Dr. Mercola is Professor of Medicine and Professor in the Stanford Cardiovascular Institute. He completed postdoctoral training at the Dana-Farber Cancer Institute and Harvard Medical School, was on the faculty in the Department of Cell Biology at Harvard Medical School for 12 years, and later at the Sanford-Burnham-Prebys Institute and Department of Bioengineering at the University of California, San Diego before relocating to Stanford in 2015.
Prof. Mercola is known for identifying many of the factors that are responsible for inducing and forming the heart, including the discovery that Wnt inhibition is a critical step in cardiogenesis that provided the conceptual basis and reagents for the large-scale production of cardiovascular tissues from pluripotent stem cells. He has collaborated with medicinal chemists, optical engineers and software developers to pioneer the use of patient iPSC-cardiomyocytes for disease modeling, safety pharmacology and drug development. His academic research is focused on developing and using quantitative assays of patient-specific cardiomyocyte function to discover druggable targets for preserving contractile function in heart failure and promoting regeneration following ischemic injury. He co-established drug screening and assay development at the Conrad Prebys Drug Discovery Center (San Diego), which operated as one of 4 large screening centers of the US National Institutes of Health (NIH) Molecular Libraries screening initiative and continues as one of the largest academic drug screening centers.
Prof. Mercola received an NIH MERIT award for his work on heart formation, and authored over 120 papers. He holds numerous patents, including describing the invention of the first engineered dominant negative protein and small molecules for stem cell and cancer applications. He serves on multiple editorial and advisory boards, including Vala Sciences, Stem Cell Theranostics, Regencor, and the Human Biomolecular Research Institute. His laboratory is funded by the NIH, California Institute for Regenerative Medicine and the Fondation Leducq.
High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells.
Science translational medicine
2017; 9 (377)
Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.
View details for DOI 10.1126/scitranslmed.aaf2584
View details for PubMedID 28202772
View details for PubMedCentralID PMC5409837
Bringing new dimensions to drug discovery screening: impact of cellular stimulation technologies.
Drug discovery today
The current mandate for the drug discovery industry is to develop more efficient drugs faster while reducing the costs associated with their development. Incorporation of cell stimulation technologies during screening assays is expected to revolutionize the discovery of novel drugs as well as safety pharmacology. In this review, we highlight 'classical' and emerging cell stimulation technologies that provide the ability to evaluate the effects of drug candidates on cells in different functional states to assess clinically relevant phenotypes.
View details for DOI 10.1016/j.drudis.2017.01.015
View details for PubMedID 28179145
miR-322/-503 cluster is expressed in the earliest cardiac progenitor cells and drives cardiomyocyte specification
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (34): 9551-9556
Understanding the mechanisms of early cardiac fate determination may lead to better approaches in promoting heart regeneration. We used a mesoderm posterior 1 (Mesp1)-Cre/Rosa26-EYFP reporter system to identify microRNAs (miRNAs) enriched in early cardiac progenitor cells. Most of these miRNA genes bear MESP1-binding sites and active histone signatures. In a calcium transient-based screening assay, we identified miRNAs that may promote the cardiomyocyte program. An X-chromosome miRNA cluster, miR-322/-503, is the most enriched in the Mesp1 lineage and is the most potent in the screening assay. It is specifically expressed in the looping heart. Ectopic miR-322/-503 mimicking the endogenous temporal patterns specifically drives a cardiomyocyte program while inhibiting neural lineages, likely by targeting the RNA-binding protein CUG-binding protein Elav-like family member 1 (Celf1). Thus, early miRNAs in lineage-committed cells may play powerful roles in cell-fate determination by cross-suppressing other lineages. miRNAs identified in this study, especially miR-322/-503, are potent regulators of early cardiac fate.
View details for DOI 10.1073/pnas.1608256113
View details for Web of Science ID 000381860800054
View details for PubMedID 27512039
- The All-Chemical Approach: A Solution for Converting Fibroblasts Into Myocytes. Circulation research 2016; 119 (4): 505-507
High throughput physiological screening of iPSC-derived cardiomyocytes for drug development
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH
2016; 1863 (7): 1717-1727
Cardiac drug discovery is hampered by the reliance on non-human animal and cellular models with inadequate throughput and physiological fidelity to accurately identify new targets and test novel therapeutic strategies. Similarly, adverse drug effects on the heart are challenging to model, contributing to costly failure of drugs during development and even after market launch. Human induced pluripotent stem cell derived cardiac tissue represents a potentially powerful means to model aspects of heart physiology relevant to disease and adverse drug effects, providing both the human context and throughput needed to improve the efficiency of drug development. Here we review emerging technologies for high throughput measurements of cardiomyocyte physiology, and comment on the promises and challenges of using iPSC-derived cardiomyocytes to model disease and introduce the human context into early stages of drug discovery. This article is part of a Special Issue entitled: Cardiomyocyte biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.
View details for DOI 10.1016/j.bbamcr.2016.03.003
View details for Web of Science ID 000378360400004
View details for PubMedID 26952934
Notch-independent RBPJ controls angiogenesis in the adult heart
Increasing angiogenesis has long been considered a therapeutic target for improving heart function after injury such as acute myocardial infarction. However, gene, protein and cell therapies to increase microvascularization have not been successful, most likely because the studies failed to achieve regulated and concerted expression of pro-angiogenic and angiostatic factors needed to produce functional microvasculature. Here, we report that the transcription factor RBPJ is a homoeostatic repressor of multiple pro-angiogenic and angiostatic factor genes in cardiomyocytes. RBPJ controls angiogenic factor gene expression independently of Notch by antagonizing the activity of hypoxia-inducible factors (HIFs). In contrast to previous strategies, the cardiomyocyte-specific deletion of Rbpj increased microvascularization of the heart without adversely affecting cardiac structure or function even into old age. Furthermore, the loss of RBPJ in cardiomyocytes increased hypoxia tolerance, improved heart function and decreased pathological remodelling after myocardial infarction, suggesting that inhibiting RBPJ might be therapeutic for ischaemic injury.
View details for DOI 10.1038/ncomms12088
View details for Web of Science ID 000379113200001
View details for PubMedID 27357444
Epicardial FSTL1 reconstitution regenerates the adult mammalian heart.
2015; 525 (7570): 479-485
The elucidation of factors that activate the regeneration of the adult mammalian heart is of major scientific and therapeutic importance. Here we found that epicardial cells contain a potent cardiogenic activity identified as follistatin-like 1 (Fstl1). Epicardial Fstl1 declines following myocardial infarction and is replaced by myocardial expression. Myocardial Fstl1 does not promote regeneration, either basally or upon transgenic overexpression. Application of the human Fstl1 protein (FSTL1) via an epicardial patch stimulates cell cycle entry and division of pre-existing cardiomyocytes, improving cardiac function and survival in mouse and swine models of myocardial infarction. The data suggest that the loss of epicardial FSTL1 is a maladaptive response to injury, and that its restoration would be an effective way to reverse myocardial death and remodelling following myocardial infarction in humans.
View details for DOI 10.1038/nature15372
View details for PubMedID 26375005
- Epicardial FSTL1 reconstitution regenerates the adult mammalian heart NATURE 2015; 525 (7570): 479-?
Developmental origin of age-related coronary artery disease.
2015; 107 (2): 287-294
Age and injury cause structural and functional changes in coronary artery smooth muscle cells (caSMCs) that influence the pathogenesis of coronary artery disease. Although paracrine signalling is widely believed to drive phenotypic changes in caSMCs, here we show that developmental origin within the fetal epicardium can have a profound effect as well.Fluorescent dye and transgene pulse-labelling techniques in mice revealed that the majority of caSMCs are derived from Wt1(+), Gata5-Cre(+) cells that migrate before E12.5, whereas a minority of cells are derived from a later-emigrating, Wt1(+), Gata5-Cre(-) population. We functionally evaluated the influence of early emigrating cells on coronary artery development and disease by Gata5-Cre excision of Rbpj, which prevents their contribution to coronary artery smooth muscle cells. Ablation of the Gata5-Cre(+) population resulted in coronary arteries consisting solely of Gata5-Cre(-) caSMCs. These coronary arteries appeared normal into early adulthood; however, by 5-8 months of age, they became progressively fibrotic, lost the adventitial outer elastin layer, were dysfunctional and leaky, and animals showed early mortality.Taken together, these data reveal heterogeneity in the fetal epicardium that is linked to coronary artery integrity, and that distortion of the coronaries epicardial origin predisposes to adult onset disease.
View details for DOI 10.1093/cvr/cvv167
View details for PubMedID 26054850
Inhibition of miR-25 improves cardiac contractility in the failing heart
2014; 508 (7497): 531-?
Heart failure is characterized by a debilitating decline in cardiac function, and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy. MicroRNAs (miRNAs) are dysregulated in heart failure but whether they control contractility or constitute therapeutic targets remains speculative. Using high-throughput functional screening of the human microRNAome, here we identify miRNAs that suppress intracellular calcium handling in heart muscle by interacting with messenger RNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a (also known as ATP2A2). Of 875 miRNAs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes in vitro and was upregulated in heart failure, both in mice and humans. Whereas adeno-associated virus 9 (AAV9)-mediated overexpression of miR-25 in vivo resulted in a significant loss of contractile function, injection of an antisense oligonucleotide (antagomiR) against miR-25 markedly halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR oligonucleotide. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggest that it might be targeted therapeutically to restore function.
View details for DOI 10.1038/nature13073
View details for Web of Science ID 000334741600038
View details for PubMedID 24670661
HDAC-regulated myomiRs control BAF60 variant exchange and direct the functional phenotype of fibro-adipogenic progenitors in dystrophic muscles
GENES & DEVELOPMENT
2014; 28 (8): 841-857
Fibro-adipogenic progenitors (FAPs) are important components of the skeletal muscle regenerative environment. Whether FAPs support muscle regeneration or promote fibro-adipogenic degeneration is emerging as a key determinant in the pathogenesis of muscular diseases, including Duchenne muscular dystrophy (DMD). However, the molecular mechanism that controls FAP lineage commitment and activity is currently unknown. We show here that an HDAC-myomiR-BAF60 variant network regulates the fate of FAPs in dystrophic muscles of mdx mice. Combinatorial analysis of gene expression microarray, genome-wide chromatin remodeling by nuclease accessibility (NA) combined with next-generation sequencing (NA-seq), small RNA sequencing (RNA-seq), and microRNA (miR) high-throughput screening (HTS) against SWI/SNF BAF60 variants revealed that HDAC inhibitors (HDACis) derepress a "latent" myogenic program in FAPs from dystrophic muscles at early stages of disease. Specifically, HDAC inhibition induces two core components of the myogenic transcriptional machinery, MYOD and BAF60C, and up-regulates the myogenic miRs (myomiRs) (miR-1.2, miR-133, and miR-206), which target the alternative BAF60 variants BAF60A and BAF60B, ultimately directing promyogenic differentiation while suppressing the fibro-adipogenic phenotype. In contrast, FAPs from late stage dystrophic muscles are resistant to HDACi-induced chromatin remodeling at myogenic loci and fail to activate the promyogenic phenotype. These results reveal a previously unappreciated disease stage-specific bipotency of mesenchimal cells within the regenerative environment of dystrophic muscles. Resolution of such bipotency by epigenetic intervention with HDACis provides a molecular rationale for the in situ reprogramming of target cells to promote therapeutic regeneration of dystrophic muscles.
View details for DOI 10.1101/gad.234468.113
View details for Web of Science ID 000334585400005
View details for PubMedID 24682306
Technical Variations in Low-Input RNA-seq Methodologies
Recent advances in RNA-seq methodologies from limiting amounts of mRNA have facilitated the characterization of rare cell-types in various biological systems. So far, however, technical variations in these methods have not been adequately characterized, vis-à-vis sensitivity, starting with reduced levels of mRNA. Here, we generated sequencing libraries from limiting amounts of mRNA using three amplification-based methods, viz. Smart-seq, DP-seq and CEL-seq, and demonstrated significant technical variations in these libraries. Reduction in mRNA levels led to inefficient amplification of the majority of low to moderately expressed transcripts. Furthermore, noise in primer hybridization and/or enzyme incorporation was magnified during the amplification step resulting in significant distortions in fold changes of the transcripts. Consequently, the majority of the differentially expressed transcripts identified were either high-expressed and/or exhibited high fold changes. High technical variations ultimately masked subtle biological differences mandating the development of improved amplification-based strategies for quantitative transcriptomics from limiting amounts of mRNA.
View details for DOI 10.1038/srep03678
View details for Web of Science ID 000329846100007
View details for PubMedID 24419370
Coordinate Nodal and BMP inhibition directs Baf60c-dependent cardiomyocyte commitment
GENES & DEVELOPMENT
2013; 27 (21): 2332-2344
A critical but molecularly uncharacterized step in heart formation and regeneration is the process that commits progenitor cells to differentiate into cardiomyocytes. Here, we show that the endoderm-derived dual Nodal/bone morphogenetic protein (BMP) antagonist Cerberus-1 (Cer1) in embryonic stem cell cultures orchestrates two signaling pathways that direct the SWI/SNF chromatin remodeling complex to cardiomyogenic loci in multipotent (KDR/Flk1+) progenitors, activating lineage-specific transcription. Transient inhibition of Nodal by Cer1 induces Brahma-associated factor 60c (Baf60c), one of three Baf60 variants (a, b, and c) that are mutually exclusively assembled into SWI/SNF. Blocking Nodal and BMP also induces lineage-specific transcription factors Gata4 and Tbx5, which interact with Baf60c. siRNA to Cer1, Baf60c, or the catalytic SWI/SNF subunit Brg1 prevented the developmental opening of chromatin surrounding the Nkx2.5 early cardiac enhancer and cardiomyocyte differentiation. Overexpression of Baf60c fully rescued these deficits, positioning Baf60c and SWI/SNF function downstream from Cer1. Thus, antagonism of Nodal and BMP coordinates induction of the myogenic Baf60c variant and interacting transcription factors to program the developmental opening of cardiomyocyte-specific loci in chromatin. This is the first demonstration that cues from the progenitor cell environment direct the subunit variant composition of SWI/SNF to remodel the transcriptional landscape for lineage-specific differentiation.
View details for DOI 10.1101/gad.225144.113
View details for Web of Science ID 000326921900005
View details for PubMedID 24186978
- Jumonji and Cardiac Fate CIRCULATION RESEARCH 2013; 113 (7): 837-839
Quantitative Transcriptomics using Designed Primer-based Amplification
We developed a novel Designed Primer-based RNA-sequencing strategy (DP-seq) that uses a defined set of heptamer primers to amplify the majority of expressed transcripts from limiting amounts of mRNA, while preserving their relative abundance. Our strategy reproducibly yielded high levels of amplification from as low as 50 picograms of mRNA while offering a dynamic range of over five orders of magnitude in RNA concentrations. We also demonstrated the potential of DP-seq to selectively suppress the amplification of the highly expressing ribosomal transcripts by more than 70% in our sequencing library. Using lineage segregation in embryonic stem cell cultures as a model of early mammalian embryogenesis, DP-seq revealed novel sets of low abundant transcripts, some corresponding to the identity of cellular progeny before they arise, reflecting the specification of cell fate prior to actual germ layer segregation.
View details for DOI 10.1038/srep01740
View details for Web of Science ID 000318145200001
View details for PubMedID 23624976
Induced Pluripotent Stem Cells in Cardiovascular Drug Discovery
2013; 112 (3): 534-548
The unexpected discovery that somatic cells can be reprogrammed to a pluripotent state yielding induced pluripotent stem cells has made it possible to produce cardiovascular cells exhibiting inherited traits and disorders. Use of these cells in high throughput analyses should broaden our insight into fundamental disease mechanisms and provide many benefits for patients, including new therapeutics and individually tailored therapies. Here we review recent progress in generating induced pluripotent stem cell-based models of cardiovascular disease and their multiple applications in drug development.
View details for DOI 10.1161/CIRCRESAHA.111.250266
View details for Web of Science ID 000314356700021
View details for PubMedID 23371902
Developing microRNA screening as a functional genomics tool for disease research.
Frontiers in physiology
2013; 4: 223-?
Originally discovered as regulators of developmental timing in C. elegans, microRNAs (miRNAs) have emerged as modulators of nearly every cellular process, from normal development to pathogenesis. With the advent of whole genome libraries of miRNA mimics suitable for high throughput screening, it is possible to comprehensively evaluate the function of each member of the miRNAome in cell-based assays. Since the relatively few microRNAs in the genome are thought to directly regulate a large portion of the proteome, miRNAome screening, coupled with the identification of the regulated proteins, might be a powerful new approach to gaining insight into complex biological processes.
View details for DOI 10.3389/fphys.2013.00223
View details for PubMedID 23986717
- CARDIOVASCULAR BIOLOGY A boost for heart regeneration NATURE 2012; 492 (7429): 360-362
BAF60 A, B, and Cs of muscle determination and renewal
GENES & DEVELOPMENT
2012; 26 (24): 2673-2683
Developmental biologists have defined many of the diffusible and transcription factors that control muscle differentiation, yet we still have only rudimentary knowledge of the mechanisms that dictate whether a myogenic progenitor cell forms muscle versus alternate lineages, including those that can be pathological in a state of disease or degeneration. Clues about the molecular basis for lineage determination in muscle progenitors are only now emerging from studies of chromatin modifications that avail myogenic genes for transcription, together with analysis of the composition and activities of the chromatin-modifying complexes themselves. Here we review recent progress on muscle determination and explore a unifying theme that environmental cues from the stem or progenitor niche control the selection of specific subunit variants of the switch/sucrose nonfermentable (SWI/SNF) chromatin-modifying complex, creating a combinatorial code that dictates whether cells adopt myogenic versus nonmyogenic cell fates. A key component of the code appears to be the mutually exclusive usage of the a, b, and c variants of the 60-kD structural subunit BAF60 (BRG1/BRM-associated factor 60), of which BAF60c is essential to activate both skeletal and cardiac muscle programs. Since chromatin remodeling governs myogenic fate, the combinatorial assembly of the SWI/SNF complex might be targeted to develop drugs aimed at the therapeutic reduction of compensatory fibrosis and fatty deposition in chronic muscular disorders.
View details for DOI 10.1101/gad.207415.112
View details for Web of Science ID 000312775700002
View details for PubMedID 23222103
Whole-genome microRNA screening identifies let-7 and mir-18 as regulators of germ layer formation during early embryogenesis
GENES & DEVELOPMENT
2012; 26 (23): 2567-2579
Tight control over the segregation of endoderm, mesoderm, and ectoderm is essential for normal embryonic development of all species, yet how neighboring embryonic blastomeres can contribute to different germ layers has never been fully explained. We postulated that microRNAs, which fine-tune many biological processes, might modulate the response of embryonic blastomeres to growth factors and other signals that govern germ layer fate. A systematic screen of a whole-genome microRNA library revealed that the let-7 and miR-18 families increase mesoderm at the expense of endoderm in mouse embryonic stem cells. Both families are expressed in ectoderm and mesoderm, but not endoderm, as these tissues become distinct during mouse and frog embryogenesis. Blocking let-7 function in vivo dramatically affected cell fate, diverting presumptive mesoderm and ectoderm into endoderm. siRNA knockdown of computationally predicted targets followed by mutational analyses revealed that let-7 and miR-18 down-regulate Acvr1b and Smad2, respectively, to attenuate Nodal responsiveness and bias blastomeres to ectoderm and mesoderm fates. These findings suggest a crucial role for the let-7 and miR-18 families in germ layer specification and reveal a remarkable conservation of function from amphibians to mammals.
View details for DOI 10.1101/gad.200758.112
View details for Web of Science ID 000311944000002
View details for PubMedID 23152446
Synthesis and SAR of b-Annulated 1,4-Dihydropyridines Define Cardiomyogenic Compounds as Novel Inhibitors of TGF beta Signaling
JOURNAL OF MEDICINAL CHEMISTRY
2012; 55 (22): 9946-9957
A medium-throughput murine embryonic stem cell (mESC)-based high-content screening of 17000 small molecules for cardiogenesis led to the identification of a b-annulated 1,4-dihydropyridine (1,4-DHP) that inhibited transforming growth factor β (TGFβ)/Smad signaling by clearing the type II TGFβ receptor from the cell surface. Because this is an unprecedented mechanism of action, we explored the series' structure-activity relationship (SAR) based on TGFβ inhibition, and evaluated SAR aspects for cell-surface clearance of TGFβ receptor II (TGFBR2) and for biological activity in mESCs. We determined a pharmacophore and generated 1,4-DHPs with IC(50)s for TGFβ inhibition in the nanomolar range (e.g., compound 28, 170 nM). Stereochemical consequences of a chiral center at the 4-position was evaluated, revealing 10- to 15-fold more potent TGFβ inhibition for the (+)- than the (-) enantiomer. This stereopreference was not observed for the low level inhibition against Activin A signaling and was reversed for effects on calcium handling in HL-1 cells.
View details for DOI 10.1021/jm301144g
View details for Web of Science ID 000311461500045
View details for PubMedID 23130626
High throughput measurement of Ca2+ dynamics for drug risk assessment in human stem cell-derived cardiomyocytes by kinetic image cytometry
JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS
2012; 66 (3): 246-256
Current methods to measure physiological properties of cardiomyocytes and predict fatal arrhythmias that can cause sudden death, such as Torsade de Pointes, lack either the automation and throughput needed for early-stage drug discovery and/or have poor predictive value. To increase throughput and predictive power of in vitro assays, we developed kinetic imaging cytometry (KIC) for automated cell-by-cell analyses via intracellular fluorescence Ca²⁺ indicators. The KIC instrument simultaneously records and analyzes intracellular calcium concentration [Ca²⁺](i) at 30-ms resolution from hundreds of individual cells/well of 96-well plates in seconds, providing kinetic details not previously possible with well averaging technologies such as plate readers. Analyses of human embryonic stem cell and induced pluripotent stem cell-derived cardiomyocytes revealed effects of known cardiotoxic and arrhythmogenic drugs on kinetic parameters of Ca²⁺ dynamics, suggesting that KIC will aid in the assessment of cardiotoxic risk and in the elucidation of pathogenic mechanisms of heart disease associated with drugs treatment and/or genetic background.
View details for DOI 10.1016/j.vascn.2012.08.167
View details for Web of Science ID 000311489100006
View details for PubMedID 22926323
Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (40): 16196-16201
Generation of human induced pluripotent stem cells (hiPSCs) by the expression of specific transcription factors depends on successful epigenetic reprogramming to a pluripotent state. Although hiPSCs and human embryonic stem cells (hESCs) display a similar epigenome, recent reports demonstrated the persistence of specific epigenetic marks from the somatic cell type of origin and aberrant methylation patterns in hiPSCs. However, it remains unknown whether the use of different somatic cell sources, encompassing variable levels of selection pressure during reprogramming, influences the level of epigenetic aberrations in hiPSCs. In this work, we characterized the epigenomic integrity of 17 hiPSC lines derived from six different cell types with varied reprogramming efficiencies. We demonstrate that epigenetic aberrations are a general feature of the hiPSC state and are independent of the somatic cell source. Interestingly, we observe that the reprogramming efficiency of somatic cell lines inversely correlates with the amount of methylation change needed to acquire pluripotency. Additionally, we determine that both shared and line-specific epigenetic aberrations in hiPSCs can directly translate into changes in gene expression in both the pluripotent and differentiated states. Significantly, our analysis of different hiPSC lines from multiple cell types of origin allow us to identify a reprogramming-specific epigenetic signature comprised of nine aberrantly methylated genes that is able to segregate hESC and hiPSC lines regardless of the somatic cell source or differentiation state.
View details for DOI 10.1073/pnas.1202352109
View details for Web of Science ID 000309611400053
View details for PubMedID 22991473
A Nodal-to-TGF beta Cascade Exerts Biphasic Control Over Cardiopoiesis
2012; 111 (7): 876-?
The transforming growth factor-β (TGFβ) family member Nodal promotes cardiogenesis, but the mechanism is unclear despite the relevance of TGFβ family proteins for myocardial remodeling and regeneration.To determine the function(s) of TGFβ family members during stem cell cardiogenesis.Murine embryonic stem cells were engineered with a constitutively active human type I Nodal receptor (caACVR1b) to mimic activation by Nodal and found to secrete a paracrine signal that promotes cardiogenesis. Transcriptome and gain- and loss-of-function studies identified the factor as TGFβ2. Both Nodal and TGFβ induced early cardiogenic progenitors in embryonic stem cell cultures at day 0 to 2 of differentiation. However, Nodal expression declines by day 4 due to feedback inhibition, whereas TGFβ persists. At later stages (days 4-6), TGFβ suppresses the formation of cardiomyocytes from multipotent Kdr(+) progenitors while promoting the differentiation of vascular smooth muscle and endothelial cells.Nodal induces TGFβ, and both stimulate the formation of multipotent cardiovascular Kdr(+) progenitors. TGFβ, however, becomes uniquely responsible for controlling subsequent lineage segregation by stimulating vascular smooth muscle and endothelial lineages and simultaneously blocking cardiomyocyte differentiation.
View details for DOI 10.1161/CIRCRESAHA.112.270272
View details for Web of Science ID 000308868800014
View details for PubMedID 22872153
APJ acts as a dual receptor in cardiac hypertrophy
2012; 488 (7411): 394-398
Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues. Here we report that genetic loss of APJ, a G-protein-coupled receptor, confers resistance to chronic pressure overload by markedly reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin-independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechanosensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate Gαi and elicits a protective response, stretch signals in an APJ-dependent, G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of β-arrestins or by pharmacological doses of apelin acting through Gαi. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.
View details for DOI 10.1038/nature11263
View details for Web of Science ID 000307501000045
View details for PubMedID 22810587
Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (32): 13016-13021
Unique insights for the reprograming of cell lineages have come from embryonic development in the ascidian Ciona, which is dependent upon the transcription factors Ci-ets1/2 and Ci-mesp to generate cardiac progenitors. We tested the idea that mammalian v-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) and mesoderm posterior (MESP) homolog may be used to convert human dermal fibroblasts into cardiac progenitors. Here we show that murine ETS2 has a critical role in directing cardiac progenitors during cardiopoiesis in embryonic stem cells. We then use lentivirus-mediated forced expression of human ETS2 to convert normal human dermal fibroblasts into replicative cells expressing the cardiac mesoderm marker KDR(+). However, although neither ETS2 nor the purported cardiac master regulator MESP1 can by themselves generate cardiac progenitors de novo from fibroblasts, forced coexpression of ETS2 and MESP1 or cell treatment with purified proteins reprograms fibroblasts into cardiac progenitors, as shown by the de novo appearance of core cardiac transcription factors, Ca(2+) transients, and sarcomeres. Our data indicate that ETS2 and MESP1 play important roles in a genetic network that governs cardiopoiesis.
View details for DOI 10.1073/pnas.1120299109
View details for Web of Science ID 000307551700041
View details for PubMedID 22826236
Small Molecule-Mediated TGF-beta Type II Receptor Degradation Promotes Cardiomyogenesis in Embryonic Stem Cells
CELL STEM CELL
2012; 11 (2): 242-252
The cellular signals controlling the formation of cardiomyocytes, vascular smooth muscle, and endothelial cells from stem cell-derived mesoderm are poorly understood. To identify these signals, a mouse embryonic stem cell (ESC)-based differentiation assay was screened against a small molecule library resulting in a 1,4-dihydropyridine inducer of type II TGF-β receptor (TGFBR2) degradation-1 (ITD-1). ITD analogs enhanced proteasomal degradation of TGFBR2, effectively clearing the receptor from the cell surface and selectively inhibiting intracellular signaling (IC(50) ~0.4-0.8 μM). ITD-1 was used to evaluate TGF-β involvement in mesoderm formation and cardiopoietic differentiation, which occur sequentially during early development, revealing an essential role in both processes in ESC cultures. ITD-1 selectively enhanced the differentiation of uncommitted mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells. ITD-1 is a highly selective TGF-β inhibitor and reveals an unexpected role for TGF-β signaling in controlling cardiomyocyte differentiation from multipotent cardiovascular precursors.
View details for DOI 10.1016/j.stem.2012.04.025
View details for Web of Science ID 000307432600014
View details for PubMedID 22862949
HNF4 alpha Antagonists Discovered by a High-Throughput Screen for Modulators of the Human Insulin Promoter
CHEMISTRY & BIOLOGY
2012; 19 (7): 806-818
Hepatocyte nuclear factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic β cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and were proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.
View details for DOI 10.1016/j.chembiol.2012.05.014
View details for Web of Science ID 000307261100007
View details for PubMedID 22840769
Wnt Inhibition Correlates with Human Embryonic Stem Cell Cardiomyogenesis: A Structure-Activity Relationship Study Based on Inhibitors for the Wnt Response
JOURNAL OF MEDICINAL CHEMISTRY
2012; 55 (2): 697-708
Human embryonic stem cell-based high-content screening of 550 known signal transduction modulators showed that one "lead" (1, a recently described inhibitor of the proteolytic degradation of Axin) stimulated cardiomyogenesis. Because Axin controls canonical Wnt signaling, we conducted an investigation to determine whether the cardiogenic activity of 1 is Wnt-dependent, and we developed a structure-activity relationship to optimize the cardiogenic properties of 1. We prepared analogues with a range of potencies (low nanomolar to inactive) for Wnt/β-catenin inhibition and for cardiogenic induction. Both functional activities correlated positively (r(2) = 0.72). The optimal compounds induced cardiogenesis 1.5-fold greater than 1 at 30-fold lower concentrations. In contrast, no correlation was observed for cardiogenesis and modulation of transforming growth factor β (TGFβ)/Smad signaling that prominently influences cardiogenesis. Taken together, these data show that Wnt signaling inhibition is essential for cardiogenic activity and that the pathway can be targeted for the design of druglike cardiogenic molecules.
View details for DOI 10.1021/jm2010223
View details for Web of Science ID 000299453300013
View details for PubMedID 22191557
Fine-Tuning of Drp1/Fis1 Availability by AKAP121/Siah2 Regulates Mitochondrial Adaptation to Hypoxia
2011; 44 (4): 532-544
Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. Here we demonstrate that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability of AKAP121 by the ubiquitin ligase Siah2 relieves Drp1 inhibition by PKA and increases its interaction with Fis1, resulting in mitochondrial fission. High AKAP121 levels, seen in cells lacking Siah2, attenuate fission and reduce apoptosis of cardiomyocytes under simulated ischemia. Infarct size and degree of cell death were reduced in Siah2(-/-) mice subjected to myocardial infarction. Inhibition of Siah2 or Drp1 in hatching C. elegans reduces their life span. Through modulating Fis1/Drp1 complex availability, our studies identify Siah2 as a key regulator of hypoxia-induced mitochondrial fission and its physiological significance in ischemic injury and nematode life span.
View details for DOI 10.1016/j.molcel.2011.08.045
View details for Web of Science ID 000297387800006
View details for PubMedID 22099302
Small-Molecule Inhibitors of the Wnt Pathway Potently Promote Cardiomyocytes From Human Embryonic Stem Cell-Derived Mesoderm
2011; 109 (4): 360-364
Human embryonic stem cells can form cardiomyocytes when cultured under differentiation conditions. Although the initiating step of mesoderm formation is well characterized, the subsequent steps that promote for cardiac lineages are poorly understood and limit the yield of cardiomyocytes.Our aim was to develop a human embryonic stem cell-based high-content screening assay to discover small molecules that drive cardiogenic differentiation after mesoderm is established to improve our understanding of the biology involved. Screening of libraries of small-molecule pathway modulators was predicted to provide insight into the cellular proteins and signaling pathways that control stem cell cardiogenesis.Approximately 550 known pathway modulators were screened in a high-content screening assay, with hits being called out by the appearance of a red fluorescent protein driven by the promoter of the cardiac-specific MYH6 gene. One potent small molecule was identified that inhibits transduction of the canonical Wnt response within the cell, which demonstrated that Wnt inhibition alone was sufficient to generate cardiomyocytes from human embryonic stem cell-derived mesoderm cells. Transcriptional profiling of inhibitor-treated compared with vehicle-treated samples further indicated that inhibition of Wnt does not induce other mesoderm lineages. Notably, several other Wnt inhibitors were very efficient in inducing cardiogenesis, including a molecule that prevents Wnts from being secreted by the cell, which confirmed that Wnt inhibition was the relevant biological activity.Pharmacological inhibition of Wnt signaling is sufficient to drive human mesoderm cells to form cardiomyocytes; this could yield novel tools for the benefit of pharmaceutical and clinical applications.
View details for DOI 10.1161/CIRCRESAHA.111.249540
View details for Web of Science ID 000293504100004
View details for PubMedID 21737789
Cardiac muscle regeneration: lessons from development
GENES & DEVELOPMENT
2011; 25 (4): 299-309
The adult human heart is an ideal target for regenerative intervention since it does not functionally restore itself after injury yet has a modest regenerative capacity that could be enhanced by innovative therapies. Adult cardiac cells with regenerative potential share gene expression signatures with early fetal progenitors that give rise to multiple cardiac cell types, suggesting that the evolutionarily conserved regulatory networks that drive embryonic heart development might also control aspects of regeneration. Here we discuss commonalities of development and regeneration, and the application of the rich developmental biology heritage to achieve therapeutic regeneration of the human heart.
View details for DOI 10.1101/gad.2018411
View details for Web of Science ID 000287365000003
View details for PubMedID 21325131
What Your Heart Doth Know
CELL STEM CELL
2011; 8 (2): 124-126
Combining embryological insight with careful analysis of early stage cardiomyocyte differentiation, Kattman et al. (2011) in this issue of Cell Stem Cell have defined minimal culture conditions to efficiently produce cardiomyocytes from hESCs and hiPSCs. The lessons learned are applicable to the derivation of other organotypic cell types.
View details for DOI 10.1016/j.stem.2011.01.003
View details for Web of Science ID 000287633400003
View details for PubMedID 21295266
Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (23): 10514-10519
Two major goals of regenerative medicine are to reproducibly transform adult somatic cells into a pluripotent state and to control their differentiation into specific cell fates. Progress toward these goals would be greatly helped by obtaining a complete picture of the RNA isoforms produced by these cells due to alternative splicing (AS) and alternative promoter selection (APS). To investigate the roles of AS and APS, reciprocal exon-exon junctions were interrogated on a genome-wide scale in differentiating mouse embryonic stem (ES) cells with a prototype Affymetrix microarray. Using a recently released open-source software package named AltAnalyze, we identified 144 genes for 170 putative isoform variants, the majority (67%) of which were predicted to alter protein sequence and domain composition. Verified alternative exons were largely associated with pathways of Wnt signaling and cell-cycle control, and most were conserved between mouse and human. To examine the functional impact of AS, we characterized isoforms for two genes. As predicted by AltAnalyze, we found that alternative isoforms of the gene Serca2 were targeted by distinct microRNAs (miRNA-200b, miRNA-214), suggesting a critical role for AS in cardiac development. Analysis of the Wnt transcription factor Tcf3, using selective knockdown of an ES cell-enriched and characterized isoform, revealed several distinct targets for transcriptional repression (Stmn2, Ccnd2, Atf3, Klf4, Nodal, and Jun) as well as distinct differentiation outcomes in ES cells. The findings herein illustrate a critical role for AS in the specification of ES cells with differentiation, and highlight the utility of global functional analyses of AS.
View details for DOI 10.1073/pnas.0912260107
View details for Web of Science ID 000278549300035
View details for PubMedID 20498046
Non-Cardiomyocytes Influence the Electrophysiological Maturation of Human Embryonic Stem Cell-Derived Cardiomyocytes During Differentiation
STEM CELLS AND DEVELOPMENT
2010; 19 (6): 783-795
Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs). We isolated pure populations of hESC-CMs from EBs by lentivirus-engineered Puromycin resistance at various stages of differentiation. Using pharmacological agents, calcium (Ca(2+)) imaging, and intracellular recording techniques, we found that intracellular Ca(2+)-cycling mechanisms developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological properties of hESC-CMs. In contrast to the development of intracellular Ca(2+)-handling proteins, ion channel development and electrophysiological maturation of hESC-CMs did not occur when hESC-CMs were isolated from EBs early and maintained in culture without further interaction with non-cardiomyocytes. Adding back non-cardiomyocytes to early-isolated hESC-CMs rescued the arrest of electrophysiological maturation, indicating that non-cardiomyocytes in EBs drive electrophysiological maturation of early hESC-CMs. Non-cardiomyocytes in EBs contain most cell types present in the embryonic heart that are known to influence early cardiac development. Our study is the first to demonstrate that non-cardiomyocytes influence electrophysiological maturation of early hESC-CMs in cultures. Defining the nature of these extrinsic signals will aid in the directed maturation of immature hESC-CMs to mitigate arrhythmogenic risks of cell-based therapies.
View details for DOI 10.1089/scd.2009.0349
View details for Web of Science ID 000279033900004
View details for PubMedID 20001453
- Electrophysiological Challenges of Cell-Based Myocardial Repair CIRCULATION 2009; 120 (24): 2496-2508
Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes
2009; 4 (4)
Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.
View details for DOI 10.1371/journal.pone.0005046
View details for Web of Science ID 000265505700004
View details for PubMedID 19352491
Deletion of Shp2 Tyrosine Phosphatase in Muscle Leads to Dilated Cardiomyopathy, Insulin Resistance, and Premature Death
MOLECULAR AND CELLULAR BIOLOGY
2009; 29 (2): 378-388
The intracellular signaling mechanisms underlying the pathogenesis of cardiac diseases are not fully understood. We report here that selective deletion of Shp2, an SH2-containing cytoplasmic tyrosine phosphatase, in striated muscle results in severe dilated cardiomyopathy in mice, leading to heart failure and premature mortality. Development of cardiomyopathy in this mouse model is coupled with insulin resistance, glucose intolerance, and impaired glucose uptake in striated muscle cells. Shp2 deficiency leads to upregulation of leukemia inhibitory factor-stimulated phosphatidylinositol 3-kinase/Akt, Erk5, and Stat3 pathways in cardiomyocytes. Insulin resistance and impaired glucose uptake in Shp2-deficient mice are at least in part due to impaired protein kinase C-zeta/lambda and AMP-kinase activities in striated muscle. Thus, we have generated a mouse line modeling human patients suffering from cardiomyopathy and insulin resistance. This study reinforces a concept that a compound disease with multiple cardiovascular and metabolic disturbances can be caused by a defect in a single molecule such as Shp2, which modulates multiple signaling pathways initiated by cytokines and hormones.
View details for DOI 10.1128/MCB.01661-08
View details for Web of Science ID 000262045800007
View details for PubMedID 19001090
Notch activates cell cycle reentry and progression in quiescent cardiomyocytes
JOURNAL OF CELL BIOLOGY
2008; 183 (1): 129-141
The inability of heart muscle to regenerate by replication of existing cardiomyocytes has engendered considerable interest in identifying developmental or other stimuli capable of sustaining the proliferative capacity of immature cardiomyocytes or stimulating division of postmitotic cardiomyocytes. Here, we demonstrate that reactivation of Notch signaling causes embryonic stem cell-derived and neonatal ventricular cardiomyocytes to enter the cell cycle. The proliferative response of neonatal ventricular cardiomyocytes declines as they mature, such that late activation of Notch triggers the DNA damage checkpoint and G2/M interphase arrest. Notch induces recombination signal-binding protein 1 for Jkappa (RBP-Jkappa)-dependent expression of cyclin D1 but, unlike other inducers, also shifts its subcellular distribution from the cytosol to the nucleus. Nuclear localization of cyclin D1 is independent of RBP-Jkappa. Thus, the influence of Notch on nucleocytoplasmic localization of cyclin D1 is an unanticipated property of the Notch intracellular domain that is likely to regulate the cell cycle in multiple contexts, including tumorigenesis as well as cardiogenesis.
View details for DOI 10.1083/jcb.200806104
View details for Web of Science ID 000259985000013
View details for PubMedID 18838555
Developmental patterning of the cardiac atrioventricular canal by Notch and Hairy-related transcription factors
2006; 133 (21): 4381-4390
Mutations in Notch2, Jagged1 or homologs of the Hairy-related transcriptional repressor Hey2 cause congenital malformations involving the non-chamber atrioventricular canal (AVC) and inner curvature (IC) regions of the heart, but the underlying mechanisms have not been investigated. By manipulating signaling directly within the developing chick heart, we demonstrated that Notch2, Hey1 and Hey2 initiate a signaling cascade that delimits the non-chamber AVC and IC regions. Specifically, misactivation of Notch2 signaling, or misexpression of either Hey1 or Hey2, repressed Bmp2. Because Jagged (also known as Serrate in non-mammalian species) ligands were found to be present in prospective chamber myocardium, these data support the model that Notch2 and Hey proteins cause the progressive restriction of Bmp2 expression to within the developing AVC and IC, where it is essential for differentiation. Misactivation or inhibition of Notch2 specifically induced or inhibited Hey1, respectively, but these manipulations did not affect Hey2, implicating Hey1 as the direct mediator of Notch2. Bmp2 within the developing AVC and IC has been shown to induce Tbx2, and we found that Tbx2 misexpression inhibited the expression of both Hey1 and Hey2. Tbx2, therefore, is envisaged to constitute a feedback loop that sharpens the border with the developing AVC and IC by delimiting Hey gene expression to within prospective chamber regions. Analysis of the loss-of-function phenotype in mouse embryos homozygous for targeted disruption of Hey2 revealed an expanded AVC domain of Bmp2. Similarly, zebrafish gridlock (Hey2 homolog) mutant embryos showed ectopic expression of Bmp4, which normally marks AVC myocardium in this species. Thus, Hey pathway regulation of cardiac Bmp appears to be an evolutionarily conserved mechanism to delimit AVC and IC fate, and provides a potential mechanistic explanation for cardiac malformations caused by mutations in Serrate/Jagged1 and Notch signaling components.
View details for DOI 10.1242/dev.02607
View details for Web of Science ID 000241217400023
View details for PubMedID 17021042