Jared M. Churko

Chemically defined generation of human cardiomyocytes

Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule–based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.Existing methodologies for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require the use of complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed a highly optimized cardiac differentiation strategy, employing a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate, and ricederived recombinant human albumin. Along with small molecule-based differentiation induction, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell, and was effective in 11 hiPSC lines tested. This is the first fully chemically defined platform for cardiac specification of hiPSCs, and allows Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Addresses for Correspondence: Joseph C. Wu, MD, PhD, Stanford University School of Medicine, Lorry I. Lokey Stem Cell Research Building, 265 Campus Drive, Room G1120B, Stanford, CA 94305-5454. joewu@stanford.edu or Paul W. Burridge, PhD, burridge@stanford.edu. Author Contributions P.W.B. conceived, performed, and interpreted the experiments and wrote the manuscript; E.M. performed cardiomyocyte immunofluorescence, single-cell RT-PCR, and electrophysiology data assessment; P.S., Z.L., and A.J.O. performed electrophysiology experiments and assessed data; S.D. provided CoMiP reprogrammed cells; B.H. performed teratoma assay; J.M.C. A.D.E, F.L., N.M.M., and J.R.P tested differentiation; B.C., J.D.G. provided experimental advice; and J.C.W. provided experimental advice, manuscript writing, and funding support. Competing Financial Interests JCW is a co-founder of Stem Cell Theranostics. Other authors declare no competing financial interests. HHS Public Access Author manuscript Nat Methods. Author manuscript; available in PMC 2015 February 01. Published in final edited form as: Nat Methods. 2014 August ; 11(8): 855–860. doi:10.1038/nmeth.2999. A uhor M anscript

Identification of a New Modulator of the Intercalated Disc in a Zebrafish Model of Arrhythmogenic Cardiomyopathy

Drug screening in a zebrafish model of arrhythmogenic cardiomyopathy identified a small molecule that remodels the intercalated disc. Fishing for Heart Healing When Judy Garland sings “Thump, thump, thump went my heart strings,” she’s celebrating a new love connection. But it remains unclear why patients with arrhythmogenic cardiomyopathy (ACM) suffer frequent heart arrhythmias. Asimaki et al. used a zebrafish model of ACM to illuminate disease mechanisms and discovered a potential drug, SB216763, that suppressed the disease phenotype in the fish model. The authors also observed SB216763-reversible, abnormal cardiac remodeling in cardiac myocytes derived from rats that carried an ACM-related mutation and from induced pluripotent stem cells from two ACM probands. These observations pinpoint aberrant trafficking of cardiac proteins as a central mechanism underlying ACM cardiomyocyte injury and electrical abnormalities. Arrhythmogenic cardiomyopathy (ACM) is characterized by frequent cardiac arrhythmias. To elucidate the underlying mechanisms and discover potential chemical modifiers, we created a zebrafish model of ACM with cardiac myocyte–specific expression of the human 2057del2 mutation in the gene encoding plakoglobin. A high-throughput screen identified SB216763 as a suppressor of the disease phenotype. Early SB216763 therapy prevented heart failure and reduced mortality in the fish model. Zebrafish ventricular myocytes that expressed 2057del2 plakoglobin exhibited 70 to 80% reductions in INa and IK1 current densities, which were normalized by SB216763. Neonatal rat ventricular myocytes that expressed 2057del2 plakoglobin recapitulated pathobiological features seen in patients with ACM, all of which were reversed or prevented by SB216763. The reverse remodeling observed with SB216763 involved marked subcellular redistribution of plakoglobin, connexin 43, and Nav1.5, but without changes in their total cellular content, implicating a defect in protein trafficking to intercalated discs. In further support of this mechanism, we observed SB216763-reversible, abnormal subcellular distribution of SAP97 (a protein known to mediate forward trafficking of Nav1.5 and Kir2.1) in rat cardiac myocytes expressing 2057del2 plakoglobin and in cardiac myocytes derived from induced pluripotent stem cells from two ACM probands with plakophilin-2 mutations. These observations pinpoint aberrant trafficking of intercalated disc proteins as a central mechanism in ACM myocyte injury and electrical abnormalities.

Implications of pannexin 1 and pannexin 3 for keratinocyte differentiation

Pannexin (Panx) 1 and Panx3 are integral membrane proteins that share some sequence homology with the innexin family of invertebrate gap junctions. They are expressed in mammalian skin. Pannexins have been reported to form functional mechanosensitive single-membrane channels, but their importance in regulating cellular function is poorly understood. In this study, Panx1 and Panx3 were detected in the epidermis of 13.5 day embryonic mice. Compared with newborn mice, there was less Panx1 expression in both thin and thick murine skin, whereas Panx3 expression was unchanged. To investigate the role of pannexins in keratinocyte differentiation, we employed rat epidermal keratinocytes (REKs) that have the capacity to differentiate into organotypic epidermis, and engineered them to overexpress Panx1, Panx1-GFP or Panx3. The expression of Panx1 or Panx3 resulted in the increased ability of REKs to take up dye, suggesting that cell-surface channels were formed. Compared with monolayer REKs, endogenous Panx1 levels remained unchanged, whereas the 70 kDa immunoreactive species of Panx3 was greatly increased in the organotypic epidermis. In monolayer cultures, ectopic Panx1 and Panx1-GFP localized to the plasma membrane, whereas Panx3 displayed both intracellular and plasma-membrane profiles. Although both pannexins reduced cell proliferation, only Panx1 disrupted the architecture of the organotypic epidermis and markedly dysregulated cytokeratin 14 expression and localization. Furthermore, ectopic expression of only Panx1 reduced the vital layer thickness of the organotypic epidermis. In summary, Panx1 and Panx3 are coexpressed in the mammalian epidermis, and the regulation of Panx1 plays a key role in keratinocyte differentiation.

Pannexin1 and Pannexin3 Delivery, Cell Surface Dynamics, and Cytoskeletal Interactions

Pannexins (Panx) are a class of integral membrane proteins that have been proposed to exhibit characteristics similar to those of connexin family members. In this study, we utilized Cx43-positive BICR-M1Rk cells to stably express Panx1, Panx3, or Panx1-green fluorescent protein (GFP) to assess their trafficking, cell surface dynamics, and interplay with the cytoskeletal network. Expression of a Sar1 dominant negative mutant revealed that endoplasmic reticulum to Golgi transport of Panx1 and Panx3 was mediated via COPII-dependent vesicles. Distinct from Cx43-GFP, fluorescence recovery after photobleaching studies revealed that both Panx1-GFP and Panx3-GFP remained highly mobile at the cell surface. Unlike Cx43, Panx1-GFP exhibited no detectable interrelationship with microtubules. Conversely, cytochalasin B-induced disruption of microfilaments caused a severe loss of cell surface Panx1-GFP, a reduction in the recoverable fraction of Panx1-GFP that remained at the cell surface, and a decrease in Panx1-GFP vesicular transport. Furthermore, co-immunoprecipitation and co-sedimentation assays revealed actin as a novel binding partner of Panx1. Collectively, we conclude that although Panx1 and Panx3 share a common endoplasmic reticulum to Golgi secretory pathway to Cx43, their ultimate cell surface residency appears to be independent of cell contacts and the need for intact microtubules. Importantly, Panx1 has an interaction with actin microfilaments that regulates its cell surface localization and mobility.

Loss of Pannexin 1 Attenuates Melanoma Progression by Reversion to a Melanocytic Phenotype

Background: Panx1 is a channel-forming glycoprotein that regulates epidermal differentiation and proliferation. Results: Depletion of Panx1 in melanomas causes cell re-differentiation into a melanocytic-like phenotype and reduced tumorigenesis. Conclusion: Panx1 is up-regulated during melanoma progression promoting tumor growth and metastasis. Significance: This is the first report of Panx1 as a proto-oncogene establishing it as a potential target for melanoma treatment. Pannexin 1 (Panx1) is a channel-forming glycoprotein expressed in different cell types of mammalian skin. We examined the role of Panx1 in melanoma tumorigenesis and metastasis since qPCR and Western blots revealed that mouse melanocytes exhibited low levels of Panx1 while increased Panx1 expression was correlated with tumor cell aggressiveness in the isogenic melanoma cell lines (B16-F0, -F10, and -BL6). Panx1 shRNA knockdown (Panx1-KD) generated stable BL6 cell lines, with reduced dye uptake, that showed a marked increase in melanocyte-like cell characteristics including higher melanin production, decreased cell migration and enhanced formation of cellular projections. Western blotting and proteomic analyses using 2D-gel/mass spectroscopy identified vimentin and β-catenin as two of the markers of malignant melanoma that were down-regulated in Panx1-KD cells. Xenograft Panx1-KD cells grown within the chorioallantoic membrane of avian embryos developed tumors that were significantly smaller than controls. Mouse-Alu qPCR of the excised avian embryonic organs revealed that tumor metastasis to the liver was significantly reduced upon Panx1 knockdown. These data suggest that while Panx1 is present in skin melanocytes it is up-regulated during melanoma tumor progression, and tumorigenesis can be inhibited by the knockdown of Panx1 raising the possibility that Panx1 may be a viable target for the treatment of melanoma.

Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes as an In Vitro Model for Coxsackievirus B3–Induced Myocarditis and Antiviral Drug Screening Platform

Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon&bgr;1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferon&bgr;1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.

Epigenetic Regulation of Phosphodiesterases 2A and 3A Underlies Compromised β-adrenergic Signaling in an iPSC Model of Dilated Cardiomyopathy

β-adrenergic signaling pathways mediate key aspects of cardiac function. Its dysregulation is associated with a range of cardiac diseases, including dilated cardiomyopathy (DCM). Previously, we established an iPSC model of familial DCM from patients with a mutation in TNNT2, a sarcomeric protein. Here, we found that the β-adrenergic agonist isoproterenol induced mature β-adrenergic signaling in iPSC-derived cardiomyocytes (iPSC-CMs) but that this pathway was blunted in DCM iPSC-CMs. Although expression levels of several β-adrenergic signaling components were unaltered between control and DCM iPSC-CMs, we found that phosphodiesterases (PDEs) 2A and PDE3A were upregulated in DCM iPSC-CMs and that PDE2A was also upregulated in DCM patient tissue. We further discovered increased nuclear localization of mutant TNNT2 and epigenetic modifications of PDE genes in both DCM iPSC-CMs and patient tissue. Notably, pharmacologic inhibition of PDE2A and PDE3A restored cAMP levels and ameliorated the impaired β-adrenergic signaling of DCM iPSC-CMs, suggesting therapeutic potential.

High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells

High-throughput screening of drugs with human induced pluripotent stem cell–derived cardiomyocytes reveals a “cardiac safety index.” Failing fast for tyrosine kinase inhibitors Discovery early in its life cycle that an anticancer drug causes heart damage (a common side effect) can halt development—saving money, time, and perhaps lives. To this end, Sharma and colleagues derived heart cells from human induced pluripotent stem cells and then examined how a battery of anticancer tyrosine kinase inhibitors altered their physiology. By measuring cell death, contraction, excitability, calcium dynamics, and signal transduction and integrating the results, they calculated a drug-specific “cardiac safety index.” This index proved highly informative, with low values corresponding to those drugs known to cause heart problems in patients. The analysis even revealed that VEGFR2-inhibiting drugs caused cells to try to compensate for the toxic effects by up-regulating protective insulin/IGF pathways, prompting the authors to devise a combination treatment that may limit the toxicity of this class of drug. This screening method is expected to reveal early on whether potential anticancer drugs are cardiotoxic. 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.

Characterization of the molecular mechanisms underlying increased ischemic damage in the aldehyde dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell model system

The decrease of function in the ALDH2*2 genotype disrupts an important cardioprotective oxidative stress regulatory circuit, thus increasing cardiac cell death after ischemic insult. Personalized Heart Healing In poetry, we welcome assaults to the heart that leave one breathless. But depriving actual heart tissue of oxygen—through decreased blood flow—can cause irreparable damage. The human genome houses ALDH2, a gene that encodes the heart-protective metabolic enzyme aldehyde dehydrogenase 2. But ~8% of the human population carries an inactivating gene polymorphism (ALDH2*2) that has been linked to enhanced severity of damage from cardiac ischemia—a shortage in the heart’s oxygen supply—and an increased risk of coronary artery disease (CAD). Now, Ebert et al. investigate the mechanisms underlying these ALDH2*2-associated maladies using a human cellular model of the ALDH2*2 genotype made with induced pluripotent stem cell–derived cardiomyocytes generated from patient fibroblasts. The authors found that ALDH2 regulated cell survival by modulating oxidative stress, a circuit that was dysfunctional in ALDH2*2 cells. This aberration induced cell cycle arrest and enhanced apoptosis in cardiomyocytes after ischemic insult, illuminating a new function for ALDH2 in cell survival decisions. Such mechanistic insights may spur the development of new diagnostic methods for and improved risk management of CAD as well as genotype-specific cardiac therapies. Now, if we can only find a cure for the poetic broken heart…. Nearly 8% of the human population carries an inactivating point mutation in the gene that encodes the cardioprotective enzyme aldehyde dehydrogenase 2 (ALDH2). This genetic polymorphism (ALDH2*2) is linked to more severe outcomes from ischemic heart damage and an increased risk of coronary artery disease (CAD), but the underlying molecular bases are unknown. We investigated the ALDH2*2 mechanisms in a human model system of induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation gave rise to elevated amounts of reactive oxygen species and toxic aldehydes, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. We established that ALDH2 controls cell survival decisions by modulating oxidative stress levels and that this regulatory circuitry was dysfunctional in the loss-of-function ALDH2*2 genotype, causing up-regulation of apoptosis in cardiomyocytes after ischemic insult. These results reveal a new function for the metabolic enzyme ALDH2 in modulation of cell survival decisions. Insight into the molecular mechanisms that mediate ALDH2*2-related increased ischemic damage is important for the development of specific diagnostic methods and improved risk management of CAD and may lead to patient-specific cardiac therapies.

Overview of High Throughput Sequencing Technologies to Elucidate Molecular Pathways in Cardiovascular Diseases

High throughput sequencing technologies have become essential in studies on genomics, epigenomics, and transcriptomics. Although sequencing information has traditionally been elucidated using a low throughput technique called Sanger sequencing, high throughput sequencing technologies are capable of sequencing multiple DNA molecules in parallel, enabling hundreds of millions of DNA molecules to be sequenced at a time. This advantage allows high throughput sequencing to be used to create large data sets, generating more comprehensive insights into the cellular genomic and transcriptomic signatures of various diseases and developmental stages. Within high throughput sequencing technologies, whole exome sequencing can be used to identify novel variants and other mutations that may underlie many genetic cardiac disorders, whereas RNA sequencing can be used to analyze how the transcriptome changes. Chromatin immunoprecipitation sequencing and methylation sequencing can be used to identify epigenetic changes, whereas ribosome sequencing can be used to determine which mRNA transcripts are actively being translated. In this review, we will outline the differences in various sequencing modalities and examine the main sequencing platforms on the market in terms of their relative read depths, speeds, and costs. Finally, we will discuss the development of future sequencing platforms and how these new technologies may improve on current sequencing platforms. Ultimately, these sequencing technologies will be instrumental in further delineating how the cardiovascular system develops and how perturbations in DNA and RNA can lead to cardiovascular disease.