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1.
Cell ; 185(26): 4937-4953.e23, 2022 12 22.
Article in English | MEDLINE | ID: mdl-36563664

ABSTRACT

To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts, which enabled optimization of in vitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. In vitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.


Subject(s)
Chromatin , Heart Defects, Congenital , Humans , Chromatin/genetics , Heart Defects, Congenital/genetics , Heart , Mutation , Single-Cell Analysis
2.
Dev Cell ; 54(6): 694-709.e9, 2020 09 28.
Article in English | MEDLINE | ID: mdl-32763147

ABSTRACT

Transposable elements (TEs) comprise nearly half of the human genome and are often transcribed or exhibit cis-regulatory properties with unknown function in specific processes such as heart development. In the case of endogenous retroviruses (ERVs), a TE subclass, experimental interrogation is constrained as many are primate-specific or human-specific. Here, we use primate pluripotent stem-cell-derived cardiomyocytes that mimic fetal cardiomyocytes in vitro to discover hundreds of ERV transcripts from the primate-specific MER41 family, some of which are regulated by the cardiogenic transcription factor TBX5. The most significant of these are located within BANCR, a long non-coding RNA (lncRNA) exclusively expressed in primate fetal cardiomyocytes. Functional studies reveal that BANCR promotes cardiomyocyte migration in vitro and ventricular enlargement in vivo. We conclude that recently evolved TE loci such as BANCR may represent potent de novo developmental regulatory elements that can be interrogated with species-matching pluripotent stem cell models.


Subject(s)
Endogenous Retroviruses/genetics , Myocytes, Cardiac/metabolism , RNA, Long Noncoding/genetics , Transcription Factors/genetics , Animals , DNA Transposable Elements/genetics , Evolution, Molecular , Gene Expression Regulation/genetics , Genome, Human , Humans , Primates/genetics , Species Specificity
3.
Circulation ; 138(23): 2666-2681, 2018 12 04.
Article in English | MEDLINE | ID: mdl-29914921

ABSTRACT

BACKGROUND: The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic "healthy" individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity. METHODS: To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a "healthy" asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene MYL3, reported by the ClinVar database to be "likely pathogenic." Human-induced pluripotent stem cells (iPSCs) were derived from the heterozygous VUS MYL3(170C>A) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected "healthy" control; (2) homozygous VUS MYL3(170C>A); (3) heterozygous frameshift mutation MYL3(170C>A/fs); and (4) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUS MYL3(170C>A), lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs). RESULTS: The heterozygous VUS MYL3(170C>A)-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUS MYL3(170C>A)- and frameshift mutation MYL3(170C>A/fs)-iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular MYL3 variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic MYL3 mutation, MYL3(170C>G), and a carrier-specific iPSC-CMs line, carrying a MYBPC3(961G>A) HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments. CONCLUSIONS: Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrier-specific manner in a dish. As such, this platform represents a promising VUS risk-assessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field.


Subject(s)
CRISPR-Cas Systems/genetics , Cardiomyopathies/pathology , Genetic Variation , Amino Acid Sequence , Calcium/metabolism , Cardiomyopathies/genetics , Cell Differentiation , Frameshift Mutation , Gene Editing/methods , Gene Expression , Heterozygote , Homozygote , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Myosin Light Chains/chemistry , Myosin Light Chains/genetics , Sequence Alignment
4.
Cell Stem Cell ; 22(3): 428-444.e5, 2018 03 01.
Article in English | MEDLINE | ID: mdl-29499155

ABSTRACT

Cardiac development requires coordinated and large-scale rearrangements of the epigenome. The roles and precise mechanisms through which specific epigenetic modifying enzymes control cardiac lineage specification, however, remain unclear. Here we show that the H3K4 methyltransferase SETD7 controls cardiac differentiation by reading H3K36 marks independently of its enzymatic activity. Through chromatin immunoprecipitation sequencing (ChIP-seq), we found that SETD7 targets distinct sets of genes to drive their stage-specific expression during cardiomyocyte differentiation. SETD7 associates with different co-factors at these stages, including SWI/SNF chromatin-remodeling factors during mesodermal formation and the transcription factor NKX2.5 in cardiac progenitors to drive their differentiation. Further analyses revealed that SETD7 binds methylated H3K36 in the bodies of its target genes to facilitate RNA polymerase II (Pol II)-dependent transcription. Moreover, abnormal SETD7 expression impairs functional attributes of terminally differentiated cardiomyocytes. Together, these results reveal how SETD7 acts at sequential steps in cardiac lineage commitment, and they provide insights into crosstalk between dynamic epigenetic marks and chromatin-modifying enzymes.


Subject(s)
Cell Differentiation , Cell Lineage , Histone-Lysine N-Methyltransferase/genetics , Myocardium/cytology , Transcriptional Activation/genetics , Calcium Signaling , Cell Differentiation/genetics , Cell Line , Chromatin/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Humans , Lysine/metabolism , Mesoderm/cytology , Methylation , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , RNA Polymerase II/metabolism , Transcription, Genetic
5.
Circ Res ; 120(10): 1561-1571, 2017 May 12.
Article in English | MEDLINE | ID: mdl-28246128

ABSTRACT

RATIONALE: Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome. OBJECTIVE: The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro. METHODS AND RESULTS: By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development. CONCLUSIONS: Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.


Subject(s)
Cardiovascular Diseases/genetics , Gene Knockout Techniques/methods , Gene Library , Genetic Engineering/methods , Induced Pluripotent Stem Cells/physiology , Base Sequence , Cardiovascular Diseases/therapy , Cells, Cultured , Gene Targeting/methods , Humans , Induced Pluripotent Stem Cells/transplantation
6.
Circ Cardiovasc Genet ; 9(2): 110-8, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26896382

ABSTRACT

BACKGROUND: The molecular regulation of heart development is regulated by cis- and trans-factors acting on the genome and epigenome. As a class of important regulatory RNAs, the role of long noncoding RNAs (lncRNAs) in human heart development is still poorly understood. Furthermore, factors that interact with lncRNAs in this process are not well characterized. METHODS AND RESULTS: Using RNA sequencing, we systematically define the contrasting lncRNA expression patterns between fetal and adult hearts. We report that lncRNAs upregulated in adult versus fetal heart have different sequence features and distributions. For example, the adult heart expresses more sense lncRNAs compared with fetal heart. We also report the coexpression of lncRNAs and neighboring coding genes that have important functions in heart development. Importantly, the regulation of lncRNA expression during fetal to adult heart development seems to be due, in part, to the coordination of specific developmental epigenetic modifications, such as H3K4me1 and H3k4me3. The expression of promoter-associated lncRNAs in adult and fetal hearts also seems to be related to these epigenetic states. Finally, transcription factor-binding analysis suggests that lncRNAs are directly regulating cardiac gene expression during development. CONCLUSIONS: We provide a systematic analysis of lncRNA control of heart development that gives clues to the roles that specific lncRNAs play in fetal and adult hearts.


Subject(s)
Fetus/metabolism , Gene Expression Regulation, Developmental , Heart/embryology , RNA, Long Noncoding/genetics , Adult , Epigenesis, Genetic , Gene Regulatory Networks , Humans , Open Reading Frames/genetics , Polymorphism, Single Nucleotide/genetics , Protein Binding , RNA, Long Noncoding/metabolism , Transcription Factors/metabolism
7.
Circ Res ; 117(7): 603-11, 2015 Sep 11.
Article in English | MEDLINE | ID: mdl-26265630

ABSTRACT

RATIONALE: Thousands of mutations across >50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited because many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and high cost. Furthermore, customization of these assays to cover emerging targets that suit individual needs is often expensive and time consuming. OBJECTIVE: We sought to develop a custom high throughput, clinical-grade next-generation sequencing assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost. METHODS AND RESULTS: We used double-stranded probes (complementary long padlock probes), an inexpensive and customizable capture technology, to efficiently capture and amplify the entire coding region and flanking intronic and regulatory sequences of 88 genes and 40 microRNAs associated with inherited cardiomyopathies, congenital heart disease, and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base pair coverage of 420, of which 97% had >20× coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms. The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was 3 days at an approximate cost of $100 per sample. CONCLUSIONS: Accurate, high-throughput detection of mutations across numerous cardiac genes is achievable with complementary long padlock probe technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and congenital heart disease genes are discovered, giving researchers a powerful new tool for DNA mutation detection and discovery.


Subject(s)
Cost-Benefit Analysis , Heart Diseases/economics , Heart Diseases/genetics , High-Throughput Nucleotide Sequencing/economics , High-Throughput Nucleotide Sequencing/methods , Base Sequence , Gene Targeting/economics , Gene Targeting/methods , Heart Diseases/diagnosis , Humans , Molecular Sequence Data , Polymorphism, Single Nucleotide/genetics , Time Factors
9.
J Cell Sci ; 127(Pt 7): 1428-40, 2014 Apr 01.
Article in English | MEDLINE | ID: mdl-24463812

ABSTRACT

The key regulators of endothelial differentiation that is induced by shear stress are mostly unclear. Human atonal homolog 6 (Hath6 or ATOH8) is an endothelial-selective and shear-stress-responsive transcription factor. In this study, we sought to elucidate the role of Hath6 in the endothelial specification of embryonic stem cells. In a stepwise human embryonic stem cell to endothelial cell (hESC-EC) induction system, Hath6 mRNA was upregulated synchronously with endothelial determination. Subsequently, gain-of-function and loss-of-function studies of Hath6 were performed using the hESC-EC induction model and endothelial cell lines. The overexpression of Hath6, which mimics shear stress treatment, resulted in an increased CD45(-)CD31(+)KDR(+) population, a higher tubular-structure-formation capacity and increased endothelial-specific gene expression. By contrast, the knockdown of Hath6 mRNA markedly decreased endothelial differentiation. Hath6 also facilitated the maturation of endothelial cells in terms of endothelial gene expression, tubular-structure formation and cell migration. We further demonstrated that the gene encoding eNOS is a direct target of Hath6 through a reporter system assay and western blot analysis, and that the inhibition of eNOS diminishes hESC-EC differentiation. These results suggest that eNOS plays a key role in linking Hath6 to the endothelial phenotype. Further in situ hybridization studies in zebrafish and mouse embryos indicated that homologs of Hath6 are involved in vasculogenesis and angiogenesis. This study provides the first confirmation of the positive impact of Hath6 on human embryonic endothelial differentiation and function. Moreover, we present a potential signaling pathway through which shear stress stimulates endothelial differentiation.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Endothelial Cells/cytology , Animals , Cell Differentiation/physiology , Endothelial Cells/metabolism , Gene Expression , Human Umbilical Vein Endothelial Cells , Humans , Mice
10.
Stem Cells ; 31(2): 259-68, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23136034

ABSTRACT

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. In this study, we analyzed global miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular, we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors, NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR-302, which we experimentally confirm by reporter luciferase assays and real-time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR-302. Importantly, higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells into iPSCs using four factors (KLF4, C-MYC, OCT4, and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as "KMOS3") when compared to using four factors ("KMOS"). Furthermore, shRNA knockdown of NR2F2 mimics the over-expression of miR-302 by also enhancing reprogramming efficiency. Interestingly, we were unable to generate iPSCs from miR-302a/b/c/d alone, which is in contrast to previous publications that have reported that miR-302 by itself can reprogram human skin cancer cells and human hair follicle cells. Taken together, these findings demonstrate that miR-302 inhibits NR2F2 and promotes pluripotency through indirect positive regulation of OCT4. This feedback loop represents an important new mechanism for understanding and inducing pluripotency in somatic cells.


Subject(s)
Adipocytes/drug effects , COUP Transcription Factor II/genetics , Induced Pluripotent Stem Cells/drug effects , MicroRNAs/genetics , Octamer Transcription Factor-3/genetics , Adipocytes/cytology , Adipocytes/metabolism , COUP Transcription Factor II/antagonists & inhibitors , COUP Transcription Factor II/metabolism , Cell Differentiation/drug effects , Cellular Reprogramming/drug effects , Cellular Reprogramming/genetics , Feedback, Physiological , Female , Gene Expression Regulation/drug effects , Genes, Reporter , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Kruppel-Like Transcription Factors/pharmacology , Luciferases , MicroRNAs/metabolism , MicroRNAs/pharmacology , Microarray Analysis , Octamer Transcription Factor-3/metabolism , Octamer Transcription Factor-3/pharmacology , Primary Cell Culture , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/pharmacology , RNA, Small Interfering/genetics , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , SOXB1 Transcription Factors/pharmacology , Signal Transduction/drug effects
11.
PLoS One ; 6(11): e27417, 2011.
Article in English | MEDLINE | ID: mdl-22110643

ABSTRACT

BACKGROUND: MicroRNAs (miRs) negatively regulate transcription and are important determinants of normal heart development and heart failure pathogenesis. Despite the significant knowledge gained in mouse studies, their functional roles in human (h) heart remain elusive. METHODS AND RESULTS: We hypothesized that miRs that figure prominently in cardiac differentiation are differentially expressed in differentiating, developing, and terminally mature human cardiomyocytes (CMs). As a first step, we mapped the miR profiles of human (h) embryonic stem cells (ESCs), hESC-derived (hE), fetal (hF) and adult (hA) ventricular (V) CMs. 63 miRs were differentially expressed between hESCs and hE-VCMs. Of these, 29, including the miR-302 and -371/372/373 clusters, were associated with pluripotency and uniquely expressed in hESCs. Of the remaining miRs differentially expressed in hE-VCMs, 23 continued to express highly in hF- and hA-VCMs, with miR-1, -133, and -499 displaying the largest fold differences; others such as miR-let-7a, -let-7b, -26b, -125a and -143 were non-cardiac specific. Functionally, LV-miR-499 transduction of hESC-derived cardiovascular progenitors significantly increased the yield of hE-VCMs (to 72% from 48% of control; p<0.05) and contractile protein expression without affecting their electrophysiological properties (p>0.05). By contrast, LV-miR-1 transduction did not bias the yield (p>0.05) but decreased APD and hyperpolarized RMP/MDP in hE-VCMs due to increased I(to), I(Ks) and I(Kr), and decreased I(f) (p<0.05) as signs of functional maturation. Also, LV-miR-1 but not -499 augmented the immature Ca(2+) transient amplitude and kinetics. Molecular pathway analyses were performed for further insights. CONCLUSION: We conclude that miR-1 and -499 play differential roles in cardiac differentiation of hESCs in a context-dependent fashion. While miR-499 promotes ventricular specification of hESCs, miR-1 serves to facilitate electrophysiological maturation.


Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Heart Ventricles/cytology , MicroRNAs/metabolism , Myocytes, Cardiac/cytology , Ventricular Function/genetics , Adult , Animals , Calcium/metabolism , Cell Line , Electrophysiological Phenomena/genetics , Fetus/cytology , Heart Ventricles/metabolism , Humans , Mice , Myocytes, Cardiac/metabolism
12.
Cancer Res ; 71(14): 5030-9, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21646469

ABSTRACT

Pluripotent stem cells, both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC), can give rise to multiple cell types and hence have tremendous potential for regenerative therapies. However, the tumorigenic potential of these cells remains a great concern, as reflected in the formation of teratomas by transplanted pluripotent cells. In clinical practice, most pluripotent cells will be differentiated into useful therapeutic cell types such as neuronal, cardiac, or endothelial cells prior to human transplantation, drastically reducing their tumorigenic potential. Our work investigated the extent to which these differentiated stem cell derivatives are truly devoid of oncogenic potential. In this study, we analyzed the gene expression patterns from three sets of hiPSC- and hESC-derivatives and the corresponding primary cells, and compared their transcriptomes with those of five different types of cancer. Our analysis revealed a significant gene expression overlap of the hiPSC- and hESC-derivatives with cancer, whereas the corresponding primary cells showed minimum overlap. Real-time quantitative PCR analysis of a set of cancer-related genes (selected on the basis of rigorous functional and pathway analyses) confirmed our results. Overall, our findings suggested that pluripotent stem cell derivatives may still bear oncogenic properties even after differentiation, and additional stringent functional assays to purify these cells should be done before they can be used for regenerative therapy.


Subject(s)
Cell Transformation, Neoplastic/pathology , Embryonic Stem Cells/physiology , Neoplasms/pathology , Neoplastic Stem Cells/physiology , Pluripotent Stem Cells/physiology , Blotting, Western , Cell Differentiation/physiology , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression , Gene Expression Profiling , Humans , Neoplasms/genetics , Neoplasms/metabolism , Neoplastic Stem Cells/cytology , Neoplastic Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Regenerative Medicine/methods , Reverse Transcriptase Polymerase Chain Reaction
13.
J Clin Invest ; 121(3): 1217-21, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21317531

ABSTRACT

Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising candidate cell sources for regenerative medicine. However, despite the common ability of hiPSCs and hESCs to differentiate into all 3 germ layers, their functional equivalence at the single cell level remains to be demonstrated. Moreover, single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here, we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs, suggesting that hiPSCs occupy an alternate, less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exercised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs, particularly when producing differentiated cells for regenerative medicine aims.


Subject(s)
Embryonic Stem Cells/cytology , Gene Expression Regulation , Induced Pluripotent Stem Cells/cytology , Transcription, Genetic , Algorithms , Animals , Cell Differentiation , Cell Lineage , Cell Separation , Collagen/chemistry , Drug Combinations , Flow Cytometry , Gene Expression Profiling , Genetic Techniques , Humans , Kinetics , Laminin/chemistry , Mice , Mice, SCID , Myocytes, Cardiac/cytology , Proteoglycans/chemistry , Teratoma/metabolism
14.
Hum Mol Genet ; 20(4): 752-62, 2011 Feb 15.
Article in English | MEDLINE | ID: mdl-21131292

ABSTRACT

Historically, our understanding of molecular genetic aspects of human germ cell development has been limited, at least in part due to inaccessibility of early stages of human development to experimentation. However, the derivation of pluripotent stem cells may provide the necessary human genetic system to study germ cell development. In this study, we compared the potential of human induced pluripotent stem cells (iPSCs), derived from adult and fetal somatic cells to form primordial and meiotic germ cells, relative to human embryonic stem cells. We found that ∼5% of human iPSCs differentiated to primordial germ cells (PGCs) following induction with bone morphogenetic proteins. Furthermore, we observed that PGCs expressed green fluorescent protein from a germ cell-specific reporter and were enriched for the expression of endogenous germ cell-specific proteins and mRNAs. In response to the overexpression of intrinsic regulators, we also observed that iPSCs formed meiotic cells with extensive synaptonemal complexes and post-meiotic haploid cells with a similar pattern of ACROSIN staining as observed in human spermatids. These results indicate that human iPSCs derived from reprogramming of adult somatic cells can form germline cells. This system may provide a useful model for molecular genetic studies of human germline formation and pathology and a novel platform for clinical studies and potential therapeutical applications.


Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Germ Cells/cytology , Induced Pluripotent Stem Cells/cytology , Bone Morphogenetic Proteins/metabolism , Bone Morphogenetic Proteins/pharmacology , Cell Line , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Germ Cells/metabolism , Haploidy , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Synaptonemal Complex/metabolism
15.
Circ Cardiovasc Genet ; 3(5): 426-35, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20733065

ABSTRACT

BACKGROUND: MicroRNAs (miRNAs) are a newly discovered endogenous class of small, noncoding RNAs that play important posttranscriptional regulatory roles by targeting messenger RNAs for cleavage or translational repression. Human embryonic stem cells are known to express miRNAs that are often undetectable in adult organs, and a growing body of evidence has implicated miRNAs as important arbiters of heart development and disease. METHODS AND RESULTS: To better understand the transition between the human embryonic and cardiac "miRNA-omes," we report here the first miRNA profiling study of cardiomyocytes derived from human embryonic stem cells. Analyzing 711 unique miRNAs, we have identified several interesting miRNAs, including miR-1, -133, and -208, that have been previously reported to be involved in cardiac development and disease and that show surprising patterns of expression across our samples. We also identified novel miRNAs, such as miR-499, that are strongly associated with cardiac differentiation and that share many predicted targets with miR-208. Overexpression of miR-499 and -1 resulted in upregulation of important cardiac myosin heavy-chain genes in embryoid bodies; miR-499 overexpression also caused upregulation of the cardiac transcription factor MEF2C. CONCLUSIONS: Taken together, our data give significant insight into the regulatory networks that govern human embryonic stem cell differentiation and highlight the ability of miRNAs to perturb, and even control, the genes that are involved in cardiac specification of human embryonic stem cells.


Subject(s)
Cell Differentiation/physiology , Embryonic Stem Cells/physiology , Gene Expression Regulation , Heart , MicroRNAs/metabolism , Animals , Cell Line , Embryoid Bodies/cytology , Embryoid Bodies/physiology , Embryonic Stem Cells/cytology , Gene Expression Profiling , Heart/embryology , Heart/growth & development , Humans , Mice , MicroRNAs/genetics , Microarray Analysis , Molecular Sequence Data , Myocytes, Cardiac/cytology , Myocytes, Cardiac/physiology , Patch-Clamp Techniques , Signal Transduction
16.
Cancer Res ; 70(13): 5539-48, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20530673

ABSTRACT

Human embryonic stem cells (hESC) present a novel platform for in vitro investigation of the early embryonic cellular response to ionizing radiation. Thus far, no study has analyzed the genome-wide transcriptional response to ionizing radiation in hESCs, nor has any study assessed their ability to form teratomas, the definitive test of pluripotency. In this study, we use microarrays to analyze the global gene expression changes in hESCs after low-dose (0.4 Gy), medium-dose (2 Gy), and high-dose (4 Gy) irradiation. We identify genes and pathways at each radiation dose that are involved in cell death, p53 signaling, cell cycling, cancer, embryonic and organ development, and others. Using Gene Set Enrichment Analysis, we also show that the expression of a comprehensive set of core embryonic transcription factors is not altered by radiation at any dose. Transplantation of irradiated hESCs to immune-deficient mice results in teratoma formation from hESCs irradiated at all doses, definitive proof of pluripotency. Further, using a bioluminescence imaging technique, we have found that irradiation causes hESCs to initially die after transplantation, but the surviving cells quickly recover by 2 weeks to levels similar to control. To conclude, we show that similar to somatic cells, irradiated hESCs suffer significant death and apoptosis after irradiation. However, they continue to remain pluripotent and are able to form all three embryonic germ layers. Studies such as this will help define the limits for radiation exposure for pregnant women and also radiotracer reporter probes for tracking cellular regenerative therapies.


Subject(s)
Embryonic Stem Cells/radiation effects , Pluripotent Stem Cells/radiation effects , Animals , Dose-Response Relationship, Radiation , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Female , Gene Expression Profiling , Humans , Mice , Mice, SCID , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/physiology , Teratoma/pathology
17.
Nat Methods ; 7(3): 197-9, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20139967

ABSTRACT

Owing to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by nonviral methods to generate induced pluripotent stem cells (iPSCs). We report the use of 'minicircle' DNA, a vector type that is free of bacterial DNA and capable of high expression in cells, for this purpose. Here we use a single minicircle vector to generate transgene-free iPSCs from adult human adipose stem cells.


Subject(s)
DNA, Circular/genetics , Genetic Vectors , Induced Pluripotent Stem Cells/metabolism , Adult , Humans , Transfection
18.
PLoS One ; 5(2): e8975, 2010 Feb 01.
Article in English | MEDLINE | ID: mdl-20126639

ABSTRACT

Human induced pluripotent stem cells (hiPSCs) generated by de-differentiation of adult somatic cells offer potential solutions for the ethical issues surrounding human embryonic stem cells (hESCs), as well as their immunologic rejection after cellular transplantation. However, although hiPSCs have been described as "embryonic stem cell-like", these cells have a distinct gene expression pattern compared to hESCs, making incomplete reprogramming a potential pitfall. It is unclear to what degree the difference in tissue of origin may contribute to these gene expression differences. To answer these important questions, a careful transcriptional profiling analysis is necessary to investigate the exact reprogramming state of hiPSCs, as well as analysis of the impression, if any, of the tissue of origin on the resulting hiPSCs. In this study, we compare the gene profiles of hiPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to their corresponding donor cells and hESCs. Our analysis elucidates the overall degree of reprogramming within each hiPSC line, as well as the "distance" between each hiPSC line and its donor cell. We further identify genes that have a similar mode of regulation in hiPSCs and their corresponding donor cells compared to hESCs, allowing us to specify core sets of donor genes that continue to be expressed in each hiPSC line. We report that residual gene expression of the donor cell type contributes significantly to the differences among hiPSCs and hESCs, and adds to the incompleteness in reprogramming. Specifically, our analysis reveals that fetal fibroblast-derived hiPSCs are closer to hESCs, followed by adipose, neonatal fibroblast, and keratinocyte-derived hiPSCs.


Subject(s)
Cell Lineage/genetics , Embryonic Stem Cells/metabolism , Gene Expression Profiling , Induced Pluripotent Stem Cells/metabolism , Adipose Tissue/cytology , Adipose Tissue/metabolism , Cell Differentiation/genetics , Cell Line , Cells, Cultured , Cluster Analysis , Embryonic Stem Cells/cytology , Fetus , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Infant, Newborn , Keratinocytes/cytology , Keratinocytes/metabolism , Oligonucleotide Array Sequence Analysis/methods , Stem Cells/cytology , Stem Cells/metabolism
19.
Proc Natl Acad Sci U S A ; 106(37): 15720-5, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19805220

ABSTRACT

Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However, most of the studies used skin fibroblasts as the starting population for reprogramming, which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore, iPS cells can be readily derived from adult hASCs in a feeder-free condition, thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion, are easy to maintain in culture, and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells.


Subject(s)
Adipocytes/cytology , Adult Stem Cells/cytology , Cell Dedifferentiation , Pluripotent Stem Cells/cytology , Adipocytes/immunology , Adipocytes/metabolism , Adult , Adult Stem Cells/immunology , Adult Stem Cells/metabolism , Aged , Alkaline Phosphatase/metabolism , Antigens, CD/metabolism , Antigens, Surface/metabolism , Cell Culture Techniques/methods , Cell Dedifferentiation/genetics , Cell Dedifferentiation/immunology , Cell Dedifferentiation/physiology , Cell Line , Cell Separation/methods , Gene Expression , Humans , Middle Aged , Pluripotent Stem Cells/immunology , Pluripotent Stem Cells/metabolism , Proteoglycans/metabolism
20.
PLoS One ; 4(9): e7040, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19753305

ABSTRACT

BACKGROUND: Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs). METHODS AND RESULTS: To induce endothelial cell differentiation, undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days, CD31(+) cells (13.7+/-2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation, these hESC-ECs expressed endothelial specific markers such as vWF (96.3+/-1.4%), CD31 (97.2+/-2.5%), and VE-cadherin (93.7+/-2.8%), form vascular-like channels, and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward, 5x10(6) hESC-ECs treated for 24 hours with nicotine (10(-8) M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 microg/ml) in the drinking water. Surprisingly, bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally, in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs). CONCLUSIONS: This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs, and enhance their angiogenic effects in vivo. Furthermore, activation of nAChRs has anti-apoptotic, angiogenic, and proliferative effects through MAPK and Akt signaling pathways.


Subject(s)
Apoptosis , Embryonic Stem Cells/cytology , Endothelial Cells/cytology , Neovascularization, Pathologic , Receptors, Nicotinic/metabolism , Animals , Cell Differentiation , Cell Proliferation , Female , Humans , Karyotyping , MAP Kinase Signaling System , Mice , Mice, SCID , Proto-Oncogene Proteins c-akt/metabolism
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