Sharon Paige

All Publications

• Patient-Specific Induced Pluripotent Stem Cells Implicate Intrinsic Impaired Contractility in Hypoplastic Left Heart Syndrome. Circulation Paige, S. L., Galdos, F. X., Lee, S., Chin, E. T., Ranjbarvaziri, S., Feyen, D. A., Darsha, A. K., Xu, S., Ryan, J. A., Beck, A. L., Qureshi, M. Y., Miao, Y., Gu, M., Bernstein, D., Nelson, T. J., Mercola, M., Rabinovitch, M., Ashley, E. A., Parikh, V. N., Wu, S. M. 2020; 142 (16): 1605–8

  View details for DOI 10.1161/CIRCULATIONAHA.119.045317

  View details for PubMedID 33074758

• Cardiac involvement in classical or hypermobile Ehlers-Danlos syndrome is uncommon. Genetics in medicine : official journal of the American College of Medical Genetics Paige, S. L., Lechich, K. M., Tierney, E. S., Collins, R. T. 2020

  Abstract

  PURPOSE: Cardiac-valvular and vascular Ehlers-Danlos syndrome (EDS) have significant cardiovascular issues. The prevalence and significance of such abnormalities in classical (cEDS) or hypermobile EDS (hEDS) remain unclear. We report the prevalence of cardiac abnormalities in patients with cEDS and hEDS.METHODS: We identified 532 pediatric patients with potential EDS evaluated at our institution from January 2014 through April 2019 by retrospective chart review. Ninety-five patients (12 cEDS and 83 hEDS patients) met 2017 EDS diagnostic criteria and had an echocardiogram. One patient was excluded due to complex congenital heart disease, and two were excluded due to lack of images. We reviewed echocardiograms for all structural abnormalities.RESULTS: Of these 95 patients, 1 had mild aortic root dilation, and 1 had mild ascending aorta dilation in the setting of a bicuspid aortic valve. Eleven patients (11.6%) had a cardiac valve abnormality, all of which were trivial to mild. None of the patients required cardiac intervention.CONCLUSION: Our results demonstrate that aortic dilation and valvular anomalies are uncommon in cEDS or hEDS patients. Given the lack of evidence, we do not recommend echocardiographic evaluation and surveillance in patients with cEDS and hEDS in the absence of clinical findings or positive family history.

  View details for DOI 10.1038/s41436-020-0856-8

  View details for PubMedID 32518415

• Risk factors associated with the development of double-inlet ventricle congenital heart disease BIRTH DEFECTS RESEARCH Paige, S. L., Yang, W., Priest, J. R., Botto, L. D., Shaw, G. M., Collins, R., Natl Birth Defects Prevention 2019; 111 (11): 640–48

  View details for DOI 10.1002/bdr2.1501

  View details for Web of Science ID 000473561000003

• Beyond Gene Panels: Whole Exome Sequencing for Diagnosis of Congenital Heart Disease. Circulation. Genomic and precision medicine Paige, S. L., Saha, P. n., Priest, J. R. 2018; 11 (3): e002097

  View details for PubMedID 29555674

• A Temporal Chromatin Signature in Human Embryonic Stem Cells Identifies Regulators of Cardiac Development CELL Paige, S. L., Thomas, S., Stoick-Cooper, C. L., Wang, H., Maves, L., Sandstrom, R., Pabon, L., Reinecke, H., Pratt, G., Keller, G., Moon, R. T., Stamatoyannopoulos, J., Murry, C. E. 2012; 151 (1): 221-232

  Abstract

  Directed differentiation of human embryonic stem cells (ESCs) into cardiovascular cells provides a model for studying molecular mechanisms of human cardiovascular development. Although it is known that chromatin modification patterns in ESCs differ markedly from those in lineage-committed progenitors and differentiated cells, the temporal dynamics of chromatin alterations during differentiation along a defined lineage have not been studied. We show that differentiation of human ESCs into cardiovascular cells is accompanied by programmed temporal alterations in chromatin structure that distinguish key regulators of cardiovascular development from other genes. We used this temporal chromatin signature to identify regulators of cardiac development, including the homeobox gene MEIS2. Using the zebrafish model, we demonstrate that MEIS2 is critical for proper heart tube formation and subsequent cardiac looping. Temporal chromatin signatures should be broadly applicable to other models of stem cell differentiation to identify regulators and provide key insights into major developmental decisions.

  View details for DOI 10.1016/j.cell.2012.08.027

  View details for Web of Science ID 000309544200022

  View details for PubMedID 22981225

  View details for PubMedCentralID PMC3462257

• Endogenous Wnt/beta-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells PLOS ONE Paige, S. L., Osugi, T., Afanasiev, O. K., Pabon, L., Reinecke, H., Murry, C. E. 2010; 5 (6)

  Abstract

  Wnt/beta-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.We tested the hypothesis that Wnt/beta-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/beta-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for beta-myosin heavy chain (beta-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/beta-catenin signaling is required for Smad1 activation by BMP4.Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/beta-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.

  View details for DOI 10.1371/journal.pone.0011134

  View details for Web of Science ID 000278775900023

  View details for PubMedID 20559569

  View details for PubMedCentralID PMC2886114

• Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome. Circulation Krane, M., DreSSen, M., Santamaria, G., My, I., Schneider, C. M., Dorn, T., Laue, S., Mastantuono, E., Berutti, R., Rawat, H., Gilsbach, R., Schneider, P., Lahm, H., Schwarz, S., Doppler, S. A., Paige, S., Puluca, N., Doll, S., Neb, I., Brade, T., Zhang, Z., Abou-Ajram, C., Northoff, B., Holdt, L. M., Sudhop, S., Sahara, M., Goedel, A., Dendorfer, A., Tjong, F. V., Rijlaarsdam, M. E., Cleuziou, J., Lang, N., Kupatt, C., Bezzina, C., Lange, R., Bowles, N. E., Mann, M., Gelb, B. D., Crotti, L., Hein, L., Meitinger, T., Wu, S., Sinnecker, D., Gruber, P. J., Laugwitz, K., Moretti, A. 2021; 144 (17): 1409-1428

  Abstract

  BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role.METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls.RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues.CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

  View details for DOI 10.1161/CIRCULATIONAHA.121.056198

  View details for PubMedID 34694888

• CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes. Scientific reports Chirikian, O., Goodyer, W. R., Dzilic, E., Serpooshan, V., Buikema, J. W., McKeithan, W., Wu, H., Li, G., Lee, S., Merk, M., Galdos, F., Beck, A., Ribeiro, A. J., Paige, S., Mercola, M., Wu, J. C., Pruitt, B. L., Wu, S. M. 2021; 11 (1): 3026

  Abstract

  Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.

  View details for DOI 10.1038/s41598-021-81860-x

  View details for PubMedID 33542270

• Purification of Pluripotent Stem Cell-Derived Cardiomyocytes Using CRISPR/Cas9-Mediated Integration of Fluorescent Reporters. Methods in molecular biology (Clifton, N.J.) Galdos, F. X., Darsha, A. K., Paige, S. L., Wu, S. M. 2021; 2158: 223–40

  Abstract

  Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have become critically important for the detailed study of cardiac development, disease modeling, and drug screening. However, directed differentiation of hiPSCs into cardiomyocytes often results in mixed populations of cardiomyocytes and other cell types, which may confound experiments that require pure populations of cardiomyocytes. Here, we detail the use of a CRISPR/Cas9 genome editing strategy to develop cardiomyocyte-specific reporters that allow for the isolation of hiPSC-derived cardiomyocytes and chamber-specific myocytes. Moreover, we describe a cardiac differentiation protocol to derive cardiomyocytes from hiPSCs, as well as a strategy to use fluorescence-activated cell sorting to isolate pure populations of fluorescently labeled cardiomyocytes for downstream applications.

  View details for DOI 10.1007/978-1-0716-0668-1_17

  View details for PubMedID 32857377

• 4HNE Impairs Myocardial Bioenergetics in Congenital Heart DiseaseInduced Right Ventricular Failure. Circulation Hwang, H. V., Sandeep, N., Paige, S. L., Ranjbarvaziri, S., Hu, D., Zhao, M., Lan, I. S., Coronado, M., Kooiker, K. B., Wu, S. M., Fajardo, G., Bernstein, D., Reddy, S. 2020

  Abstract

  Background: In patients with complex congenital heart disease, such as those with tetralogy of Fallot, the right ventricle (RV) is subject to pressure overload stress, leading to RV hypertrophy and eventually RV failure. The role of lipid peroxidation, a potent form of oxidative stress, in mediating RV hypertrophy and failure in congenital heart disease is unknown. Methods: Lipid peroxidation and mitochondrial function and structure were assessed in RV myocardium collected from patients with RV hypertrophy with normal RV systolic function (RV FAC 47.3±3.8%) and in patients with RV failure showing decreased RV systolic function (RV FAC 26.6±3.1%). The mechanism of the effect of lipid peroxidation, mediated by 4-hydroxynonenal (4HNE; a byproduct of lipid peroxidation) on mitochondrial function and structure was assessed in HL1 murine cardiomyocytes and human induced pluripotent stem cellderived cardiomyocytes. Results: RV failure was characterized by an increase in 4HNE adduction of metabolic and mitochondrial proteins (16/27 identified proteins), in particular electron transport chain proteins. Sarcomeric (myosin) and cytoskeletal proteins (desmin, tubulin) also underwent 4HNEadduction. RV failure showed lower oxidative phosphorylation [moderate RV hypertrophy 287.6±19.75 vs. RV failure 137.8±11.57 pmol/(sec*ml), p=0.0004], and mitochondrial structural damage. Using a cell model, we show that 4HNE decreases cell number and oxidative phosphorylation (control 388.1±23.54 vs. 4HNE 143.7±11.64 pmol/(sec*ml), p<0.0001). Carvedilol, a known antioxidant did not decrease 4HNE adduction of metabolic and mitochondrial proteins and did not improve oxidative phosphorylation. Conclusions: Metabolic, mitochondrial, sarcomeric and cytoskeletal proteins are susceptible to 4HNE-adduction in patients with RV failure. 4HNE decreases mitochondrial oxygen consumption by inhibiting electron transport chain complexes. Carvedilol did not improve the 4HNE-mediated decrease in oxygen consumption. Strategies to decrease lipid peroxidation could improve mitochondrial energy generation and cardiomyocyte survival and improve RV failure in patients with congenital heart disease.

  View details for DOI 10.1161/CIRCULATIONAHA.120.045470

  View details for PubMedID 32806952

• Intrinsic Endocardial Defects Contribute to Hypoplastic Left Heart Syndrome. Cell stem cell Miao, Y., Tian, L., Martin, M., Paige, S. L., Galdos, F. X., Li, J., Klein, A., Zhang, H., Ma, N., Wei, Y., Stewart, M., Lee, S., Moonen, J., Zhang, B., Grossfeld, P., Mital, S., Chitayat, D., Wu, J. C., Rabinovitch, M., Nelson, T. J., Nie, S., Wu, S. M., Gu, M. 2020

  Abstract

  Hypoplastic left heart syndrome (HLHS) is a complex congenital heart disease characterized by abnormalities in the left ventricle, associated valves, and ascending aorta. Studies have shown intrinsic myocardial defects but do not sufficiently explain developmental defects in the endocardial-derived cardiac valve, septum, and vasculature. Here, we identify a developmentally impaired endocardial population in HLHS through single-cell RNA profiling of hiPSC-derived endocardium and human fetal heart tissue with an underdeveloped left ventricle. Intrinsic endocardial defects contribute to abnormal endothelial-to-mesenchymal transition, NOTCH signaling, and extracellular matrix organization, key factors in valve formation. Endocardial abnormalities cause reduced cardiomyocyte proliferation and maturation by disrupting fibronectin-integrin signaling, consistent with recently described de novo HLHS mutations associated with abnormal endocardial gene and fibronectin regulation. Together, these results reveal a critical role for endocardium in HLHS etiology and provide a rationale for considering endocardial function in regenerative strategies.

  View details for DOI 10.1016/j.stem.2020.07.015

  View details for PubMedID 32810435

• Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes. Cell stem cell Buikema, J. W., Lee, S. n., Goodyer, W. R., Maas, R. G., Chirikian, O. n., Li, G. n., Miao, Y. n., Paige, S. L., Lee, D. n., Wu, H. n., Paik, D. T., Rhee, S. n., Tian, L. n., Galdos, F. X., Puluca, N. n., Beyersdorf, B. n., Hu, J. n., Beck, A. n., Venkamatran, S. n., Swami, S. n., Wijnker, P. n., Schuldt, M. n., Dorsch, L. M., van Mil, A. n., Red-Horse, K. n., Wu, J. Y., Geisen, C. n., Hesse, M. n., Serpooshan, V. n., Jovinge, S. n., Fleischmann, B. K., Doevendans, P. A., van der Velden, J. n., Garcia, K. C., Wu, J. C., Sluijter, J. P., Wu, S. M. 2020; 27 (1): 50–63.e5

  Abstract

  Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3β inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.

  View details for DOI 10.1016/j.stem.2020.06.001

  View details for PubMedID 32619518

• Multi-disciplinary evaluation of a 5-month-old with hypertrophic cardiomyopathy related to a functional adrenocortical tumor JOURNAL OF PEDIATRIC ENDOCRINOLOGY & METABOLISM Nally, L. M., Conner, E., Paige, S., Mooney, K. L., Naber, U., Richards, R., Wright, G. 2018; 31 (12): 1371–76

  View details for DOI 10.1515/jpem-2018-0340

  View details for Web of Science ID 000452393300012

• Cardiac Regeneration Lessons From Development CIRCULATION RESEARCH Galdos, F. X., Guo, Y., Paige, S. L., VanDusen, N. J., Wu, S. M., Pu, W. T. 2017; 120 (6): 941-959

  Abstract

  Palliative surgery for congenital heart disease has allowed patients with previously lethal heart malformations to survive and, in most cases, to thrive. However, these procedures often place pressure and volume loads on the heart, and over time, these chronic loads can cause heart failure. Current therapeutic options for initial surgery and chronic heart failure that results from failed palliation are limited, in part, by the mammalian heart's low inherent capacity to form new cardiomyocytes. Surmounting the heart regeneration barrier would transform the treatment of congenital, as well as acquired, heart disease and likewise would enable development of personalized, in vitro cardiac disease models. Although these remain distant goals, studies of heart development are illuminating the path forward and suggest unique opportunities for heart regeneration, particularly in fetal and neonatal periods. Here, we review major lessons from heart development that inform current and future studies directed at enhancing cardiac regeneration.

  View details for DOI 10.1161/CIRCRESAHA.116.309040

  View details for Web of Science ID 000397330700007

  View details for PubMedID 28302741

• Nkx2.5+ Cardiomyoblasts Contribute to Cardiomyogenesis in the Neonatal Heart. Scientific reports Serpooshan, V. n., Liu, Y. H., Buikema, J. W., Galdos, F. X., Chirikian, O. n., Paige, S. n., Venkatraman, S. n., Kumar, A. n., Rawnsley, D. R., Huang, X. n., Pijnappels, D. A., Wu, S. M. 2017; 7 (1): 12590

  Abstract

  During normal lifespan, the mammalian heart undergoes limited renewal of cardiomyocytes. While the exact mechanism for this renewal remains unclear, two possibilities have been proposed: differentiated myocyte replication and progenitor/immature cell differentiation. This study aimed to characterize a population of cardiomyocyte precursors in the neonatal heart and to determine their requirement for cardiac development. By tracking the expression of an embryonic Nkx2.5 cardiac enhancer, we identified cardiomyoblasts capable of differentiation into striated cardiomyocytes in vitro. Genome-wide expression profile of neonatal Nkx2.5+ cardiomyoblasts showed the absence of sarcomeric gene and the presence of cardiac transcription factors. To determine the lineage contribution of the Nkx2.5+ cardiomyoblasts, we generated a doxycycline suppressible Cre transgenic mouse under the regulation of the Nkx2.5 enhancer and showed that neonatal Nkx2.5+ cardiomyoblasts mature into cardiomyocytes in vivo. Ablation of neonatal cardiomyoblasts resulted in ventricular hypertrophy and dilation, supporting a functional requirement of the Nkx2.5+ cardiomyoblasts. This study provides direct lineage tracing evidence that a cardiomyoblast population contributes to cardiogenesis in the neonatal heart. The cell population identified here may serve as a promising therapeutic for pediatric cardiac regeneration.

  View details for PubMedID 28974782

• Comparison of Human Embryonic Stem Cell-Derived Cardiomyocytes, Cardiovascular Progenitors, and Bone Marrow Mononuclear Cells for Cardiac Repair STEM CELL REPORTS Fernandes, S., Chong, J. J., Paige, S. L., Iwata, M., Torok-Storb, B., Keller, G., Reinecke, H., Murry, C. E. 2015; 5 (5): 753-762

  Abstract

  Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) can improve the contractility of injured hearts.We hypothesized that mesodermal cardiovascular progenitors (hESC-CVPs), capable of generating vascular cells in addition to cardiomyocytes, would provide superior repair by contributing to multiple components of myocardium. We performed a head-to-head comparison of hESC-CMs and hESC-CVPs and compared these with the most commonly used clinical cell type, human bone marrow mononuclear cells (hBMMNCs). In a nude rat model of myocardial infarction, hESC-CMs and hESC-CVPs generated comparable grafts. Both similarly improved systolic function and ventricular dilation. Furthermore, only rare human vessels formed from hESC-CVPs. hBM-MNCs attenuated ventricular dilation and enhanced host vascularization without engrafting long-term or improving contractility. Thus, hESC-CMs and CVPs show similar efficacy for cardiac repair, and both are more efficient than hBM-MNCs. However, hESC-CVPs do not form larger grafts or more significant numbers of human vessels in the infarcted heart.

  View details for DOI 10.1016/j.stemcr.2015.09.011

  View details for Web of Science ID 000364991000008

  View details for PubMedID 26607951

  View details for PubMedCentralID PMC4649260

• Mechanical Stress Promotes Maturation of Human Myocardium From Pluripotent Stem Cell-Derived Progenitors STEM CELLS Ruan, J., Tulloch, N. L., Saiget, M., Paige, S. L., Razumova, M. V., Regnier, M., Tung, K. C., Keller, G., Pabon, L., Reinecke, H., Murry, C. E. 2015; 33 (7): 2148-2157

  Abstract

  Recent advances in pluripotent stem cell biology and directed differentiation have identified a population of human cardiovascular progenitors that give rise to cardiomyocytes, smooth muscle, and endothelial cells. Because the heart develops from progenitors in 3D under constant mechanical load, we sought to test the effects of a 3D microenvironment and mechanical stress on differentiation and maturation of human cardiovascular progenitors into myocardial tissue. Progenitors were derived from embryonic stem cells, cast into collagen hydrogels, and left unstressed or subjected to static or cyclic mechanical stress. Compared to 2D culture, the unstressed 3D environment increased cardiomyocyte numbers and decreased smooth muscle numbers. Additionally, 3D culture suppressed smooth muscle α-actin content, suggesting diminished cell maturation. Cyclic stress-conditioning increased expression of several cardiac markers, including β-myosin heavy chain and cardiac troponin T, and the tissue showed enhanced calcium dynamics and force production. There was no effect of mechanical loading on cardiomyocyte or smooth muscle specification. Thus, 3D growth conditions favor cardiac differentiation from cardiovascular progenitors, whereas 2D conditions promote smooth muscle differentiation. Mechanical loading promotes cardiomyocyte structural and functional maturation. Culture in 3-D facilitates understanding how cues such as mechanical stress affect the differentiation and morphogenesis of distinct cardiovascular cell populations into organized, functional human cardiovascular tissue. Stem Cells 2015;33:2148-2157.

  View details for DOI 10.1002/stem.2036

  View details for Web of Science ID 000356668200007

  View details for PubMedID 25865043

  View details for PubMedCentralID PMC4478130

• Molecular Regulation of Cardiomyocyte Differentiation CIRCULATION RESEARCH Paige, S. L., Plonowska, K., Xu, A., Wu, S. M. 2015; 116 (2): 341-353

  Abstract

  The heart is the first organ to form during embryonic development. Given the complex nature of cardiac differentiation and morphogenesis, it is not surprising that some form of congenital heart disease is present in ≈1 percent of newborns. The molecular determinants of heart development have received much attention over the past several decades. This has been driven in large part by an interest in understanding the causes of congenital heart disease coupled with the potential of using knowledge from developmental biology to generate functional cells and tissues that could be used for regenerative medicine purposes. In this review, we highlight the critical signaling pathways and transcription factor networks that regulate cardiomyocyte lineage specification in both in vivo and in vitro models. Special focus will be given to epigenetic regulators that drive the commitment of cardiomyogenic cells from nascent mesoderm and their differentiation into chamber-specific myocytes, as well as regulation of myocardial trabeculation.

  View details for DOI 10.1161/CIRCRESAHA.116.302752

  View details for Web of Science ID 000347939000019

  View details for PubMedCentralID PMC4299877

• Engineered Biomaterials Control Differentiation and Proliferation of Human-Embryonic-Stem-Cell-Derived Cardiomyocytes via Timed Notch Activation STEM CELL REPORTS Tung, J. C., Paige, S. L., Ratner, B. D., Murry, C. E., Giachelli, C. M. 2014; 2 (3): 271-281

  Abstract

  For cell-based treatments of myocardial infarction, a better understanding of key developmental signaling pathways and more robust techniques for producing cardiomyocytes are required. Manipulation of Notch signaling has promise as it plays an important role during cardiovascular development, but previous studies presented conflicting results that Notch activation both positively and negatively regulates cardiogenesis. We developed surface- and microparticle-based Notch-signaling biomaterials that function in a time-specific activation-tunable manner, enabling precise investigation of Notch activation at specific developmental stages. Using our technologies, a biphasic effect of Notch activation on cardiac differentiation was found: early activation in undifferentiated human embryonic stem cells (hESCs) promotes ectodermal differentiation, activation in specified cardiovascular progenitor cells increases cardiac differentiation. Signaling also induces cardiomyocyte proliferation, and repeated doses of Notch-signaling microparticles further enhance cardiomyocyte population size. These results highlight the diverse effects of Notch activation during cardiac development and provide approaches for generating large quantities of cardiomyocytes.

  View details for DOI 10.1016/j.stemcr.2014.01.011

  View details for Web of Science ID 000336647700004

  View details for PubMedID 24672751

  View details for PubMedCentralID PMC3964284

• Transmembrane protein 88: a Wnt regulatory protein that specifies cardiomyocyte development DEVELOPMENT Palpant, N. J., Pabon, L., Rabinowitz, J. S., Hadland, B. K., Stoick-Cooper, C. L., Paige, S. L., Bernstein, I. D., Moon, R. T., Murry, C. E. 2013; 140 (18): 3799-3808

  Abstract

  Genetic regulation of the cell fate transition from lateral plate mesoderm to the specification of cardiomyocytes requires suppression of Wnt/β-catenin signaling, but the mechanism for this is not well understood. By analyzing gene expression and chromatin dynamics during directed differentiation of human embryonic stem cells (hESCs), we identified a suppressor of Wnt/β-catenin signaling, transmembrane protein 88 (TMEM88), as a potential regulator of cardiovascular progenitor cell (CVP) specification. During the transition from mesoderm to the CVP, TMEM88 has a chromatin signature of genes that mediate cell fate decisions, and its expression is highly upregulated in advance of key cardiac transcription factors in vitro and in vivo. In early zebrafish embryos, tmem88a is expressed broadly in the lateral plate mesoderm, including the bilateral heart fields. Short hairpin RNA targeting of TMEM88 during hESC cardiac differentiation increases Wnt/β-catenin signaling, confirming its role as a suppressor of this pathway. TMEM88 knockdown has no effect on NKX2.5 or GATA4 expression, but 80% of genes most highly induced during CVP development have reduced expression, suggesting adoption of a new cell fate. In support of this, analysis of later stage cell differentiation showed that TMEM88 knockdown inhibits cardiomyocyte differentiation and promotes endothelial differentiation. Taken together, TMEM88 is crucial for heart development and acts downstream of GATA factors in the pre-cardiac mesoderm to specify lineage commitment of cardiomyocyte development through inhibition of Wnt/β-catenin signaling.

  View details for DOI 10.1242/dev.094789

  View details for Web of Science ID 000323698100009

  View details for PubMedID 23924634

  View details for PubMedCentralID PMC3754478

• Developmental Fate and Cellular Maturity Encoded in Human Regulatory DNA Landscapes CELL Stergachis, A. B., Neph, S., Reynolds, A., Humbert, R., Miller, B., Paige, S. L., Vernot, B., Cheng, J. B., Thurman, R. E., Sandstrom, R., Haugen, E., Heimfeld, S., Murry, C. E., Akey, J. M., Stamatoyannopoulos, J. A. 2013; 154 (4): 888-903

  Abstract

  Cellular-state information between generations of developing cells may be propagated via regulatory regions. We report consistent patterns of gain and loss of DNase I-hypersensitive sites (DHSs) as cells progress from embryonic stem cells (ESCs) to terminal fates. DHS patterns alone convey rich information about cell fate and lineage relationships distinct from information conveyed by gene expression. Developing cells share a proportion of their DHS landscapes with ESCs; that proportion decreases continuously in each cell type as differentiation progresses, providing a quantitative benchmark of developmental maturity. Developmentally stable DHSs densely encode binding sites for transcription factors involved in autoregulatory feedback circuits. In contrast to normal cells, cancer cells extensively reactivate silenced ESC DHSs and those from developmental programs external to the cell lineage from which the malignancy derives. Our results point to changes in regulatory DNA landscapes as quantitative indicators of cell-fate transitions, lineage relationships, and dysfunction.

  View details for DOI 10.1016/j.cell.2013.07.020

  View details for Web of Science ID 000323202500018

  View details for PubMedID 23953118

  View details for PubMedCentralID PMC3962256

• Cardiogenesis From Human Embryonic Stem Cells - Mechanisms and Applications CIRCULATION JOURNAL Mignone, J. L., Kreutziger, K. L., Paige, S. L., Murry, C. E. 2010; 74 (12): 2517-2526

  Abstract

  Over the past decade, the ability to culture and differentiate human embryonic stem cells (ESCs) has offered researchers a novel therapeutic that may, for the first time, repair regions of the damaged heart. Studies of cardiac development in lower organisms have led to identification of the transforming growth factor-β superfamily (eg, activin A and bone morphogenic protein 4) and the Wnt/β-catenin pathway as key inducers of mesoderm and cardiovascular differentiation. These factors act in a context-specific manner (eg, Wnt/β-catenin is required initially to form mesoderm but must be antagonized thereafter to make cardiac muscle). Different lines of ESCs produce different levels of agonists and antagonists for these pathways, but with careful optimization, highly enriched populations of immature cardiomyocytes can be generated. These cardiomyocytes survive transplantation to infarcted hearts of experimental animals, where they create new human myocardial tissue and improve heart function. The grafts generated by cell transplantation have been small, however, leading to an exploration of tissue engineering as an alternate strategy. Engineered tissue generated from preparations of human cardiomyocytes survives poorly after transplantation, most likely because of ischemia. Creation of pre-organized vascular networks in the tissue markedly enhances survival, with human capillaries anastomosed to the host coronary circulation. Thus, pathways controlling formation of the human cardiovascular system are emerging, yielding the building blocks for tissue regeneration that may address the root causes of heart failure.

  View details for DOI 10.1253/circj.CJ-10-0958

  View details for Web of Science ID 000284861400002

  View details for PubMedID 21084757

  View details for PubMedCentralID PMC3938118

• Chromatin remodeling during mouse and human embryonic stem cell differentiation DEVELOPMENTAL DYNAMICS Golob, J. L., Paige, S. L., Muskheli, V., Pabon, L., Murry, C. E. 2008; 237 (5): 1389-1398

  Abstract

  Embryonic stem cell (ESC) differentiation is an excellent model to study chromatin changes at developmentally regulated loci. Differentiating mouse and human ESCs increase genome-wide acetylation (euchromatic) and tri-methylation (heterochromatic) of lysine 9 on histone H3. The Oct4 locus is euchromatic when expressed in undifferentiated ESCs and heterochromatic after differentiation. Brachyury T, a mesoderm-specific transcription factor, is not yet expressed in undifferentiated cells, where its locus has "bivalent" tri-methyl lysine 4 and lysine 27 modifications. During directed differentiation to pre-cardiac mesoderm, the activated brachyury locus has high levels of tri-methyl lysine 4 (euchromatin), switching to heterochromatin after gene silencing. Thus, ESC differentiation is accompanied by genome-wide commitment to euchromatin or heterochromatin. Undifferentiated hESCs bivalently modify the brachyury locus, activate it to euchromatin during mesoderm induction, and subsequently repress it to heterochromatin, demonstrating, to our knowledge, the first analysis of chromatin dynamics at a locus essential for mesoderm and endoderm differentiation.

  View details for DOI 10.1002/dvdy.21545

  View details for Web of Science ID 000255842900015

  View details for PubMedID 18425849

  View details for PubMedCentralID PMC3075915