24836851

ABSTRACT

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.


Subject(s)
Cell Differentiation , Cellular Reprogramming , Induced Pluripotent Stem Cells/cytology , Myocytes, Cardiac/cytology , Proviruses/genetics , Virus Integration/genetics , Blotting, Southern , Cell Proliferation , Cells, Cultured , Dental Pulp/cytology , Dental Pulp/metabolism , Dermis/cytology , Dermis/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Flow Cytometry , Fluorescent Antibody Technique , High-Throughput Screening Assays , Humans , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/metabolism
7.
Expert Opin Drug Saf ; 11(2): 285-98, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22103746

ABSTRACT

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.


Subject(s)
Arrhythmias, Cardiac/chemically induced , Biological Assay , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Induced Pluripotent Stem Cells/drug effects , Myocytes, Cardiac/drug effects , Toxicity Tests/methods , Action Potentials , Animals , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/physiopathology , Cells, Cultured , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/genetics , Ether-A-Go-Go Potassium Channels/metabolism , Genetic Predisposition to Disease , Humans , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/metabolism , Patch-Clamp Techniques , Reproducibility of Results , Risk Assessment , Risk Factors
8.
Nat Methods ; 8(12): 1037-40, 2011 Oct 23.
Article in English | MEDLINE | ID: mdl-22020065

ABSTRACT

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.


Subject(s)
Cell Separation/methods , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Green Fluorescent Proteins/metabolism , Homeodomain Proteins/metabolism , Myoblasts, Cardiac/cytology , Myocytes, Cardiac/cytology , Transcription Factors/metabolism , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Biomarkers/analysis , Cell Differentiation , Gene Expression Profiling , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/genetics , Humans , Myoblasts, Cardiac/metabolism , Myocytes, Cardiac/metabolism , Polymerase Chain Reaction , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Transcription Factors/genetics , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolism
9.
Trends Mol Med ; 17(9): 475-84, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21703926

ABSTRACT

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.


Subject(s)
Cardiomyopathies/genetics , Drug Discovery , Heart Defects, Congenital/genetics , Heart Defects, Congenital/metabolism , Myocytes, Cardiac/cytology , Pluripotent Stem Cells/cytology , Action Potentials , Animals , Cardiomyopathies/metabolism , Cardiomyopathies/pathology , Cell Line , Disease Models, Animal , Electrophysiology , Humans , Mice , Myocytes, Cardiac/metabolism , Pluripotent Stem Cells/metabolism , Signal Transduction
11.
Ann N Y Acad Sci ; 1188: 52-7, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20201886

ABSTRACT

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.


Subject(s)
Amides/pharmacology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , rho-Associated Kinases/antagonists & inhibitors , Cell Differentiation , Cell Line , Cell Separation , Cell Survival , Embryonic Stem Cells/enzymology , Humans , Myocytes, Cardiac/enzymology , rho-Associated Kinases/metabolism
12.
J Proteome Res ; 9(3): 1610-8, 2010 Mar 05.
Article in English | MEDLINE | ID: mdl-20088484

ABSTRACT

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.


Subject(s)
Biomarkers/analysis , Embryonic Stem Cells/metabolism , Membrane Proteins/analysis , Myocytes, Cardiac/metabolism , Proteomics/methods , Antibodies/metabolism , Biomarkers/metabolism , Blotting, Western , Cell Differentiation , Cell Separation , Embryonic Stem Cells/cytology , Fibrillins , Glycoproteins/metabolism , Humans , Isotope Labeling , Mass Spectrometry , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Microscopy, Fluorescence , Myocytes, Cardiac/cytology
13.
Methods Mol Biol ; 584: 413-23, 2010.
Article in English | MEDLINE | ID: mdl-19907990

ABSTRACT

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.


Subject(s)
Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Transfer Techniques , Animals , Cell Culture Techniques/methods , Cell Differentiation , Cell Line , Cell Separation , Cryopreservation , Culture Media , Culture Media, Conditioned , Electroporation/methods , Flow Cytometry , Humans , Mice , Plasmids/genetics , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , RNA, Small Interfering/genetics , Transduction, Genetic/methods , Transfection/methods
14.
Stem Cell Res ; 4(2): 107-16, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20034863

ABSTRACT

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.


Subject(s)
Drug-Related Side Effects and Adverse Reactions , Embryonic Stem Cells/cytology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Animals , Cell Line , Drug Evaluation, Preclinical , Electrophysiology , Humans , Lidocaine/toxicity , Long QT Syndrome/chemically induced , Mice , Patch-Clamp Techniques , Quinidine/toxicity , Sotalol/toxicity
15.
Trends Pharmacol Sci ; 30(10): 536-45, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19762090

ABSTRACT

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.


Subject(s)
Biomedical Research , Drug Discovery/methods , Myocytes, Cardiac/metabolism , Pluripotent Stem Cells , Cell Differentiation , Cells, Cultured , Humans , Myocytes, Cardiac/cytology , Myocytes, Cardiac/transplantation , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Regenerative Medicine/methods
16.
Cell Stem Cell ; 5(2): 214-26, 2009 Aug 07.
Article in English | MEDLINE | ID: mdl-19664995

ABSTRACT

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.


Subject(s)
Embryonic Stem Cells/metabolism , Phosphoproteins/metabolism , Pluripotent Stem Cells/metabolism , Proteome/metabolism , Bone Morphogenetic Proteins/pharmacology , CDC2 Protein Kinase/metabolism , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cyclin-Dependent Kinase 2/metabolism , Embryonic Stem Cells/drug effects , HeLa Cells , Humans , Phosphoproteins/drug effects , Phosphorylation/drug effects , Phosphorylation/physiology , Pluripotent Stem Cells/drug effects , Proteome/drug effects , SOXB1 Transcription Factors/antagonists & inhibitors , SOXB1 Transcription Factors/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology
17.
Nat Protoc ; 3(9): 1435-43, 2008.
Article in English | MEDLINE | ID: mdl-18772870

ABSTRACT

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.


Subject(s)
Cell Culture Techniques/methods , Embryonic Stem Cells/physiology , Culture Media, Conditioned , Genetic Techniques , Humans
18.
Stem Cells ; 26(11): 2777-81, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18703662

ABSTRACT

Human embryonic stem cells (hESCs) are often cocultured on mitotically inactive fibroblast feeder cells to maintain their undifferentiated state. Under these growth conditions, hESCs form multilayered colonies of morphologically heterogeneous cells surrounded by flattened mesenchymal cells. In contrast, hESCs grown in feeder cell-conditioned medium on Matrigel instead tend to grow as monolayers with uniform morphology. Using mass spectrometry and immunofluorescence microscopy, we showed that hESCs under these conditions primarily express proteins belonging to epithelium-related cell-cell adhesion complexes, including adherens junctions, tight junctions, desmosomes, and gap junctions. This indicates that monolayers of hESCs cultured under feeder-free conditions retain a homogeneous epithelial phenotype similar to that of the upper central cell layer of colonies maintained on feeder cells. Notably, feeder-free hESCs also coexpressed vimentin, which is usually associated with mesenchyme, suggesting that these cells may have undergone epithelium-to-mesenchyme transitions, indicating differentiation. However, if grown on a "soft" substrate (Hydrogel), intracellular vimentin levels were substantially reduced. Moreover, when hESCs were transferred back to feeder cells, expression of vimentin was again absent from the epithelial cell population. These results imply that on tissue culture substrates, vimentin expression is most likely a stress-induced response, unrelated to differentiation. Disclosure of potential conflicts of interest is found at the end of this article.


Subject(s)
Cell Membrane/metabolism , Embryonic Stem Cells/cytology , Epithelial Cells/cytology , Membrane Proteins/metabolism , Antigens, Differentiation/metabolism , Cell Adhesion , Cell Differentiation , Cells, Cultured , Coculture Techniques/methods , Collagen/metabolism , Drug Combinations , Embryonic Stem Cells/metabolism , Epithelial Cells/metabolism , Humans , Laminin/metabolism , Mass Spectrometry , Microscopy, Fluorescence , Proteoglycans/metabolism
19.
Stem Cells ; 26(9): 2257-65, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18599809

ABSTRACT

Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel, a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here, we defined ECM requirements of hESC by analyzing integrin expression and ECM production and determined integrin function using blocking antibodies. hESC expressed all major ECM proteins and corresponding integrins. We then systematically replaced Matrigel with defined medium supplements and ECM proteins. Cells attached efficiently to natural human vitronectin, fibronectin, and Matrigel but poorly to laminin + entactin and collagen IV. Integrin-blocking antibodies demonstrated that alphaVbeta5 integrins mediated adhesion to vitronectin, alpha5beta1 mediated adhesion to fibronectin, and alpha6beta1 mediated adhesion to laminin + entactin. Fibronectin in feeder cell-conditioned medium partially supported growth on all natural matrices, but in defined, nonconditioned medium only Matrigel or (natural and recombinant) vitronectin was effective. Recombinant vitronectin was the only defined functional alternative to Matrigel, supporting sustained self-renewal and pluripotency in three independent hESC lines.


Subject(s)
Embryonic Stem Cells/cytology , Receptors, Vitronectin/physiology , Vitronectin/pharmacology , Animals , Cell Adhesion , Cell Differentiation , Cell Proliferation , Cells, Cultured , Collagen , Culture Media , Drug Combinations , Embryonic Stem Cells/metabolism , Extracellular Matrix Proteins/biosynthesis , Humans , Laminin , Mice , Proteoglycans , Recombinant Proteins/pharmacology
20.
J Proteome Res ; 7(7): 2936-51, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18489135

ABSTRACT

Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired chromosomal abnormalities in culture are essentially indistinguishable from hECC. Direct comparison of karyotypically normal hESCs with hECCs could lead to understanding differences between their mechanisms of growth control and contribute to implementing safe therapeutic use of stem cells without the development of germ cell cancer. While several comparisons of hECCs and hESCs have been reported, their cell surface proteomes are largely unknown, partly because plasma membrane proteomics is still a major challenge. Here, we present a strategy for the identification of plasma membrane proteins that has been optimized for application to the relatively small numbers of stem cells normally available, and that does not require tedious cell fractionation. The method led to the identification of 237 and 219 specific plasma membrane proteins in the hESC line HUES-7 and the hECC line NT2/D1, respectively. In addition to known stemness-associated cell surface markers like ALP, CD9, and CTNNB, a large number of receptors, transporters, signal transducers, and cell-cell adhesion proteins were identified. Our study revealed that several Hedgehog and Wnt pathway members are differentially expressed in hESCs and hECCs including NPC1, FZD2, FZD6, FZD7, LRP6, and SEMA4D, which play a pivotal role in stem cell self-renewal and cancer growth. Various proteins encoded on chromosome 12p, duplicated in testicular cancer, were uniquely identified in hECCs. These included GAPDH, LDHB, YARS2, CLSTN3, CSDA, LRP6, NDUFA9, and NOL1, which are known to be upregulated in testicular cancer. Distinct HLA molecules were revealed on the surface of hESCs and hECCs, despite their low abundance. Results were compared with genomic and proteomic data sets reported previously for mouse ESCs, hECCs, and germ cell tumors. Our data provides a surface signature for HUES-7 and NT2/D1 cells and distinguishes normal hESCs from hECCs, helping explain their 'benign' versus 'malignant' nature.


Subject(s)
Embryonic Stem Cells/metabolism , Membrane Proteins/metabolism , Animals , Cell Membrane/metabolism , Chromatography, Liquid , Embryonal Carcinoma Stem Cells/metabolism , Gene Expression Profiling , Humans , Membrane Proteins/genetics , Mice , Organelles/metabolism , Proteomics , Species Specificity , Tandem Mass Spectrometry
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