Sean M. Wu

All Publications

• Cardiac ACTN2 enhancer regulates cardiometabolism and maturation. Nature cardiovascular research Galdos, F. X., Lee, C., Wu, S. M. 2024; 3 (6): 616-618

  View details for DOI 10.1038/s44161-024-00483-3

  View details for PubMedID 39196224

  View details for PubMedCentralID 7199445

• The sum of the parts is greater than the whole: current research models for congenital heart disease. Nature cardiovascular research Samad, T., Wu, S. M. 2023; 2 (8): 708-710

  View details for DOI 10.1038/s44161-023-00308-9

  View details for PubMedID 39195960

  View details for PubMedCentralID 4099249

• Combined lineage tracing and scRNA-seq reveals unexpected first heart field predominance of human iPSC differentiation. eLife Galdos, F. X., Lee, C., Lee, S., Paige, S., Goodyer, W., Xu, S., Samad, T., Escobar, G. V., Darsha, A., Beck, A., Bak, R. O., Porteus, M. H., Wu, S. 2023; 12

  Abstract

  During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation stage human embryos. Human induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF- progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.

  View details for DOI 10.7554/eLife.80075

  View details for PubMedID 37284748

• Sex differences in ICI myocarditis: Hormones to the rescue SCIENCE TRANSLATIONAL MEDICINE Nguyen, P. K., Wu, S. M. 2022; 14 (669)

  View details for Web of Science ID 000909177500007

• devCellPy is a machine learning-enabled pipeline for automated annotation of complex multilayered single-cell transcriptomic data. Nature communications Galdos, F. X., Xu, S., Goodyer, W. R., Duan, L., Huang, Y. V., Lee, S., Zhu, H., Lee, C., Wei, N., Lee, D., Wu, S. M. 2022; 13 (1): 5271

  Abstract

  A major informatic challenge in single cell RNA-sequencing analysis is the precise annotation of datasets where cells exhibit complex multilayered identities or transitory states. Here, we present devCellPy a highly accurate and precise machine learning-enabled tool that enables automated prediction of cell types across complex annotation hierarchies. To demonstrate the power of devCellPy, we construct a murine cardiac developmental atlas from published datasets encompassing 104,199 cells from E6.5-E16.5 and train devCellPy to generate a cardiac prediction algorithm. Using this algorithm, we observe a high prediction accuracy (>90%) across multiple layers of annotation and across de novo murine developmental data. Furthermore, we conduct a cross-species prediction of cardiomyocyte subtypes from in vitro-derived human induced pluripotent stem cells and unexpectedly uncover a predominance of left ventricular (LV) identity that we confirmed by an LV-specific TBX5 lineage tracing system. Together, our results show devCellPy to be a useful tool for automated cell prediction across complex cellular hierarchies, species, and experimental systems.

  View details for DOI 10.1038/s41467-022-33045-x

  View details for PubMedID 36071107

• In vivo visualization and molecular targeting of the cardiac conduction system. The Journal of clinical investigation Goodyer, W. R., Beyersdorf, B. M., Duan, L., van den Berg, N. S., Mantri, S., Galdos, F. X., Puluca, N., Buikema, J. W., Lee, S., Salmi, D., Robinson, E. R., Rogalla, S., Cogan, D. P., Khosla, C., Rosenthal, E. L., Wu, S. M. 2022

  Abstract

  Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects to CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab, that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo providing a proof-of-principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging and arrhythmia management.

  View details for DOI 10.1172/JCI156955

  View details for PubMedID 35951416

• Identification of Pathogenic Immune Cell Subsets Associated With Checkpoint Inhibitor-Induced Myocarditis. Circulation Zhu, H., Galdos, F. X., Lee, D., Waliany, S., Vivian Huang, Y., Ryan, J., Dang, K., Neal, J. W., Wakelee, H. A., Reddy, S. A., Srinivas, S., Lin, L. L., Witteles, R. M., Maecker, H. T., Davis, M. M., Nguyen, P. K., Wu, S. M. 2022: 101161CIRCULATIONAHA121056730

  Abstract

  Immune checkpoint inhibitors (ICIs) are monoclonal antibodies used to activate the immune system against tumor cells. Despite therapeutic benefits, ICIs have the potential to cause immune-related adverse events such as myocarditis, a rare but serious side effect with up to 50% mortality in affected patients. Histologically, patients with ICI myocarditis have lymphocytic infiltrates in the heart, implicating T cell-mediated mechanisms. However, the precise pathological immune subsets and molecular changes in ICI myocarditis are unknown.To identify immune subset(s) associated with ICI myocarditis, we performed time-of-flight mass cytometry on peripheral blood mononuclear cells from 52 individuals: 29 patients with autoimmune adverse events (immune-related adverse events) on ICI, including 8 patients with ICI myocarditis, and 23 healthy control subjects. We also used multiomics single-cell technology to immunophenotype 30 patients/control subjects using single-cell RNA sequencing, single-cell T-cell receptor sequencing, and cellular indexing of transcriptomes and epitopes by sequencing with feature barcoding for surface marker expression confirmation. To correlate between the blood and the heart, we performed single-cell RNA sequencing/T-cell receptor sequencing/cellular indexing of transcriptomes and epitopes by sequencing on MRL/Pdcd1-/- (Murphy Roths large/programmed death-1-deficient) mice with spontaneous myocarditis.Using these complementary approaches, we found an expansion of cytotoxic CD8+ T effector cells re-expressing CD45RA (Temra CD8+ cells) in patients with ICI myocarditis compared with control subjects. T-cell receptor sequencing demonstrated that these CD8+ Temra cells were clonally expanded in patients with myocarditis compared with control subjects. Transcriptomic analysis of these Temra CD8+ clones confirmed a highly activated and cytotoxic phenotype. Longitudinal study demonstrated progression of these Temra CD8+ cells into an exhausted phenotype 2 months after treatment with glucocorticoids. Differential expression analysis demonstrated elevated expression levels of proinflammatory chemokines (CCL5/CCL4/CCL4L2) in the clonally expanded Temra CD8+ cells, and ligand receptor analysis demonstrated their interactions with innate immune cells, including monocytes/macrophages, dendritic cells, and neutrophils, as well as the absence of key anti-inflammatory signals. To complement the human study, we performed single-cell RNA sequencing/T-cell receptor sequencing/cellular indexing of transcriptomes and epitopes by sequencing in Pdcd1-/- mice with spontaneous myocarditis and found analogous expansions of cytotoxic clonal effector CD8+ cells in both blood and hearts of such mice compared with controls.Clonal cytotoxic Temra CD8+ cells are significantly increased in the blood of patients with ICI myocarditis, corresponding to an analogous increase in effector cytotoxic CD8+ cells in the blood/hearts of Pdcd1-/- mice with myocarditis. These expanded effector CD8+ cells have unique transcriptional changes, including upregulation of chemokines CCL5/CCL4/CCL4L2, which may serve as attractive diagnostic/therapeutic targets for reducing life-threatening cardiac immune-related adverse events in ICI-treated patients with cancer.

  View details for DOI 10.1161/CIRCULATIONAHA.121.056730

  View details for PubMedID 35762356

• The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans. Science (New York, N.Y.) Jones, R. C., Karkanias, J., Krasnow, M. A., Pisco, A. O., Quake, S. R., Salzman, J., Yosef, N., Bulthaup, B., Brown, P., Harper, W., Hemenez, M., Ponnusamy, R., Salehi, A., Sanagavarapu, B. A., Spallino, E., Aaron, K. A., Concepcion, W., Gardner, J. M., Kelly, B., Neidlinger, N., Wang, Z., Crasta, S., Kolluru, S., Morri, M., Pisco, A. O., Tan, S. Y., Travaglini, K. J., Xu, C., Alcántara-Hernández, M., Almanzar, N., Antony, J., Beyersdorf, B., Burhan, D., Calcuttawala, K., Carter, M. M., Chan, C. K., Chang, C. A., Chang, S., Colville, A., Crasta, S., Culver, R. N., Cvijović, I., D'Amato, G., Ezran, C., Galdos, F. X., Gillich, A., Goodyer, W. R., Hang, Y., Hayashi, A., Houshdaran, S., Huang, X., Irwin, J. C., Jang, S., Juanico, J. V., Kershner, A. M., Kim, S., Kiss, B., Kolluru, S., Kong, W., Kumar, M. E., Kuo, A. H., Leylek, R., Li, B., Loeb, G. B., Lu, W. J., Mantri, S., Markovic, M., McAlpine, P. L., de Morree, A., Morri, M., Mrouj, K., Mukherjee, S., Muser, T., Neuhöfer, P., Nguyen, T. D., Perez, K., Phansalkar, R., Pisco, A. O., Puluca, N., Qi, Z., Rao, P., Raquer-McKay, H., Schaum, N., Scott, B., Seddighzadeh, B., Segal, J., Sen, S., Sikandar, S., Spencer, S. P., Steffes, L. C., Subramaniam, V. R., Swarup, A., Swift, M., Travaglini, K. J., Van Treuren, W., Trimm, E., Veizades, S., Vijayakumar, S., Vo, K. C., Vorperian, S. K., Wang, W., Weinstein, H. N., Winkler, J., Wu, T. T., Xie, J., Yung, A. R., Zhang, Y., Detweiler, A. M., Mekonen, H., Neff, N. F., Sit, R. V., Tan, M., Yan, J., Bean, G. R., Charu, V., Forgó, E., Martin, B. A., Ozawa, M. G., Silva, O., Tan, S. Y., Toland, A., Vemuri, V. N., Afik, S., Awayan, K., Botvinnik, O. B., Byrne, A., Chen, M., Dehghannasiri, R., Detweiler, A. M., Gayoso, A., Granados, A. A., Li, Q., Mahmoudabadi, G., McGeever, A., de Morree, A., Olivieri, J. E., Park, M., Pisco, A. O., Ravikumar, N., Salzman, J., Stanley, G., Swift, M., Tan, M., Tan, W., Tarashansky, A. J., Vanheusden, R., Vorperian, S. K., Wang, P., Wang, S., Xing, G., Xu, C., Yosef, N., Alcántara-Hernández, M., Antony, J., Chan, C. K., Chang, C. A., Colville, A., Crasta, S., Culver, R., Dethlefsen, L., Ezran, C., Gillich, A., Hang, Y., Ho, P. Y., Irwin, J. C., Jang, S., Kershner, A. M., Kong, W., Kumar, M. E., Kuo, A. H., Leylek, R., Liu, S., Loeb, G. B., Lu, W. J., Maltzman, J. S., Metzger, R. J., de Morree, A., Neuhöfer, P., Perez, K., Phansalkar, R., Qi, Z., Rao, P., Raquer-McKay, H., Sasagawa, K., Scott, B., Sinha, R., Song, H., Spencer, S. P., Swarup, A., Swift, M., Travaglini, K. J., Trimm, E., Veizades, S., Vijayakumar, S., Wang, B., Wang, W., Winkler, J., Xie, J., Yung, A. R., Artandi, S. E., Beachy, P. A., Clarke, M. F., Giudice, L. C., Huang, F. W., Huang, K. C., Idoyaga, J., Kim, S. K., Krasnow, M., Kuo, C. S., Nguyen, P., Quake, S. R., Rando, T. A., Red-Horse, K., Reiter, J., Relman, D. A., Sonnenburg, J. L., Wang, B., Wu, A., Wu, S. M., Wyss-Coray, T. 2022; 376 (6594): eabl4896

  Abstract

  Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

  View details for DOI 10.1126/science.abl4896

  View details for PubMedID 35549404

• Molecular hallmarks of heterochronic parabiosis at single-cell resolution. Nature Palovics, R., Keller, A., Schaum, N., Tan, W., Fehlmann, T., Borja, M., Kern, F., Bonanno, L., Calcuttawala, K., Webber, J., McGeever, A., Tabula Muris Consortium, Luo, J., Pisco, A. O., Karkanias, J., Neff, N. F., Darmanis, S., Quake, S. R., Wyss-Coray, T., Almanzar, N., Antony, J., Baghel, A. S., Bakerman, I., Bansal, I., Barres, B. A., Beachy, P. A., Berdnik, D., Bilen, B., Brownfield, D., Cain, C., Chan, C. K., Chen, M. B., Clarke, M. F., Conley, S. D., Demers, A., Demir, K., de Morree, A., Divita, T., du Bois, H., Ebadi, H., Espinoza, F. H., Fish, M., Gan, Q., George, B. M., Gillich, A., Gomez-Sjoberg, R., Green, F., Genetiano, G., Gu, X., Gulati, G. S., Hahn, O., Haney, M. S., Hang, Y., Harris, L., He, M., Hosseinzadeh, S., Huang, A., Huang, K. C., Iram, T., Isobe, T., Ives, F., Jones, R. C., Kao, K. S., Karnam, G., Kershner, A. M., Khoury, N., Kim, S. K., Kiss, B. M., Kong, W., Krasnow, M. A., Kumar, M. E., Kuo, C. S., Lam, J., Lee, D. P., Lee, S. E., Lehallier, B., Leventhal, O., Li, G., Li, Q., Liu, L., Lo, A., Lu, W., Lugo-Fagundo, M. F., Manjunath, A., May, A. P., Maynard, A., McKay, M., McNerney, M. W., Merrill, B., Metzger, R. J., Mignardi, M., Min, D., Nabhan, A. N., Ng, K. M., Nguyen, P. K., Noh, J., Nusse, R., Patkar, R., Peng, W. C., Penland, L., Pollard, K., Puccinelli, R., Qi, Z., Rando, T. A., Rulifson, E. J., Segal, J. M., Sikandar, S. S., Sinha, R., Sit, R. V., Sonnenburg, J., Staehli, D., Szade, K., Tan, M., Tato, C., Tellez, K., Torrez Dulgeroff, L. B., Travaglini, K. J., Tropini, C., Tsui, M., Waldburger, L., Wang, B. M., van Weele, L. J., Weinberg, K., Weissman, I. L., Wosczyna, M. N., Wu, S. M., Xiang, J., Xue, S., Yamauchi, K. A., Yang, A. C., Yerra, L. P., Youngyunpipatkul, J., Yu, B., Zanini, F., Zardeneta, M. E., Zee, A., Zhao, C., Zhang, F., Zhang, H., Zhang, M. J., Zhou, L., Zou, J. 2022

  Abstract

  The ability to slow or reverse biological ageing would have major implications for mitigating disease risk and maintaining vitality1. Although an increasing number of interventions show promise for rejuvenation2, their effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. Here we performed single-cell RNA sequencing on 20 organs to reveal cell-type-specific responses to young and aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, haematopoietic stem cells and hepatocytes are among those cell types that are especially responsive. On the pathway level, young blood invokes new gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. We observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it in select cell types. Together, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity.

  View details for DOI 10.1038/s41586-022-04461-2

  View details for PubMedID 35236985

• Cell types of origin of the cell-free transcriptome. Nature biotechnology Vorperian, S. K., Moufarrej, M. N., Tabula Sapiens Consortium, Quake, S. R., Jones, R. C., Karkanias, J., Krasnow, M., Pisco, A. O., Quake, S. R., Salzman, J., Yosef, N., Bulthaup, B., Brown, P., Harper, W., Hemenez, M., Ponnusamy, R., Salehi, A., Sanagavarapu, B. A., Spallino, E., Aaron, K. A., Concepcion, W., Gardner, J. M., Kelly, B., Neidlinger, N., Wang, Z., Crasta, S., Kolluru, S., Morri, M., Tan, S. Y., Travaglini, K. J., Xu, C., Alcantara-Hernandez, M., Almanzar, N., Antony, J., Beyersdorf, B., Burhan, D., Calcuttawala, K., Carter, M. M., Chan, C. K., Chang, C. A., Chang, S., Colville, A., Culver, R. N., Cvijovic, I., D'Amato, G., Ezran, C., Galdos, F. X., Gillich, A., Goodyer, W. R., Hang, Y., Hayashi, A., Houshdaran, S., Huang, X., Irwin, J. C., Jang, S., Juanico, J. V., Kershner, A. M., Kim, S., Kiss, B., Kong, W., Kumar, M. E., Kuo, A. H., Leylek, R., Li, B., Loeb, G. B., Lu, W., Mantri, S., Markovic, M., McAlpine, P. L., de Morree, A., Mrouj, K., Mukherjee, S., Muser, T., Neuhofer, P., Nguyen, T. D., Perez, K., Phansalkar, R., Puluca, N., Qi, Z., Rao, P., Raquer-McKay, H., Schaum, N., Scott, B., Seddighzadeh, B., Segal, J., Sen, S., Sikandar, S., Spencer, S. P., Steffes, L., Subramaniam, V. R., Swarup, A., Swift, M., Van Treuren, W., Trimm, E., Veizades, S., Vijayakumar, S., Vo, K. C., Vorperian, S. K., Wang, W., Weinstein, H. N., Winkler, J., Wu, T. T., Xie, J., Yung, A. R., Zhang, Y., Detweiler, A. M., Mekonen, H., Neff, N. F., Sit, R. V., Tan, M., Yan, J., Bean, G. R., Charu, V., Forgo, E., Martin, B. A., Ozawa, M. G., Silva, O., Toland, A., Vemuri, V. N., Afik, S., Awayan, K., Bierman, R., Botvinnik, O. B., Byrne, A., Chen, M., Dehghannasiri, R., Gayoso, A., Granados, A. A., Li, Q., Mahmoudabadi, G., McGeever, A., Olivieri, J. E., Park, M., Ravikumar, N., Stanley, G., Tan, W., Tarashansky, A. J., Vanheusden, R., Wang, P., Wang, S., Xing, G., Xu, C., Yosef, N., Culver, R., Dethlefsen, L., Ho, P., Liu, S., Maltzman, J. S., Metzger, R. J., Sasagawa, K., Sinha, R., Song, H., Wang, B., Artandi, S. E., Beachy, P. A., Clarke, M. F., Giudice, L. C., Huang, F. W., Huang, K. C., Idoyaga, J., Kim, S. K., Kuo, C. S., Nguyen, P., Rando, T. A., Red-Horse, K., Reiter, J., Relman, D. A., Sonnenburg, J. L., Wu, A., Wu, S. M., Wyss-Coray, T. 2022

  Abstract

  Cell-free RNA from liquid biopsies can be analyzed to determine disease tissue of origin. We extend this concept to identify cell types of origin using the Tabula Sapiens transcriptomic cell atlas as well as individual tissue transcriptomic cell atlases in combination with the Human Protein Atlas RNA consensus dataset. We define cell type signature scores, which allow the inference of cell types that contribute to cell-free RNA for a variety of diseases.

  View details for DOI 10.1038/s41587-021-01188-9

  View details for PubMedID 35132263

• 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

• 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

• A single-cell transcriptomic atlas characterizes ageing tissues in the mouse. Nature 2020

  Abstract

  Ageing is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death1. Despite rapid advances over recent years, many of the molecular and cellular processes that underlie the progressive loss of healthy physiology are poorly understood2. To gain a better insight into these processes, here we generate a single-cell transcriptomic atlas across the lifespan of Mus musculus that includes data from 23 tissues and organs. We found cell-specific changes occurring across multiple cell types and organs, as well as age-related changes in the cellular composition of different organs. Using single-cell transcriptomic data, we assessed cell-type-specific manifestations of different hallmarks of ageing-such as senescence3, genomic instability4 and changes in the immune system2. This transcriptomic atlas-which we denote Tabula Muris Senis, or 'Mouse Ageing Cell Atlas'-provides molecular information about how the most important hallmarks of ageing are reflected in a broad range of tissues and cell types.

  View details for DOI 10.1038/s41586-020-2496-1

  View details for PubMedID 32669714

• Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo. Cell stem cell Miao, Y. n., Ha, A. n., de Lau, W. n., Yuki, K. n., Santos, A. J., You, C. n., Geurts, M. H., Puschhof, J. n., Pleguezuelos-Manzano, C. n., Peng, W. C., Senlice, R. n., Piani, C. n., Buikema, J. W., Gbenedio, O. M., Vallon, M. n., Yuan, J. n., de Haan, S. n., Hemrika, W. n., Rösch, K. n., Dang, L. T., Baker, D. n., Ott, M. n., Depeille, P. n., Wu, S. M., Drost, J. n., Nusse, R. n., Roose, J. P., Piehler, J. n., Boj, S. F., Janda, C. Y., Clevers, H. n., Kuo, C. J., Garcia, K. C. 2020

  Abstract

  Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.

  View details for DOI 10.1016/j.stem.2020.07.020

  View details for PubMedID 32818433

• 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

• Ageing hallmarks exhibit organ-specific temporal signatures. Nature Schaum, N. n., Lehallier, B. n., Hahn, O. n., Pálovics, R. n., Hosseinzadeh, S. n., Lee, S. E., Sit, R. n., Lee, D. P., Losada, P. M., Zardeneta, M. E., Fehlmann, T. n., Webber, J. T., McGeever, A. n., Calcuttawala, K. n., Zhang, H. n., Berdnik, D. n., Mathur, V. n., Tan, W. n., Zee, A. n., Tan, M. n., Pisco, A. O., Karkanias, J. n., Neff, N. F., Keller, A. n., Darmanis, S. n., Quake, S. R., Wyss-Coray, T. n. 2020

  Abstract

  Ageing is the single greatest cause of disease and death worldwide, and understanding the associated processes could vastly improve quality of life. Although major categories of ageing damage have been identified-such as altered intercellular communication, loss of proteostasis and eroded mitochondrial function1-these deleterious processes interact with extraordinary complexity within and between organs, and a comprehensive, whole-organism analysis of ageing dynamics has been lacking. Here we performed bulk RNA sequencing of 17 organs and plasma proteomics at 10 ages across the lifespan of Mus musculus, and integrated these findings with data from the accompanying Tabula Muris Senis2-or 'Mouse Ageing Cell Atlas'-which follows on from the original Tabula Muris3. We reveal linear and nonlinear shifts in gene expression during ageing, with the associated genes clustered in consistent trajectory groups with coherent biological functions-including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Notably, these gene sets show similar expression across tissues, differing only in the amplitude and the age of onset of expression. Widespread activation of immune cells is especially pronounced, and is first detectable in white adipose depots during middle age. Single-cell RNA sequencing confirms the accumulation of T cells and B cells in adipose tissue-including plasma cells that express immunoglobulin J-which also accrue concurrently across diverse organs. Finally, we show how gene expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to the ageing of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of ageing, providing a foundation from which to track systemic sources of declining health at old age.

  View details for DOI 10.1038/s41586-020-2499-y

  View details for PubMedID 32669715

• Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution. Circulation research Goodyer, W. R., Beyersdorf, B., Paik, D. T., Tian, L., Li, G., Buikema, J. W., Chirikian, O., Choi, S., Venkatraman, S., Adams, E. L., Tessier-Lavigne, M., Wu, J. C., Wu, S. M. 2019

  Abstract

  RATIONALE: The cardiac conduction system (CCS) consists of distinct components including the sinoatrial node (SAN), atrioventricular node (AVN), His bundle, bundle branches (BB) and Purkinje fibers (PF). Despite an essential role for the CCS in heart development and function, the CCS has remained challenging to interrogate due to inherent obstacles including small cell numbers, large cell type heterogeneity, complex anatomy and difficulty in isolation. Single-cell RNA-sequencing (scRNA-seq) allows for genome-wide analysis of gene expression at single-cell resolution.OBJECTIVE: Assess the transcriptional landscape of the entire CCS at single-cell resolution by scRNA-seq within the developing mouse heart.METHODS AND RESULTS: Wild-type, embryonic day 16.5 mouse hearts (n=6 per zone) were harvested and three zones of microdissection were isolated, including: Zone I - SAN region; Zone II - AVN/His region; and Zone III - BB/PF region. Tissue was digested into single cell suspensions, isolated, reverse transcribed and barcoded prior to high-throughput sequencing and bioinformatics analyses. scRNA-seq was performed on over 22,000 cells and all major cell types of the murine heart were successfully captured including bona fide clusters of cells consistent with each major component of the CCS. Unsupervised weighted gene co-expression network analysis led to the discovery of a host of novel CCS genes, a subset of which were validated using fluorescent in situ hybridization as well as whole mount immunolabelling with volume imaging (iDISCO+) in three-dimensions on intact mouse hearts. Further, subcluster analysis unveiled isolation of distinct CCS cell subtypes, including the clinically-relevant but poorly characterized "transitional cells" that bridge the CCS and surrounding myocardium.CONCLUSIONS: Our study represents the first comprehensive assessment of the transcriptional profiles from the entire CCS at single-cell resolution and provides a gene atlas for facilitating future efforts in conduction cell identification, isolation and characterization in the context of development and disease.

  View details for DOI 10.1161/CIRCRESAHA.118.314578

  View details for PubMedID 31284824

• Prometheus Unbound in Ya(p) Heart DEVELOPMENTAL CELL Buikema, J. W., Wu, S. M. 2019; 48 (6): 741–42

  View details for DOI 10.1016/j.devcel.2019.03.007

  View details for Web of Science ID 000462154900001

• Single-cell analysis of early progenitor cells that build coronary arteries NATURE Su, T., Stanley, G., Sinha, R., D'Amato, G., Das, S., Rhee, S., Chang, A. H., Poduri, A., Raftrey, B., Thanh Theresa Dinh, Roper, W. A., Li, G., Quinn, K. E., Caron, K. M., Wu, S., Miquerol, L., Butcher, E. C., Weissman, I., Quake, S., Red-Horse, K. 2018; 559 (7714): 356-+

  View details for DOI 10.1038/s41586-018-0288-7

  View details for Web of Science ID 000439059800048

• Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris NATURE The Tabula Muris Consortium, .. 2018; 562: 367–372

  View details for DOI 10.1038/s41586-018-0590-4

• Transcriptomic Profiling Maps Anatomically Patterned Subpopulations among Single Embryonic Cardiac Cells DEVELOPMENTAL CELL Li, G., Xu, A., Sim, S., Priest, J. R., Tian, X., Khan, T., Quertermous, T., Zhou, B., Tsao, P. S., Quake, S. R., Wu, S. M. 2016; 39 (4): 491-507

  Abstract

  Embryonic gene expression intricately reflects anatomical context, developmental stage, and cell type. To address whether the precise spatial origins of cardiac cells can be deduced solely from their transcriptional profiles, we established a genome-wide expression database from 118, 949, and 1,166 single murine heart cells at embryonic day 8.5 (e8.5), e9.5, and e10.5, respectively. We segregated these cells by type using unsupervised bioinformatics analysis and identified chamber-specific genes. Using a random forest algorithm, we reconstructed the spatial origin of single e9.5 and e10.5 cardiomyocytes with 92.0% ± 3.2% and 91.2% ± 2.8% accuracy, respectively (99.4% ± 1.0% and 99.1% ± 1.1% if a ±1 zone margin is permitted) and predicted the second heart field distribution of Isl-1-lineage descendants. When applied to Nkx2-5(-/-) cardiomyocytes from murine e9.5 hearts, we showed their transcriptional alteration and lack of ventricular phenotype. Our database and zone classification algorithm will enable the discovery of novel mechanisms in early cardiac development and disease.

  View details for DOI 10.1016/j.devcel.2016.10.014

  View details for Web of Science ID 000389162800013

  View details for PubMedID 27840109

• Lift NIH restrictions on chimera research. Science (New York, N.Y.) Sharma, A. n., Sebastiano, V. n., Scott, C. T., Magnus, D. n., Koyano-Nakagawa, N. n., Garry, D. J., Witte, O. N., Nakauchi, H. n., Wu, J. C., Weissman, I. L., Wu, S. M. 2015; 350 (6261): 640

  View details for PubMedID 26542560

• Harnessing the potential of induced pluripotent stem cells for regenerative medicine NATURE CELL BIOLOGY Wu, S. M., Hothedlinger, K. 2011; 13 (5): 497-505

  Abstract

  The discovery of methods to convert somatic cells into induced pluripotent stem cells (iPSCs) through expression of a small combination of transcription factors has raised the possibility of producing custom-tailored cells for the study and treatment of numerous diseases. Indeed, iPSCs have already been derived from patients suffering from a large variety of disorders. Here we review recent progress that has been made in establishing iPSC-based disease models, discuss associated technical and biological challenges, and highlight possible solutions to overcome these barriers. We believe that a better understanding of the molecular basis of pluripotency, cellular reprogramming and lineage-specific differentiation of iPSCs is necessary for progress in regenerative medicine.

  View details for DOI 10.1038/ncb0511-497

  View details for Web of Science ID 000290148700004

  View details for PubMedID 21540845

  View details for PubMedCentralID PMC3617981

• Generation of Functional Ventricular Heart Muscle from Mouse Ventricular Progenitor Cells SCIENCE Domian, I. J., Chiravuri, M., van der Meer, P., Feinberg, A. W., Shi, X., Shao, Y., Wu, S. M., Parker, K. K., Chien, K. R. 2009; 326 (5951): 426-429

  Abstract

  The mammalian heart is formed from distinct sets of first and second heart field (FHF and SHF, respectively) progenitors. Although multipotent progenitors have previously been shown to give rise to cardiomyocytes, smooth muscle, and endothelial cells, the mechanism governing the generation of large numbers of differentiated progeny remains poorly understood. We have employed a two-colored fluorescent reporter system to isolate FHF and SHF progenitors from developing mouse embryos and embryonic stem cells. Genome-wide profiling of coding and noncoding transcripts revealed distinct molecular signatures of these progenitor populations. We further identify a committed ventricular progenitor cell in the Islet 1 lineage that is capable of limited in vitro expansion, differentiation, and assembly into functional ventricular muscle tissue, representing a combination of tissue engineering and stem cell biology.

  View details for DOI 10.1126/science.1177350

  View details for Web of Science ID 000270818600053

  View details for PubMedID 19833966

  View details for PubMedCentralID PMC2895998

• Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart NATURE Zhou, B., Ma, Q., Rajagopal, S., Wu, S. M., Domian, I., Rivera-Feliciano, J., Jiang, D., von Gise, A., Ikeda, S., Chien, K. R., Pu, W. T. 2008; 454 (7200): 109-U5

  Abstract

  The heart is formed from cardiogenic progenitors expressing the transcription factors Nkx2-5 and Isl1 (refs 1 and 2). These multipotent progenitors give rise to cardiomyocyte, smooth muscle and endothelial cells, the major lineages of the mature heart. Here we identify a novel cardiogenic precursor marked by expression of the transcription factor Wt1 and located within the epicardium-an epithelial sheet overlying the heart. During normal murine heart development, a subset of these Wt1(+) precursors differentiated into fully functional cardiomyocytes. Wt1(+) proepicardial cells arose from progenitors that express Nkx2-5 and Isl1, suggesting that they share a developmental origin with multipotent Nkx2-5(+) and Isl1(+) progenitors. These results identify Wt1(+) epicardial cells as previously unrecognized cardiomyocyte progenitors, and lay the foundation for future efforts to harness the cardiogenic potential of these progenitors for cardiac regeneration and repair.

  View details for DOI 10.1038/nature07060

  View details for Web of Science ID 000257308300047

  View details for PubMedID 18568026

  View details for PubMedCentralID PMC2574791

• Origins and fates of cardiovascular progenitor cells CELL Wu, S. M., Chien, K. R., Mummery, C. 2008; 132 (4): 537-543

  Abstract

  Multipotent cardiac progenitor cells are found in the fetal and adult heart of many mammalian species including humans and form as intermediates during the differentiation of embryonic stem cells. Despite similar biological properties, the molecular identities of these different cardiac progenitor cell populations appear to be distinct. Elucidating the origins and lineage relationships of these cell populations will accelerate clinical applications such as drug screening and cell therapy as well as shedding light on the pathogenic mechanisms underlying cardiac diseases.

  View details for DOI 10.1016/j.cell.2008.02.002

  View details for Web of Science ID 000253817900012

  View details for PubMedID 18295570

  View details for PubMedCentralID PMC2507768

• Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart CELL Wu, S. M., Fujiwara, Y., Cibulsky, S. M., Clapham, D. E., Lien, C., Schultheiss, T. M., Orkin, S. H. 2006; 127 (6): 1137-1150

  Abstract

  Despite recent advances in delineating the mechanisms involved in cardiogenesis, cellular lineage specification remains incompletely understood. To explore the relationship between developmental fate and potential, we isolated a cardiac-specific Nkx2.5(+) cell population from the developing mouse embryo. The majority of these cells differentiated into cardiomyocytes and conduction system cells. Some, surprisingly, adopted a smooth muscle fate. To address the clonal origin of these lineages, we isolated Nkx2.5(+) cells from in vitro differentiated murine embryonic stem cells and found approximately 28% of these cells expressed c-kit. These c-kit(+) cells possessed the capacity for long-term in vitro expansion and differentiation into both cardiomyocytes and smooth muscle cells from a single cell. We confirmed these findings by isolating c-kit(+)Nkx2.5(+) cells from mouse embryos and demonstrated their capacity for bipotential differentiation in vivo. Taken together, these results support the existence of a common precursor for cardiovascular lineages in the mammalian heart.

  View details for DOI 10.1016/j.cell.2006.10.028

  View details for Web of Science ID 000242991000013

  View details for PubMedID 17123591

• Cardiac Development at a Single-Cell Resolution. Advances in experimental medicine and biology Wei, N., Lee, C., Duan, L., Galdos, F. X., Samad, T., Raissadati, A., Goodyer, W. R., Wu, S. M. 2024; 1441: 253-268

  Abstract

  Mammalian cardiac development is a complex, multistage process. Though traditional lineage tracing studies have characterized the broad trajectories of cardiac progenitors, the advent and rapid optimization of single-cell RNA sequencing methods have yielded an ever-expanding toolkit for characterizing heterogeneous cell populations in the developing heart. Importantly, they have allowed for a robust profiling of the spatiotemporal transcriptomic landscape of the human and mouse heart, revealing the diversity of cardiac cells-myocyte and non-myocyte-over the course of development. These studies have yielded insights into novel cardiac progenitor populations, chamber-specific developmental signatures, the gene regulatory networks governing cardiac development, and, thus, the etiologies of congenital heart diseases. Furthermore, single-cell RNA sequencing has allowed for the exquisite characterization of distinct cardiac populations such as the hard-to-capture cardiac conduction system and the intracardiac immune population. Therefore, single-cell profiling has also resulted in new insights into the regulation of cardiac regeneration and injury repair. Single-cell multiomics approaches combining transcriptomics, genomics, and epigenomics may uncover an even more comprehensive atlas of human cardiac biology. Single-cell analyses of the developing and adult mammalian heart offer an unprecedented look into the fundamental mechanisms of cardiac development and the complex diseases that may arise from it.

  View details for DOI 10.1007/978-3-031-44087-8_14

  View details for PubMedID 38884716

  View details for PubMedCentralID 3784811

• Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration. Proceedings of the National Academy of Sciences of the United States of America Lee, S., Vander Roest, A. S., Blair, C. A., Kao, K., Bremner, S. B., Childers, M. C., Pathak, D., Heinrich, P., Lee, D., Chirikian, O., Mohran, S. E., Roberts, B., Smith, J. E., Jahng, J. W., Paik, D. T., Wu, J. C., Gunawardane, R. N., Ruppel, K. M., Mack, D. L., Pruitt, B. L., Regnier, M., Wu, S. M., Spudich, J. A., Bernstein, D. 2024; 121 (19): e2318413121

  Abstract

  Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the β-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

  View details for DOI 10.1073/pnas.2318413121

  View details for PubMedID 38683993

• The cardiac conduction system: History, development, and disease. Current topics in developmental biology Lee, C., Xu, S., Samad, T., Goodyer, W. R., Raissadati, A., Heinrich, P., Wu, S. M. 2024; 156: 157-200

  Abstract

  The heart is the first organ to form during embryonic development, establishing the circulatory infrastructure necessary to sustain life and enable downstream organogenesis. Critical to the heart's function is its ability to initiate and propagate electrical impulses that allow for the coordinated contraction and relaxation of its chambers, and thus, the movement of blood and nutrients. Several specialized structures within the heart, collectively known as the cardiac conduction system (CCS), are responsible for this phenomenon. In this review, we discuss the discovery and scientific history of the mammalian cardiac conduction system as well as the key genes and transcription factors implicated in the formation of its major structures. We also describe known human diseases related to CCS development and explore existing challenges in the clinical context.

  View details for DOI 10.1016/bs.ctdb.2024.02.006

  View details for PubMedID 38556422

• Osimertinib-Associated Cardiomyopathy In Patients With Non-Small Cell Lung Cancer: A Case Series JACC: CardioOncology Franquiz, M., Waliany, S., Xu, A., Hnatiuk, A., Wu, S., Cheng, P., Wakelee, H., Neal, J., Witteles, R., Zhu, H. 2023: 839-841

  View details for DOI 10.1016/j.jaccao.2023.07.006

  View details for PubMedCentralID PMC10774774

• Massive expansion of functional human iPSC-derived cardiomyocytes by concomitant glycogen synthase kinase-3 beta inhibition and removal of cell-cell contact Buikema, J. W., Lee, S., Maas, R. J., Van der Velden, J., Sluijter, J. G., Wu, S. M. OXFORD UNIV PRESS. 2023

  View details for Web of Science ID 001115619405078

• Impact of Troponin Monitoring on Cardiac Outcomes in Patients Receiving Immune Checkpoint Inhibitors Ivanovic, M., Chan, A., Franquiz, M., Xu, S., Lee, C., Fazal, M., You, J., Witteles, R., Neal, J., Wu, S., Waliany, S., Zhu, H. WILEY. 2023: 2087-2089

  View details for Web of Science ID 001190014302060

• Notch and retinoic acid signals regulate macrophage formation from endocardium downstream of Nkx2-5. Nature communications Liu, N., Kawahira, N., Nakashima, Y., Nakano, H., Iwase, A., Uchijima, Y., Wang, M., Wu, S. M., Minamisawa, S., Kurihara, H., Nakano, A. 2023; 14 (1): 5398

  Abstract

  Hematopoietic progenitors are enriched in the endocardial cushion and contribute, in a Nkx2-5-dependent manner, to tissue macrophages required for the remodeling of cardiac valves and septa. However, little is known about the molecular mechanism of endocardial-hematopoietic transition. In the current study, we identified the regulatory network of endocardial hematopoiesis. Signal network analysis from scRNA-seq datasets revealed that genes in Notch and retinoic acid (RA) signaling are significantly downregulated in Nkx2-5-null endocardial cells. In vivo and ex vivo analyses validate that the Nkx2-5-Notch axis is essential for the generation of both hemogenic and cushion endocardial cells, and the suppression of RA signaling via Dhrs3 expression plays important roles in further differentiation into macrophages. Genetic ablation study revealed that these macrophages are essential in cardiac valve remodeling. In summary, the study demonstrates that the Nkx2-5/Notch/RA signaling plays a pivotal role in macrophage differentiation from hematopoietic progenitors.

  View details for DOI 10.1038/s41467-023-41039-6

  View details for PubMedID 37669937

• Mechanisms in cardiac development and regeneration ZEITSCHRIFT FUR HERZ THORAX UND GEFASSCHIRURGIE Deutsch, M., Doppler, S. A., Gummert, J. F., Wu, S. M., Krane, M., Lange, R. 2023

  View details for DOI 10.1007/s00398-023-00600-7

  View details for Web of Science ID 001044180200001

• The Z-disc: Mechanosensor at the interface between myosin biomechanics and hypertrophic signaling Giri, P., Roest, A., Lee, S., Heinrich, P., Dunn, A. R., Wu, S., Bernstein, D. CELL PRESS. 2023: 404A

  View details for Web of Science ID 000989629702211

• Effects of changes in myosin biomechanics on canonical and non-canonical signaling and HCM phenotypes. Biophysical journal Heinrich, P., Wu, S. M. 2023; 122 (3S1): 148a

  View details for DOI 10.1016/j.bpj.2022.11.1015

  View details for PubMedID 36782681

• Changes in myosin biomechanics influence growth and maturation of iPSC-cardiomyocytes. Biophysical journal Bernstein, D., Vander Roest, A. S., Wu, S., Pruitt, B., Zhao, M., Fajardo, G., Ruppel, K., Spudich, J. A. 2023; 122 (3S1): 148a

  View details for DOI 10.1016/j.bpj.2022.11.1014

  View details for PubMedID 36782680

• The potential of auto-antigen-guided treatment of immune checkpoint inhibitor-mediated myocarditis. Med (New York, N.Y.) Zhu, H., Huang, Y. V., Wu, S. M. 2023; 4 (1): 13-14

  Abstract

  Immune checkpoint inhibitor (ICI)-mediated myocarditis is a rare but devastating side effect of cancer immunotherapy with up to 40% mortality and long-term cardiac issues such as arrhythmias and heart failure in affected patients.1 Recently, Axelrod et al.2 suggested an auto-antigen-driven mechanism as the immunological basis for this disease.

  View details for DOI 10.1016/j.medj.2022.12.005

  View details for PubMedID 36640753

• Harnessing developmental cues for cardiomyocyte production. Development (Cambridge, England) Maas, R. G., van den Dolder, F. W., Yuan, Q., van der Velden, J., Wu, S. M., Sluijter, J. P., Buikema, J. W. 2023; 150 (15)

  Abstract

  Developmental research has attempted to untangle the exact signals that control heart growth and size, with knockout studies in mice identifying pivotal roles for Wnt and Hippo signaling during embryonic and fetal heart growth. Despite this improved understanding, no clinically relevant therapies are yet available to compensate for the loss of functional adult myocardium and the absence of mature cardiomyocyte renewal that underlies cardiomyopathies of multiple origins. It remains of great interest to understand which mechanisms are responsible for the decline in proliferation in adult hearts and to elucidate new strategies for the stimulation of cardiac regeneration. Multiple signaling pathways have been identified that regulate the proliferation of cardiomyocytes in the embryonic heart and appear to be upregulated in postnatal injured hearts. In this Review, we highlight the interaction of signaling pathways in heart development and discuss how this knowledge has been translated into current technologies for cardiomyocyte production.

  View details for DOI 10.1242/dev.201483

  View details for PubMedID 37560977

• The Role of Single-Cell Profiling and Deep Immunophenotyping in Understanding Immune Therapy Cardiotoxicity. JACC. CardioOncology Huang, Y. V., Waliany, S., Lee, D., Galdos, F. X., Witteles, R. M., Neal, J. W., Fan, A. C., Maecker, H. T., Nguyen, P. K., Wu, S. M., Zhu, H. 2022; 4 (5): 629-634

  Abstract

  • ICIs used in cancer therapy can cause serious cardiac immune-related side effects. • Single-cell multi-omics are powerful tools in understanding cell subsets/phenotypes. • Multi-omics technology can elucidate disease mechanisms in ICI-induced myocarditis.

  View details for DOI 10.1016/j.jaccao.2022.08.012

  View details for PubMedID 36636436

  View details for PubMedCentralID PMC9830194

• Late-Onset Immunotherapy-Induced Myocarditis 2 Years After Checkpoint Inhibitor Initiation. JACC. CardioOncology Nguyen, A. T., Berry, G. J., Witteles, R. M., Le, D. T., Wu, S. M., Fisher, G. A., Zhu, H. 2022; 4 (5): 727-730

  View details for DOI 10.1016/j.jaccao.2022.04.007

  View details for PubMedID 36636432

  View details for PubMedCentralID PMC9830192

• KMT2D-NOTCH Mediates Coronary Abnormalities in Hypoplastic Left Heart Syndrome. Circulation research Yu, Z., Zhou, X., Liu, Z., Pastrana-Gomez, V., Liu, Y., Guo, M., Tian, L., Nelson, T. J., Wang, N., Mital, S., Chitayat, D., Wu, J. C., Rabinovitch, M., Wu, S. M., Snyder, M. P., Miao, Y., Gu, M. 2022: 101161CIRCRESAHA122320783

  View details for DOI 10.1161/CIRCRESAHA.122.320783

  View details for PubMedID 35762338

• NOVEL REGULATORY MECHANISM OF HEMOGENIC ENDOCARDIUM DURING CARDIOVASCULAR DEVELOPMENT Liu, N., Kawahira, N., Nakano, H., Iwase, A., Uchijima, Y., Wu, S., Minamisawa, S., Kurihara, H., Nakano, A. ELSEVIER SCIENCE INC. 2022: S106

  View details for Web of Science ID 000890643400173

• 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

• RNA splicing programs define tissue compartments and cell types at single cell resolution. eLife Olivieri, J. E., Dehghannasiri, R., Wang, P. L., Jang, S., de Morree, A., Tan, S. Y., Ming, J., Ruohao Wu, A., Tabula Sapiens Consortium, Quake, S. R., Krasnow, M. A., Salzman, J. 2021; 10

  Abstract

  The extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach, to detect cell-type-specific splicing in >110K cells from 12 human tissues. Using 10x data for discovery, 9.1% of genes with computable SpliZ scores are cell-type-specifically spliced, including ubiquitously expressed genes MYL6 and RPS24. These results are validated with RNA FISH, single-cell PCR, and Smart-seq2. SpliZ analysis reveals 170 genes with regulated splicing during human spermatogenesis, including examples conserved in mouse and mouse lemur. The SpliZ allows model-based identification of subpopulations indistinguishable based on gene expression, illustrated by subpopulation-specific splicing of classical monocytes involving an ultraconserved exon in SAT1. Together, this analysis of differential splicing across multiple organs establishes that splicing is regulated cell-type-specifically.

  View details for DOI 10.7554/eLife.70692

  View details for PubMedID 34515025

• Molecular Profiling of the Cardiac Conduction System: the Dawn of a New Era. Current cardiology reports Mantri, S., Wu, S. M., Goodyer, W. R. 2021; 23 (8): 103

  Abstract

  PURPOSE OF REVIEW: Recent technological advances have led to an increased ability to define the gene expression profile of the cardiac conduction system (CCS). Here, we review the most salient studies to emerge in recent years and discuss existing gaps in our knowledge as well as future areas of investigation.RECENT FINDINGS: Molecular profiling of the CCS spans several decades. However, the advent of high-throughput sequencing strategies has allowed for the discovery of unique transcriptional programs of the many diverse CCS cell types. The CCS, a diverse structure with significant inter- and intra-component cellular heterogeneity, is essential to the normal function of the heart. Progress in transcriptomic profiling has improved the resolution and depth of characterization of these unique and clinically relevant CCS cell types. Future studies leveraging this big data will play a crucial role in improving our understanding of CCS development and function as well as translating these findings into tangible translational tools for the improved detection, prevention, and treatment of cardiac arrhythmias.

  View details for DOI 10.1007/s11886-021-01536-w

  View details for PubMedID 34196831

• Overexpression of human BAG3P209L in mice causes restrictive cardiomyopathy. Nature communications Kimura, K., Ooms, A., Graf-Riesen, K., Kuppusamy, M., Unger, A., Schuld, J., Daerr, J., Lother, A., Geisen, C., Hein, L., Takahashi, S., Li, G., Roll, W., Bloch, W., van der Ven, P. F., Linke, W. A., Wu, S. M., Huesgen, P. F., Hohfeld, J., Furst, D. O., Fleischmann, B. K., Hesse, M. 2021; 12 (1): 3575

  Abstract

  An amino acid exchange (P209L) in the HSPB8 binding site of the human co-chaperone BAG3 gives rise to severe childhood cardiomyopathy. To phenocopy the disease in mice and gain insight into its mechanisms, we generated humanized transgenic mouse models. Expression of human BAG3P209L-eGFP in mice caused Z-disc disintegration and formation of protein aggregates. This was accompanied by massive fibrosis resulting in early-onset restrictive cardiomyopathy with increased mortality as observed in patients. RNA-Seq and proteomics revealed changes in the protein quality control system and increased autophagy in hearts from hBAG3P209L-eGFP mice. The mutation renders hBAG3P209L less soluble in vivo and induces protein aggregation, but does not abrogate hBAG3 binding properties. In conclusion, we report a mouse model mimicking the human disease. Our data suggest that the disease mechanism is due to accumulation of hBAG3P209L and mouse Bag3, causing sequestering of components of the protein quality control system and autophagy machinery leading to sarcomere disruption.

  View details for DOI 10.1038/s41467-021-23858-7

  View details for PubMedID 34117258

• Single cell RNA sequencing approaches to cardiac development and congenital heart disease. Seminars in cell & developmental biology Samad, T., Wu, S. M. 2021

  Abstract

  The development of single cell RNA sequencing technologies has accelerated the ability of scientists to understand healthy and disease states of the cardiovascular system. Congenital heart defects occur in approximately 40,000 births each year and 1 out of 4 children are born with critical congenital heart disease requiring surgical interventions and a lifetime of monitoring. An understanding of how the normal heart develops and how each cell contributes to normal and pathological anatomy is an important goal in pediatric cardiovascular research. Single cell sequencing has provided the tools to increase the ability to discover rare cell types and novel genes involved in normal cardiac development. Knowledge of gene expression of single cells within cardiac tissue has contributed to the understanding of how each cell type contributes to the anatomic structures of the heart. In this review, we summarize how single cell RNA sequencing has been utilized to understand cardiac developmental processes and congenital heart disease. We discuss the advantages and disadvantages of whole cell versus single nuclei RNA sequencing and describe the approaches to analyze the interactomes, transcriptomes, and differentiation trajectory from single cell data. We summarize the currently available single cell RNA sequencing technologies and technical aspects of performing single cell analysis and how to overcome common obstacles. We also review data from the recently published human and mouse fetal heart atlases and advancements that have occurred within the field due to the application of these single cell tools. Finally we highlight the potential for single cell technologies to uncover novel mechanisms of disease pathogenesis by leveraging findings from genome wide association studies.

  View details for DOI 10.1016/j.semcdb.2021.04.023

  View details for PubMedID 34006454

• Massive expansion and cryopreservation of functional human induced pluripotent stem cell-derived cardiomyocytes. STAR protocols Maas, R. G., Lee, S., Harakalova, M., Snijders Blok, C. J., Goodyer, W. R., Hjortnaes, J., Doevendans, P. A., Van Laake, L. W., van der Velden, J., Asselbergs, F. W., Wu, J. C., Sluijter, J. P., Wu, S. M., Buikema, J. W. 2021; 2 (1): 100334

  Abstract

  Since the discovery of human induced pluripotent stem cells (hiPSCs), numerous strategies have been established to efficiently derive cardiomyocytes from hiPSCs (hiPSC-CMs). Here, we describe a cost-effective strategy for the subsequent massive expansion (>250-fold) of high-purity hiPSC-CMs relying on two aspects: removal of cell-cell contacts and small-molecule inhibition with CHIR99021. The protocol maintains CM functionality, allows cryopreservation, and the cells can be used in downstream assays such as disease modeling, drug and toxicity screening, and cell therapy. For complete details on the use and execution of this protocol, please refer to Buikema (2020).

  View details for DOI 10.1016/j.xpro.2021.100334

  View details for PubMedID 33615277

• Myocarditis Surveillance with High-Sensitivity Troponin I During Cancer Treatment with Immune Checkpoint Inhibitors. JACC. CardioOncology Waliany, S., Neal, J. W., Reddy, S., Wakelee, H., Shah, S. A., Srinivas, S., Padda, S. K., Fan, A. C., Colevas, A. D., Wu, S. M., Witteles, R. M., Zhu, H. 2021; 3 (1): 137–39

  View details for DOI 10.1016/j.jaccao.2021.01.004

  View details for PubMedID 33796869

• 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

• Myocardial Disease and Long-Distance Space Travel: Solving the Radiation Problem. Frontiers in cardiovascular medicine Meerman, M., Bracco Gartner, T. C., Buikema, J. W., Wu, S. M., Siddiqi, S., Bouten, C. V., Grande-Allen, K. J., Suyker, W. J., Hjortnaes, J. 2021; 8: 631985

  Abstract

  Radiation-induced cardiovascular disease is a well-known complication of radiation exposure. Over the last few years, planning for deep space missions has increased interest in the effects of space radiation on the cardiovascular system, as an increasing number of astronauts will be exposed to space radiation for longer periods of time. Research has shown that exposure to different types of particles found in space radiation can lead to the development of diverse cardiovascular disease via fibrotic myocardial remodeling, accelerated atherosclerosis and microvascular damage. Several underlying mechanisms for radiation-induced cardiovascular disease have been identified, but many aspects of the pathophysiology remain unclear. Existing pharmacological compounds have been evaluated to protect the cardiovascular system from space radiation-induced damage, but currently no radioprotective compounds have been approved. This review critically analyzes the effects of space radiation on the cardiovascular system, the underlying mechanisms and potential countermeasures to space radiation-induced cardiovascular disease.

  View details for DOI 10.3389/fcvm.2021.631985

  View details for PubMedID 33644136

• Immune checkpoint inhibitor cardiotoxicity: Breaking barriers in the cardiovascular immune landscape. Journal of molecular and cellular cardiology Zhu, H., Ivanovic, M., Nguyen, A., Nguyen, P. K., Wu, S. M. 2021

  Abstract

  Immune checkpoint inhibitors (ICI) have changed the landscape of cancer therapy, but their use carries a high risk of cardiac immune related adverse events (iRAEs). With the expanding utilization of ICI therapy, there is a growing need to understand the underlying mechanisms behind their anti-tumor activity as well as their immune-mediated toxicities. In this review, we will focus on clinical characteristics and immune pathways of ICI cardiotoxicity, with an emphasis on single-cell technologies used to gain insights in this field. We will focus on three key areas of ICI-mediated immune pathways, including the anti-tumor immune response, the augmentation of the immune response by ICIs, and the pathologic "autoimmune" response in some individuals leading to immune-mediated toxicity, as well as local factors in the myocardial immune environment predisposing to autoimmunity. Discerning the underlying mechanisms of these immune pathways is necessary to inform the development of targeted therapies for ICI cardiotoxicities and reduce treatment related morbidity and mortality.

  View details for DOI 10.1016/j.yjmcc.2021.07.006

  View details for PubMedID 34303670

• Molecular hallmarks of heterochronic parabiosis at single cell resolution Nature Palovics, R., Keller, A., Schaum, N., Tan, W., Fehlmann, T., , Borja, M., Kern, F., Bonanno, L., Calcuttawala, K., Webber, J., McGeever, A., Muris Consortium, T., Luo, J., Pisco, A., Karkanias, J., Neff, N. F., Darmanis, S., Quake, S. R., Wyss-Coray, T. 2021
• 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

• Single-Cell RNA-seq Unveils Unique Transcriptomic Signatures of Organ-Specific Endothelial Cells. Circulation Paik, D. T., Tian, L., Williams, I. M., Rhee, S., Zhang, H., Liu, C., Mishra, R., Wu, S. M., Red-Horse, K., Wu, J. C. 2020

  Abstract

  Background: Endothelial cells (ECs) display considerable functional heterogeneity depending on the vessel and tissue in which they are located. While these functional differences are presumably imprinted in the transcriptome, the pathways and networks which sustain EC heterogeneity have not been fully delineated. Methods: To investigate the transcriptomic basis of EC specificity, we analyzed single-cell RNA-sequencing (scRNA-seq) data from tissue-specific mouse ECs generated by the Tabula Muris consortium. We employed a number of bioinformatics tools to uncover markers and sources of EC heterogeneity from scRNA-seq data. Results: We found a strong correlation between tissue-specific EC transcriptomic measurements generated by either scRNA-seq or bulk RNA-seq, thus validating the approach. Using a graph-based clustering algorithm, we found that certain tissue-specific ECs cluster strongly by tissue (e.g. liver, brain) whereas others (i.e. adipose, heart) have considerable transcriptomic overlap with ECs from other tissues. We identified novel markers of tissue-specific ECs and signaling pathways that may be involved in maintaining their identity. Sex was a considerable source of heterogeneity in the endothelial transcriptome and we discovered Lars2 to be a gene that is highly enriched in ECs from male mice. In addition, we found that markers of heart and lung ECs in mice were conserved in human fetal heart and lung ECs. Finally, we identified potential angiocrine interactions between tissue-specific ECs and other cell types by analyzing ligand and receptor expression patterns. Conclusions: In summary, we use scRNA-seq data generated by the Tabula Muris consortium to uncover transcriptional networks that maintain tissue-specific EC identity and to identify novel angiocrine and functional relationships between tissue-specific ECs.

  View details for DOI 10.1161/CIRCULATIONAHA.119.041433

  View details for PubMedID 32929989

• 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

• Proceedings From the 2019 Stanford Single Ventricle Scientific Summit: Advancing Science for Single Ventricle Patients: From Discovery to Clinical Applications. Journal of the American Heart Association Reddy, S., Handler, S. S., Wu, S., Rabinovitch, M., Wright, G. 2020; 9 (7): e015871

  Abstract

  Abstracts Because of remarkable advances in survival over the past 40years, the worldwide population of individuals with single ventricle heart disease living with Fontan circulation has grown to 70000, with nearly half aged >18years. Survival to at least 30years of age is now achievable for 75% of Fontan patients. On the other hand, single ventricle patients account for the largest group of the 6000 to 8000 children hospitalized with circulation failure, with or without heart failure annually in the United States, with the highest in-hospital mortality. Because there is little understanding of the underlying mechanisms of heart failure, arrhythmias, pulmonary and lymphatic vascular abnormalities, and other morbidities, there are no specific treatments to maintain long-term myocardial performance or to optimize overall patient outcomes.

  View details for DOI 10.1161/JAHA.119.015871

  View details for PubMedID 32188306

• Immune Checkpoint Inhibitor Cardiotoxicity: Understanding Basic Mechanisms and Clinical Characteristics and Finding a Cure. Annual review of pharmacology and toxicology Waliany, S. n., Lee, D. n., Witteles, R. M., Neal, J. W., Nguyen, P. n., Davis, M. M., Salem, J. E., Wu, S. M., Moslehi, J. J., Zhu, H. n. 2020

  Abstract

  Immune checkpoint inhibitors (ICIs) attenuate mechanisms of self-tolerance in the immune system, enabling T cell responses to cancerous tissues and revolutionizing care for cancer patients. However, by lowering barriers against self-reactivity, ICIs often result in varying degrees of autoimmunity. Cardiovascular complications, particularly myocarditis but also arrhythmias, pericarditis, and vasculitis, have emerged as significant complications associated with ICIs. In this review, we examine the clinical aspects and basic science principles that underlie ICI-associated myocarditis and other cardiovascular toxicities. In addition, we discuss current therapeutic approaches. We believe a better mechanistic understanding of ICI-associated toxicities can lead to improved patient outcomes by reducing treatment-related morbidity. Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 61 is January 8, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  View details for DOI 10.1146/annurev-pharmtox-010919-023451

  View details for PubMedID 32776859

• Cardiovascular Complications in Patients with COVID-19: Consequences of Viral Toxicities and Host Immune Response Curr Cardiol Rep Zhu, H., Rhee, J., Cheng, P., Waliany, S., Chang, A., Witteles, R. M., Maecker, H., Davis, M. M., Nguyen, P. K., Wu, S. M. 2020; 22 (5)

  View details for DOI 10.1007/s11886-020-01292-3

• Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils. Journal of visualized experiments : JoVE Lee, S. n., Yang, H. n., Chen, C. n., Venkatraman, S. n., Darsha, A. n., Wu, S. M., Wu, J. C., Seeger, T. n. 2020

  Abstract

  Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

  View details for DOI 10.3791/60888

  View details for PubMedID 32310234

• Cardiovascular Risks in Patients with COVID-19: Potential Mechanisms and Areas of Uncertainty. Current cardiology reports Cheng, P. n., Zhu, H. n., Witteles, R. M., Wu, J. C., Quertermous, T. n., Wu, S. M., Rhee, J. W. 2020; 22 (5): 34

  Abstract

  COronaVirus Disease 2019 (COVID-19) has spread at unprecedented speed and scale into a global pandemic with cardiovascular risk factors and complications emerging as important disease modifiers. We aim to review available clinical and biomedical literature on cardiovascular risks of COVID-19.SARS-CoV2, the virus responsible for COVID-19, enters the cell via ACE2 expressed in select organs. Emerging epidemiological evidence suggest cardiovascular risk factors are associated with increased disease severity and mortality in COVID-19 patients. Patients with a more severe form of COVID-19 are also more likely to develop cardiac complications such as myocardial injury and arrhythmia. The true incidence of and mechanism underlying these events remain elusive. Cardiovascular diseases appear intricately linked with COVID-19, with cardiac complications contributing to the elevated morbidity/mortality of COVID-19. Robust epidemiologic and biologic studies are urgently needed to better understand the mechanism underlying these associations to develop better therapies.

  View details for DOI 10.1007/s11886-020-01293-2

  View details for PubMedID 32350632

• Levitating Cells to Sort the Fit and the Fat. Advanced biosystems Puluca, N. n., Durmus, N. G., Lee, S. n., Belbachir, N. n., Galdos, F. X., Ogut, M. G., Gupta, R. n., Hirano, K. I., Krane, M. n., Lange, R. n., Wu, J. C., Wu, S. M., Demirci, U. n. 2020: e1900300

  Abstract

  Density is a core material property and varies between different cell types, mainly based on differences in their lipid content. Sorting based on density enables various biomedical applications such as multi-omics in precision medicine and regenerative repair in medicine. However, a significant challenge is sorting cells of the same type based on density differences. Here, a new method for real-time monitoring and sorting of single cells based on their inherent levitation profiles driven by their lipid content is reported. As a model system, human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) from a patient with neutral lipid storage disease (NLSD) due to loss of function of adipose triglyceride lipase (ATGL) resulting in abnormal lipid storage in cardiac muscle are used. This levitation-based strategy detects subpopulations within ATGL-deficient hiPSC-CMs with heterogenous lipid content, equilibrating at different levitation heights due to small density differences. In addition, sorting of these differentially levitating subpopulations are monitored in real time. Using this approach, sorted healthy and diseased hiPSC-CMs maintain viability and function. Pixel-tracking technologies show differences in contraction between NLSD and healthy hiPSC-CMs. Overall, this is a unique approach to separate diseased cell populations based on their intracellular lipid content that cannot be achieved using traditional flow cytometry techniques.

  View details for DOI 10.1002/adbi.201900300

  View details for PubMedID 32352239

• Single Cell Analysis of Endothelial Cells Identified Organ-Specific Molecular Signatures and Heart-Specific Cell Populations and Molecular Features. Frontiers in cardiovascular medicine Feng, W., Chen, L., Nguyen, P. K., Wu, S. M., Li, G. 2019; 6: 165

  Abstract

  Endothelial cells line the inner surface of vasculature and play an important role in normal physiology and disease progression. Although most tissue is known to have a heterogeneous population of endothelial cells, transcriptional differences in organ specific endothelial cells have not been systematically analyzed at the single cell level. The Tabula Muris project profiled mouse single cells from 20 organs. We found 10 of the organs profiled by this Consortium have endothelial cells. Unsupervised analysis of these endothelial cells revealed that they were mainly grouped by organs, and organ-specific cells were further partially correlated by germ layers. Unexpectedly, we found all lymphatic endothelial cells grouped together regardless of their resident organs. To further understand the cellular heterogeneity in organ-specific endothelial cells, we used the heart as an example. As a pump of the circulation system, the heart has multiple types of endothelial cells. Detailed analysis of these cells identified an endocardial endothelial cell population, a coronary vascular endothelial cell population, and an aorta-specific cell population. Through integrated analysis of the single cell data from another two studies analyzing the aorta, we identified conserved cell populations and molecular markers across the datasets. In summary, by reanalyzing the existing endothelial cell single-cell data, we identified organ-specific molecular signatures and heart-specific subpopulations and molecular markers. We expect these findings will pave the way for a deeper understanding of vascular biology and endothelial cell-related diseases.

  View details for DOI 10.3389/fcvm.2019.00165

  View details for PubMedID 31850371

  View details for PubMedCentralID PMC6901932

• Effects of Spaceflight on Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Structure and Function. Stem cell reports Wnorowski, A., Sharma, A., Chen, H., Wu, H., Shao, N., Sayed, N., Liu, C., Countryman, S., Stodieck, L. S., Rubins, K. H., Wu, S. M., Lee, P. H., Wu, J. C. 2019

  Abstract

  With extended stays aboard the International Space Station (ISS) becoming commonplace, there is a need to better understand the effects of microgravity on cardiac function. We utilized human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of microgravity on cell-level cardiac function and gene expression. The hiPSC-CMs were cultured aboard the ISS for 5.5weeks and their gene expression, structure, and functions were compared with ground control hiPSC-CMs. Exposure to microgravity on the ISS caused alterations in hiPSC-CM calcium handling. RNA-sequencing analysis demonstrated that 2,635 genes were differentially expressed among flight, post-flight, and ground control samples, including genes involved in mitochondrial metabolism. This study represents the first use of hiPSC technology to model the effects of spaceflight on human cardiomyocyte structure and function.

  View details for DOI 10.1016/j.stemcr.2019.10.006

  View details for PubMedID 31708475

• Myopathy Causing Bag3P209L Protein Leads to Restrictive Cardiomyopathy Caused by Aggregate Formation and Sarcomere Disruption in Cardiomyocytes Graf-Riesen, K., Kimura, K., Unger, A., Lother, A., Hein, L., Daerr, J., Braune, J., Ooms, A., Li, G., Wu, S. M., Hohfeld, J., Linke, W. A., Furst, D., Fleischmann, B. K., Hesse, M. LIPPINCOTT WILLIAMS & WILKINS. 2019

  View details for Web of Science ID 000511467800268

• Single-Cell Delineation of Who's on First and Second Heart Fields During Development CIRCULATION RESEARCH Galdos, F. X., Wu, S. M. 2019; 125 (4): 411–13

  View details for DOI 10.1161/CIRCRESAHA.119.315576

  View details for Web of Science ID 000478066400010

• Hypertrophic Cardiomyopathy Mutations With Opposite Effects on [latin sharp s]-myosin Biomechanics Show Similar Structural and Biomechanical Phenotypes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (hipsc-cms) Schroer, A., Jung, G., Kooiker, K., Adhikari, A., Song Linda, Liu Chao, Ruppel, K., Wu Sean, Pruitt, B., Spudich, J., Bernstein, D. LIPPINCOTT WILLIAMS & WILKINS. 2019

  View details for Web of Science ID 000511467800091

• Single cell expression analysis reveals anatomical and cell cycle-dependent transcriptional shifts during heart development. Development (Cambridge, England) Li, G., Tian, L., Goodyer, W., Kort, E. J., Buikema, J. W., Xu, A., Wu, J., Jovinge, S., Wu, S. M. 2019

  Abstract

  The heart is a complex organ composed of multiple cell and tissue types. Cardiac cells from different regions of the growing embryonic heart exhibit distinct patterns of gene expression, which are thought to contribute to heart development and morphogenesis. Single cell RNA sequencing allows genome-wide analysis of gene expression at the single cell level. Here, we analyzed cardiac cells derived from early stage developing hearts by single cell RNA-seq and identified cell cycle gene expression as a major determinant of transcriptional variation. Within cell cycle stage-matched CMs from a given heart chamber, we found that CMs in the G2/M phase downregulated sarcomeric and cytoskeletal markers. We also identified cell location-specific signaling molecules that may influence the proliferation of other nearby cell types. Our data highlight how variations in cell cycle activity selectively promote cardiac chamber growth during development, reveal profound chamber-specific cell cycle-linked transcriptional shifts, and open the way to deeper understanding of pathogenesis of congenital heart disease.

  View details for DOI 10.1242/dev.173476

  View details for PubMedID 31142541

• Bioprinting Approaches to Engineering Vascularized 3D Cardiac Tissues. Current cardiology reports Puluca, N. n., Lee, S. n., Doppler, S. n., Münsterer, A. n., Dreßen, M. n., Krane, M. n., Wu, S. M. 2019; 21 (9): 90

  Abstract

  3D bioprinting technologies hold significant promise for the generation of engineered cardiac tissue and translational applications in medicine. To generate a clinically relevant sized tissue, the provisioning of a perfusable vascular network that provides nutrients to cells in the tissue is a major challenge. This review summarizes the recent vascularization strategies for engineering 3D cardiac tissues.Considerable steps towards the generation of macroscopic sizes for engineered cardiac tissue with efficient vascular networks have been made within the past few years. Achieving a compact tissue with enough cardiomyocytes to provide functionality remains a challenging task. Achieving perfusion in engineered constructs with media that contain oxygen and nutrients at a clinically relevant tissue sizes remains the next frontier in tissue engineering. The provisioning of a functional vasculature is necessary for maintaining a high cell viability and functionality in engineered cardiac tissues. Several recent studies have shown the ability to generate tissues up to a centimeter scale with a perfusable vascular network. Future challenges include improving cell density and tissue size. This requires the close collaboration of a multidisciplinary teams of investigators to overcome complex challenges in order to achieve success.

  View details for DOI 10.1007/s11886-019-1179-8

  View details for PubMedID 31352612

• Tissue Engineering of 3D Organotypic Microtissues by Acoustic Assembly ORGANOIDS Zhu, Y., Serpooshan, V., Wu, S., Demirci, U., Chen, P., Guven, S., Turksen, K. 2019; 1576: 301-312

  View details for DOI 10.1007/7651_2017_68

  View details for Web of Science ID 000685760200026

• Cardiovascular Regenerative Medicine: Challenges, Perspectives, and Future Directions Cardiovasscular Regenerative Medicine Wu, S. M., Serpooshan, V. Springer Nature. 2019: 223–225
• Cardiovascular Regenerative Medicine edited by Serpooshan, V., Wu, S. M. Springer Nature. 2019

  View details for DOI 10.1007/9783030200473

• Modelling inherited cardiac disease using human induced pluripotent stem cell-derived cardiomyocytes: progress, pitfalls, and potential CARDIOVASCULAR RESEARCH van Mil, A., Balk, G., Neef, K., Buikema, J., Asselbergs, F. W., Wu, S. M., Doevendans, P. A., Sluijter, J. G. 2018; 114 (14): 1828–42

  View details for DOI 10.1093/cvr/cvy208

  View details for Web of Science ID 000455186400009

• Cardiovascular tissue bioprinting: Physical and chemical processes. Applied physics reviews Hu, J. B., Tomov, M. L., Buikema, J. W., Chen, C., Mahmoudi, M., Wu, S. M., Serpooshan, V. 2018; 5 (4): 041106

  Abstract

  Three-dimensional (3D) cardiac tissue bioprinting occupies a critical crossroads position between the fields of materials engineering, cardiovascular biology, 3D printing, and rational organ replacement design. This complex area of research therefore requires expertise from all those disciplines and it poses some unique considerations that must be accounted for. One of the chief hurdles is that there is a relatively limited systematic organization of the physical and chemical characteristics of bioinks that would make them applicable to cardiac bioprinting. This is of great significance, as heart tissue is functionally complex and the in vivo extracellular niche is under stringent controls with little room for variability before a cardiomyopathy manifests. This review explores the critical parameters that are necessary for biologically relevant bioinks to successfully be leveraged for functional cardiac tissue engineering, which can have applications in in vitro heart tissue models, cardiotoxicity studies, and implantable constructs that can be used to treat a range of cardiomyopathies, or in regenerative medicine.

  View details for DOI 10.1063/1.5048807

  View details for PubMedID 32550960

  View details for PubMedCentralID PMC7187889

• Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells CIRCULATION RESEARCH Paik, D. T., Tian, L., Lee, J., Sayed, N., Chen, I. Y., Rhee, S., Rhee, J., Kim, Y., Wirka, R. C., Buikema, J. W., Wu, S. M., Red-Horse, K., Quertermous, T., Wu, J. C. 2018; 123 (4): 443–50

  View details for DOI 10.1161/CIRCRESAHA.118.312913

  View details for Web of Science ID 000440670800010

• Fates Aligned: Origins and Mechanisms of Ventricular Conduction System and Ventricular Wall Development Goodyer, W. R., Wu, S. M. SPRINGER. 2018: 1090–98

  View details for DOI 10.1007/s00246-018-1869-9

  View details for Web of Science ID 000441721000004

• Reassessment of c-Kit in Cardiac Cells A Complex Interplay Between Expression, Fate, and Function CIRCULATION RESEARCH Zhou, B., Wu, S. M. 2018; 123 (1): 9–11

  View details for DOI 10.1161/CIRCRESAHA.118.313215

  View details for Web of Science ID 000435962900002

• Genome Editing Redefines Precision Medicine in the Cardiovascular Field. Stem cells international Dzilic, E., Lahm, H., Dreßen, M., Deutsch, M. A., Lange, R., Wu, S. M., Krane, M., Doppler, S. A. 2018; 2018: 4136473

  Abstract

  Genome editing is a powerful tool to study the function of specific genes and proteins important for development or disease. Recent technologies, especially CRISPR/Cas9 which is characterized by convenient handling and high precision, revolutionized the field of genome editing. Such tools have enormous potential for basic science as well as for regenerative medicine. Nevertheless, there are still several hurdles that have to be overcome, but patient-tailored therapies, termed precision medicine, seem to be within reach. In this review, we focus on the achievements and limitations of genome editing in the cardiovascular field. We explore different areas of cardiac research and highlight the most important developments: (1) the potential of genome editing in human pluripotent stem cells in basic research for disease modelling, drug screening, or reprogramming approaches and (2) the potential and remaining challenges of genome editing for regenerative therapies. Finally, we discuss social and ethical implications of these new technologies.

  View details for DOI 10.1155/2018/4136473

  View details for PubMedID 29731778

  View details for PubMedCentralID PMC5872631

• Stage-specific Effects of Bioactive Lipids on Human iPSC Cardiac Differentiation and Cardiomyocyte Proliferation. Scientific reports Sharma, A. n., Zhang, Y. n., Buikema, J. W., Serpooshan, V. n., Chirikian, O. n., Kosaric, N. n., Churko, J. M., Dzilic, E. n., Shieh, A. n., Burridge, P. W., Wu, J. C., Wu, S. M. 2018; 8 (1): 6618

  Abstract

  Bioactive lipids such as sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) regulate diverse processes including cell proliferation, differentiation, and migration. However, their roles in cardiac differentiation and cardiomyocyte proliferation have not been explored. Using a 96-well differentiation platform for generating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) we found that S1P and LPA can independently enhance cardiomyocyte generation when administered at an early stage of differentiation. We showed that the combined S1P and LPA treatment of undifferentiated hiPSCs resulted in increased nuclear accumulation of β-catenin, the canonical Wnt signaling pathway mediator, and synergized with CHIR99021, a glycogen synthase kinase 3 beta inhibitor, to enhance mesodermal induction and subsequent cardiac differentiation. At later stages of cardiac differentiation, the addition of S1P and LPA resulted in cell cycle initiation in hiPSC-CMs, an effect mediated through increased ERK signaling. Although the addition of S1P and LPA alone was insufficient to induce cell division, it was able to enhance β-catenin-mediated hiPSC-CM proliferation. In summary, we demonstrated a developmental stage-specific effect of bioactive lipids to enhance hiPSC-CM differentiation and proliferation via modulating the effect of canonical Wnt/β-catenin and ERK signaling. These findings may improve hiPSC-CM generation for cardiac disease modeling, precision medicine, and regenerative therapies.

  View details for PubMedID 29700394

• 4D Printing of Actuating Cardiac Tissue 3D PRINTING APPLICATIONS IN CARDIOVASCULAR MEDICINE Serpooshan, V., Hu, J. B., Chirikian, O., Hu, D. A., Mahmoudi, M., Wu, S. M., AlAref, S. J., Mosadegh, B., Dunham, S., Min, J. K. 2018: 153–62

  View details for DOI 10.1016/B978-0-12-803917-5.00008-0

  View details for Web of Science ID 000488516400010

• Big bottlenecks in cardiovascular tissue engineering COMMUNICATIONS BIOLOGY Huang, N. F., Serpooshan, V., Morris, V. B., Sayed, N., Pardon, G., Abilez, O. J., Nakayama, K. H., Pruitt, B. L., Wu, S. M., Yoon, Y., Zhang, J., Wu, J. C. 2018; 1

  View details for DOI 10.1038/s42003-018-0202-8

  View details for Web of Science ID 000461126500237

• Myocardial Development Reference Modules in Biomedical Sciences Galdos, F. X., Wu, S. M. Elsevier. 2018; 1
• Reactivation of the Nkx2.5 cardiac enhancer after myocardial infarction does not presage myogenesis. Cardiovascular research Deutsch, M. A., Doppler, S. A., Li, X. n., Lahm, H. n., Santamaria, G. n., Cuda, G. n., Eichhorn, S. n., Ratschiller, T. n., Dzilic, E. n., Dreßen, M. n., Eckart, A. n., Stark, K. n., Massberg, S. n., Bartels, A. n., Rischpler, C. n., Gilsbach, R. n., Hein, L. n., Fleischmann, B. K., Wu, S. M., Lange, R. n., Krane, M. n. 2018

  Abstract

  The contribution of resident stem or progenitor cells to cardiomyocyte renewal after injury in adult mammalian hearts remains a matter of considerable debate. We evaluated a cell population in the adult mouse heart induced by myocardial infarction (MI) and characterized by an activated Nkx2.5 enhancer element that is specific for multipotent cardiac progenitor cells during embryonic development. We hypothesized that these MI induced cells (MICs) harbor cardiomyogenic properties similar to their embryonic counterparts.MICs reside in the heart and mainly localize to the infarction area and border zone. Interestingly, gene expression profiling of purified MICs one week after infarction revealed increased expression of stem cell markers and embryonic cardiac transcription factors in these cells as compared to the non-mycoyte cell fraction of adult hearts. A subsequent global transcriptome comparison with embryonic cardiac progenitor cells and fibroblasts and in vitro culture of MICs unveiled that (myo-) fibroblastic features predominated and that cardiac transcription factors were only expressed at background levels.Adult injury induced reactivation of a cardiac-specific Nkx2.5 enhancer element known to specifically mark myocardial progenitor cells during embryonic development does not reflect hypothesized embryonic cardiomyogenic properties. Our data suggest a decreasing plasticity of cardiac progenitor (-like) cell populations with increasing age. A re-expression of embryonic, stem or progenitor cell features in the adult heart must be interpreted very carefully with respect to the definition of cardiac resident progenitor cells. Albeit, the abundance of scar formation after cardiac injury suggests a potential to target predestinated activated profibrotic cells to push them towards cardiomyogenic differentiation to improve regeneration.

  View details for PubMedID 29579159

• Bioengineering of vascular myocardial tissue; a 3D bioprinting approach Hu, J. B., Hu, D. A., Buikema, J. W., Chirikian, O., Venkatraman, S., Serpooshan, V., Wu, S. M. MARY ANN LIEBERT, INC. 2017: S158–S159

  View details for Web of Science ID 000416247300609

• Bioacoustic-enabled patterning of human iPSC-derived cardiomyocytes into 3D cardiac tissue BIOMATERIALS Serpooshan, V., Chen, P., Wu, H., Lee, S., Sharma, A., Hu, D. A., Venkatraman, S., Ganesan, A. V., Usta, O. B., Yarmush, M., Yang, F., Wu, J. C., Demirci, U., Wu, S. M. 2017; 131: 47-57

  Abstract

  The creation of physiologically-relevant human cardiac tissue with defined cell structure and function is essential for a wide variety of therapeutic, diagnostic, and drug screening applications. Here we report a new scalable method using Faraday waves to enable rapid aggregation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) into predefined 3D constructs. At packing densities that approximate native myocardium (10(8)-10(9) cells/ml), these hiPSC-CM-derived 3D tissues demonstrate significantly improved cell viability, metabolic activity, and intercellular connection when compared to constructs with random cell distribution. Moreover, the patterned hiPSC-CMs within the constructs exhibit significantly greater levels of contractile stress, beat frequency, and contraction-relaxation rates, suggesting their improved maturation. Our results demonstrate a novel application of Faraday waves to create stem cell-derived 3D cardiac tissue that resembles the cellular architecture of a native heart tissue for diverse basic research and clinical applications.

  View details for DOI 10.1016/j.biomaterials.2017.03.037

  View details for PubMedID 28376365

• Contractile force generation by 3D hiPSC-derived cardiac tissues is enhanced by rapid establishment of cellular interconnection in matrix with muscle-mimicking stiffness BIOMATERIALS Lee, S., Serpooshan, V., Tong, X., Venkatraman, S., Lee, M., Lee, J., Chirikian, O., Wu, J. C., Wu, S. M., Yang, F. 2017; 131: 111-120

  Abstract

  Engineering 3D human cardiac tissues is of great importance for therapeutic and pharmaceutical applications. As cardiac tissue substitutes, extracellular matrix-derived hydrogels have been widely explored. However, they exhibit premature degradation and their stiffness is often orders of magnitude lower than that of native cardiac tissue. There are no reports on establishing interconnected cardiomyocytes in 3D hydrogels at physiologically-relevant cell density and matrix stiffness. Here we bioengineer human cardiac microtissues by encapsulating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in chemically-crosslinked gelatin hydrogels (1.25 × 10(8)/mL) with tunable stiffness and degradation. In comparison to the cells in high stiffness (16 kPa)/slow degrading hydrogels, hiPSC-CMs in low stiffness (2 kPa)/fast degrading and intermediate stiffness (9 kPa)/intermediate degrading hydrogels exhibit increased intercellular network formation, α-actinin and connexin-43 expression, and contraction velocity. Only the 9 kPa microtissues exhibit organized sarcomeric structure and significantly increased contractile stress. This demonstrates that muscle-mimicking stiffness together with robust cellular interconnection contributes to enhancement in sarcomeric organization and contractile function of the engineered cardiac tissue. This study highlights the importance of intercellular connectivity, physiologically-relevant cell density, and matrix stiffness to best support 3D cardiac tissue engineering.

  View details for DOI 10.1016/j.biomaterials.2017.03.039

  View details for PubMedID 28384492

• YY1 Expression is Sufficient for the Maintenance of Cardiac Progenitor Cell State. Stem cells Gregoire, S., Li, G., Sturzu, A. C., Schwartz, R. J., Wu, S. M. 2017

  Abstract

  During cardiac development, DNA binding transcription factors and epigenetic modifiers regulate gene expression in cardiac progenitor cells (CPCs). We have previously shown that Yin Yang 1 (YY1) is essential for the commitment of mesodermal precursors into CPCs. However, the role of YY1 in the maintenance of CPC phenotype and their differentiation into cardiomyocytes is unknown. In this study, we found, by genome-wide transcriptional profiling and phenotypic assays, that YY1 overexpression prevents cardiomyogenic differentiation and maintains the proliferative capacity of CPCs. We show further that the ability of YY1 to regulate CPC phenotype is associated with its ability to modulate histone modifications specifically at a developmentally critical enhancer of Nkx2-5 and other key cardiac transcription factor such as Tbx5. Specifically, YY1 overexpression helps to maintain markers of gene activation such as the acetylation of histone H3 at lysine 9 (H3K9Ac) and lysine 27 (H3K27Ac) as well as trimethylation at lysine 4 (H3K4Me3) at the Nkx2-5 cardiac enhancer. Furthermore, transcription factors associated proteins such as PoIII, p300, and Brg1 are also enriched at the Nkx2-5 enhancer with YY1 overexpression. The biological activities of YY1 in CPCs appear to be cell autonomous, based coculture assays in differentiating embryonic stem cells. Altogether, these results demonstrate that YY1 overexpression is sufficient to maintain a CPC phenotype through its ability to sustain the presence of activating epigenetic/chromatin marks at key cardiac enhancers. Stem Cells 2017.

  View details for DOI 10.1002/stem.2646

  View details for PubMedID 28580685

• Untangling the Biology of Genetic Cardiomyopathies with Pluripotent Stem Cell Disease Models CURRENT CARDIOLOGY REPORTS Buikema, J. W., Wu, S. M. 2017; 19 (4)

  Abstract

  Recently, the discovery of strategies to reprogram somatic cells into induced pluripotent stem (iPS) cells has led to a major paradigm change in developmental and stem cell biology. The application of iPS cells and their cardiac progeny has opened novel directions to study cardiomyopathies at a cellular and molecular level. This review discusses approaches currently undertaken to unravel known inherited cardiomyopathies in a dish.With improved efficiency for mutation correction by genome editing, human iPS cells have now provided a platform to untangle the biology of cardiomyopathies. Multiple studies have derived pluripotent stem cells lines from patients with genetic heart diseases. The generation of cardiomyocytes from these cells lines has, for the first time, enable the study of cardiomyopathies using cardiomyocytes harboring patient-specific mutations and their corrected isogenic counterpart. The molecular analyses, functional assays, and drug tests of these lines have led to new molecular insights in the early pathophysiology of left ventricular non-compaction cardiomyopathy (LVNC), hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), and others. The advent of iPS cells offers an exceptional opportunity for creating disease-specific cellular models to investigate their underlying mechanisms and to optimize future therapy through drug and toxicity screening. Thus far, the iPS cell model has improved our understanding of the genetic and molecular pathophysiology of patients with various genetic cardiomyopathies. It is hoped that the new discoveries arising from using these novel platforms for cardiomyopathy research will lead to new diagnostic and therapeutic approaches to prevent and treat these diseases.

  View details for DOI 10.1007/s11886-017-0842-1

  View details for Web of Science ID 000399238000003

  View details for PubMedID 28315121

• Partial Reprogramming of Pluripotent Stem Cell-Derived Cardiomyocytes into Neurons SCIENTIFIC REPORTS Chuang, W., Sharma, A., Shukla, P., Li, G., Mall, M., Rajarajan, K., Abilez, O. J., Hamaguchi, R., Wu, J. C., Wernig, M., Wu, S. M. 2017; 7

  Abstract

  Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs.

  View details for DOI 10.1038/srep44840

  View details for Web of Science ID 000396983300001

  View details for PubMedID 28327614

  View details for PubMedCentralID PMC5361100

• 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

• Strategies for the acquisition of transcriptional and epigenetic information in single cells. Journal of thoracic disease Li, G., Dzilic, E., Flores, N., Shieh, A., Wu, S. M. 2017; 9: S9-S16

  Abstract

  As the basic unit of living organisms, each single cell has unique molecular signatures and functions. Our ability to uncover the transcriptional and epigenetic signature of single cells has been hampered by the lack of tools to explore this area of research. The advent of microfluidic single cell technology along with single cell genome-wide DNA amplification methods had greatly improved our understanding of the expression variation in single cells. Transcriptional expression profile by multiplex qPCR or genome-wide RNA sequencing has enabled us to examine genes expression in single cells in different tissues. With the new tools, the identification of new cellular heterogeneity, novel marker genes, unique subpopulations, and spatial locations of each single cell can be acquired successfully. Epigenetic modifications for each single cell can also be obtained via similar methods. Based on single cell genome sequencing, single cell epigenetic information including histone modifications, DNA methylation, and chromatin accessibility have been explored and provided valuable insights regarding gene regulation and disease prognosis. In this article, we review the development of strategies to obtain single cell transcriptional and epigenetic data. Furthermore, we discuss ways in which single cell studies may help to provide greater understanding of the mechanisms of basic cardiovascular biology that will eventually lead to improvement in our ability to diagnose disease and develop new therapies.

  View details for DOI 10.21037/jtd.2016.08.17

  View details for PubMedID 28446964

• Mammalian Heart Regeneration: The Race to the Finish Line. Circulation research Doppler, S. A., Deutsch, M., Serpooshan, V., Li, G., Dzilic, E., Lange, R., Krane, M., Wu, S. M. 2017; 120 (4): 630-632

  View details for DOI 10.1161/CIRCRESAHA.116.310051

  View details for PubMedID 28209796

• High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells. Science translational medicine Sharma, A., Burridge, P. W., McKeithan, W. L., Serrano, R., Shukla, P., Sayed, N., Churko, J. M., Kitani, T., Wu, H., Holmström, A., Matsa, E., Zhang, Y., Kumar, A., Fan, A. C., Del Álamo, J. C., Wu, S. M., Moslehi, J. J., Mercola, M., Wu, J. C. 2017; 9 (377)

  Abstract

  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

• The relationship between cardiac endothelium and fibroblasts: it's complicated. The Journal of clinical investigation Karra, R. n., Walter, A. O., Wu, S. M. 2017

  Abstract

  Coronary revascularization is an effective means of treating ischemic heart disease; however, current therapeutic revascularization strategies are limited to large caliber vessels. Because the mammalian heart scars following cardiac injury, recent work showing that cardiac fibroblasts can transdifferentiate into new coronary endothelium raises a new and exciting approach to promoting endogenous revascularization following cardiac injury. In this issue of the JCI, He et al. report on their employment of a battery of lineage-tracing tools to address the developmental origins of fibroblasts that give rise to new endothelial cells. Surprisingly, cardiac fibroblasts did not appear to contribute appreciably to regeneration of cardiac endothelium. Instead, cardiac endothelial cells were likely to proliferate and generate new endothelium following injury. As these conclusions diverge from prior findings, additional work will be required to understand the sources that generate cardiac endothelium in new blood vessels after injury. Clarification of the origins of coronary endothelial cells during cardiac repair is essential for identifying improved approaches to revascularizing damaged myocardium in patients with ischemic heart disease.

  View details for PubMedID 28650344

• Bioengineering cardiac constructs using 3D printing Journal of 3D Printing in Medicine Serpooshan, V., Mahmoudi, M., Hu, D. A., Hu, J. B., Wu, S. M. 2017; 1 (2): 1-8

  View details for DOI 10.2217/3dp-2016-0009

• Tissue Engineering of 3D Organotypic Microtissues by Acoustic Assembly. Methods in molecular biology (Clifton, N.J.) Zhu, Y. n., Serpooshan, V. n., Wu, S. n., Demirci, U. n., Chen, P. n., Güven, S. n. 2017

  Abstract

  There is a rapidly growing interest in generation of 3D organotypic microtissues with human physiologically relevant structure, function, and cell population in a wide range of applications including drug screening, in vitro physiological/pathological models, and regenerative medicine. Here, we provide a detailed procedure to generate structurally defined 3D organotypic microtissues from cells or cell spheroids using acoustic waves as a biocompatible and scaffold-free tissue engineering tool.

  View details for PubMedID 28921421

• In vivo rescue of the hematopoietic niche by pluripotent stem cell complementation of defective osteoblast compartments. Stem cells (Dayton, Ohio) Chubb, R. n., Oh, J. n., Riley, A. K., Kimura, T. n., Wu, S. M., Wu, J. Y. 2017

  Abstract

  Bone-forming osteoblasts play critical roles in supporting bone marrow hematopoiesis. Pluripotent stem cells (PSCs), including embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, are capable of differentiating into osteoblasts. To determine the capacity of stem cells needed to rescue aberrant skeletal development and bone marrow hematopoiesis in vivo, we employed a skeletal complementation model. Mice deficient in Runx2, a master transcription factor for osteoblastogenesis, fail to form a mineralized skeleton and bone marrow. Wild-type GFP(+) ES and YFP(+) iPS cells were introduced into Runx2-null blastocyst-stage embryos. We assessed GFP/YFP(+) cell contribution by whole-mount fluorescence and histological analysis and found that the proportion of PSCs in the resulting chimeric embryos is directly correlated with the degree of mineralization in the skull. Moreover, PSC contribution to long bones successfully restored bone marrow hematopoiesis. We validated this finding in a separate model with diphtheria toxin A-mediated ablation of hypertrophic chondrocytes and osteoblasts. Remarkably, chimeric embryos harboring as little as 37.5% wild-type PSCs revealed grossly normal skeletal morphology, suggesting a near-complete rescue of skeletogenesis. In summary, we demonstrate that fractional contribution of PSCs in vivo is sufficient to complement and reconstitute an osteoblast-deficient skeleton and hematopoietic marrow. Further investigation using genetically modified PSCs with conditional loss of gene function in osteoblasts will enable us to address the specific roles of signaling mediators to regulate bone formation and hematopoietic niches in vivo. This article is protected by copyright. All rights reserved.

  View details for PubMedID 28741855

• 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

• Identification of a hybrid myocardial zone in the mammalian heart after birth. Nature communications Tian, X. n., Li, Y. n., He, L. n., Zhang, H. n., Huang, X. n., Liu, Q. n., Pu, W. n., Zhang, L. n., Li, Y. n., Zhao, H. n., Wang, Z. n., Zhu, J. n., Nie, Y. n., Hu, S. n., Sedmera, D. n., Zhong, T. P., Yu, Y. n., Zhang, L. n., Yan, Y. n., Qiao, Z. n., Wang, Q. D., Wu, S. M., Pu, W. T., Anderson, R. H., Zhou, B. n. 2017; 8 (1): 87

  Abstract

  Noncompaction cardiomyopathy is characterized by the presence of extensive trabeculations, which could lead to heart failure and malignant arrhythmias. How trabeculations resolve to form compact myocardium is poorly understood. Elucidation of this process is critical to understanding the pathophysiology of noncompaction disease. Here we use genetic lineage tracing to mark the Nppa(+) or Hey2(+) cardiomyocytes as trabecular and compact components of the ventricular wall. We find that Nppa(+) and Hey2(+) cardiomyocytes, respectively, from the endocardial and epicardial zones of the ventricular wall postnatally. Interposed between these two postnatal layers is a hybrid zone, which is composed of cells derived from both the Nppa(+) and Hey2(+) populations. Inhibition of the fetal Hey2(+) cell contribution to the hybrid zone results in persistence of excessive trabeculations in postnatal heart. Our findings indicate that the expansion of Hey2(+) fetal compact component, and its contribution to the hybrid myocardial zone, are essential for normal formation of the ventricular walls.Fetal trabecular muscles in the heart undergo a poorly described morphogenetic process that results into a solidified compact myocardium after birth. Tian et al. show that cardiomyocytes in the fetal compact layer also contribute to this process, forming a hybrid myocardial zone that is composed of cells derived from both trabecular and compact layers.

  View details for PubMedID 28729659

• Integrative Analysis of PRKAG2 Cardiomyopathy iPS and Microtissue Models Identifies AMPK as a Regulator of Metabolism, Survival, and Fibrosis CELL REPORTS Hinson, J. T., Chopra, A., Lowe, A., Sheng, C. C., Gupta, R. M., Kuppusamy, R., O'Sullivan, J., Rowe, G., Wakimoto, H., Gorham, J., Zhang, K., Musunuru, K., Gerszten, R. E., Wu, S. M., Chen, C. S., Seidman, J. G., Seidman, C. E. 2016; 17 (12): 3292-3304

  Abstract

  AMP-activated protein kinase (AMPK) is a metabolic enzyme that can be activated by nutrient stress or genetic mutations. Missense mutations in the regulatory subunit, PRKAG2, activate AMPK and cause left ventricular hypertrophy, glycogen accumulation, and ventricular pre-excitation. Using human iPS cell models combined with three-dimensional cardiac microtissues, we show that activating PRKAG2 mutations increase microtissue twitch force by enhancing myocyte survival. Integrating RNA sequencing with metabolomics, PRKAG2 mutations that activate AMPK remodeled global metabolism by regulating RNA transcripts to favor glycogen storage and oxidative metabolism instead of glycolysis. As in patients with PRKAG2 cardiomyopathy, iPS cell and mouse models are protected from cardiac fibrosis, and we define a crosstalk between AMPK and post-transcriptional regulation of TGFβ isoform signaling that has implications in fibrotic forms of cardiomyopathy. Our results establish critical connections among metabolic sensing, myocyte survival, and TGFβ signaling.

  View details for DOI 10.1016/j.celrep.2016.11.066

  View details for Web of Science ID 000390895600019

  View details for PubMedID 28009297

• Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos. Cell stem cell Masaki, H., Kato-Itoh, M., Takahashi, Y., Umino, A., Sato, H., Ito, K., Yanagida, A., Nishimura, T., Yamaguchi, T., Hirabayashi, M., Era, T., Loh, K. M., Wu, S. M., Weissman, I. L., Nakauchi, H. 2016; 19 (5): 587-592

  Abstract

  Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17(+) endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17(+) endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.

  View details for DOI 10.1016/j.stem.2016.10.013

  View details for PubMedID 27814480

• iPSC-derived cardiomyocytes reveal abnormal TGF-ß signalling in left ventricular non-compaction cardiomyopathy. Nature cell biology Kodo, K., Ong, S., Jahanbani, F., Termglinchan, V., Hirono, K., Inanloorahatloo, K., Ebert, A. D., Shukla, P., Abilez, O. J., Churko, J. M., Karakikes, I., Jung, G., Ichida, F., Wu, S. M., Snyder, M. P., Bernstein, D., Wu, J. C. 2016; 18 (10): 1031-1042

  Abstract

  Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and this was associated with perturbed transforming growth factor beta (TGF-β) signalling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGF-β signalling. TBX20 regulates the expression of TGF-β signalling modifiers including one known to be a genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGF-β signalling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC.

  View details for DOI 10.1038/ncb3411

  View details for PubMedID 27642787

• Endocardium Minimally Contributes to Coronary Endothelium in the Embryonic Ventricular Free Walls CIRCULATION RESEARCH Zhang, H., Pu, W., Li, G., Huang, X., He, L., Tian, X., Liu, Q., Zhang, L., Wu, S. M., Sucov, H. M., Zhou, B. 2016; 118 (12): 1880-?

  Abstract

  There is persistent uncertainty regarding the developmental origins of coronary vessels, with two principal sources suggested as ventricular endocardium or sinus venosus (SV). These two proposed origins implicate fundamentally distinct mechanisms of vessel formation. Resolution of this controversy is critical for deciphering the programs that result in the formation of coronary vessels, and has implications for research on therapeutic angiogenesis.To resolve the controversy over the developmental origin of coronary vessels.We first generatedNfatc1-CreandNfatc1-Drelineage tracers for endocardium labeling. We found that Nfatc1 recombinases also label a significant portion of SV endothelial cells in addition to endocardium. Therefore, restricted endocardial lineage tracing requires a specific marker that distinguishes endocardium from SV. By single cell gene expression analysis, we identified a novel endocardial gene natriuretic peptide receptor 3 (Npr3). Npr3 is expressed in the entirety of the endocardium but not in the SV. Genetic lineage tracing based onNpr3-CreERshowed that endocardium contributes to a minority of coronary vessels in the free walls of embryonic heart. Intersectional genetic lineage tracing experiments demonstrated that endocardium minimally contributes to coronary endothelium in the embryonic ventricular free walls.Our study suggested that SV, but not endocardium, is the major origin for coronary endothelium in the embryonic ventricular free walls. This work thus resolves the recent controversy over the developmental origin of coronary endothelium, providing the basis for studying coronary vessel formation and regeneration after injury.

  View details for DOI 10.1161/CIRCRESAHA.116.308749

  View details for Web of Science ID 000377885100009

  View details for PubMedID 27056912

• Distilling complexity to advance cardiac tissue engineering SCIENCE TRANSLATIONAL MEDICINE Ogle, B. M., Bursac, N., Domian, I., Huang, N. F., Menasche, P., Murry, C. E., Pruitt, B., Radisic, M., Wu, J. C., Wu, S. M., Zhang, J., Zimmermann, W., Vunjak-Novakovic, G. 2016; 8 (342)

  Abstract

  The promise of cardiac tissue engineering is in the ability to recapitulate in vitro the functional aspects of a healthy heart and disease pathology as well as to design replacement muscle for clinical therapy. Parts of this promise have been realized; others have not. In a meeting of scientists in this field, five central challenges or "big questions" were articulated that, if addressed, could substantially advance the current state of the art in modeling heart disease and realizing heart repair.

  View details for DOI 10.1126/scitranslmed.aad2304

  View details for Web of Science ID 000377443800001

  View details for PubMedID 27280684

• Regenerative Medicine: Potential Mechanisms of Cardiac Recovery in Takotsubo Cardiomyopathy. Current treatment options in cardiovascular medicine Chang, A. Y., Kittle, J. T., Wu, S. M. 2016; 18 (3): 20-?

  Abstract

  Takotsubo cardiomyopathy is an increasingly reported cause of acute chest pain and acute heart failure and is often associated with significant hemodynamic compromise. The illness is remarkable for the reversibility in systolic dysfunction seen in the disease course. While the pathophysiology of takotsubo syndrome is not completely elucidated, research suggests the presence of a cytoprotective process that allows the myocardium to recover following the inciting insult. Here, we summarize molecular and histologic studies exploring the response to injury in takotsubo disease and provide some discussion on how they may contribute to further investigations in cardiac recovery and regeneration.

  View details for DOI 10.1007/s11936-016-0443-0

  View details for PubMedID 26874708

• Cardioprotective Actions of TGF beta RI Inhibition Through Stimulating Autocrine/Paracrine of Survivin and Inhibiting Wnt in Cardiac Progenitors STEM CELLS Ho, Y., Tsai, W., Lin, F., Huang, W., Lin, L., Wu, S. M., Liu, Y., Chen, W. 2016; 34 (2): 445-455

  Abstract

  Heart failure due to myocardial infarction (MI) is a major cause of morbidity and mortality in the world. We found previously that A83-01, a TGFβRI inhibitor, could facilitate cardiac repair in post-MI mice and induce the expansion of a Nkx2.5+ cardiomyoblast population. The present study aimed to investigate the key autocrine/paracrine factors regulated by A83-01 in the injured heart and the mechanism of cardioprotection by this molecule. Using a previously described transgenic Nkx2.5 enhancer-GFP reporter mice, we isolated cardiac progenitor cells (CPC) including Nkx2.5-GFP+ (Nkx2.5+), sca1+ and Nkx2.5+/sca1+ cells. A83-01 was found to induce proliferation of these three subpopulations mainly through increasing Birc5 expression in the MEK/ERK-dependent pathway. Survivin, encoded by Birc5, could also directly proliferate Nkx2.5+ cells and enhance cultured cardiomyocytes viability. A83-01 could also reverse the down-regulation of Birc5 in post-injured mice hearts (n=6) to expand CPCs. Moreover, the increased Wnt3a in post-injured hearts could decrease CPCs, which could be reversed by A83-01 via inhibiting Fzd6 and WISP1 expressions in CPCs. Next, we used inducible αMHC-cre/mTmG mice to label cardiomyocytes with GFP and non-myocytes with RFP. We found A83-01 preserved more GFP+ myocytes (68.6±3.1% vs 80.9±3.0%; P<0.05, n=6) and fewer renewed RFP+ myocytes (0.026±0.005 vs 0.062±0.008%; P<0.05, n=6) in parallel with less cardiac fibrosis in isoprenaline-injected mice treated with A83-01. TGFβRI inhibition in an injured adult heart could both stimulate the autocrine/paracrine activity of survivin and inhibit Wnt in CPCs to mediate cardioprotection and improve cardiac function. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/stem.2216

  View details for Web of Science ID 000370353200017

  View details for PubMedID 26418219

• Harnessing the Induction of Cardiomyocyte Proliferation for Cardiac Regenerative Medicine. Current treatment options in cardiovascular medicine Sharma, A., Zhang, Y., Wu, S. M. 2015; 17 (10): 404-?

  Abstract

  Adult human cardiomyocytes are terminally differentiated and have limited capacity for cell division. Hence, they are not naturally replaced following ischemic injury to the heart. As such, cardiac function is often permanently compromised after an event such as myocardial infarction. In recent years, investigators have focused intensively on ways to reactivate cardiomyocyte mitotic activity in both in vitro cell culture systems and in vivo animal models. In parallel, advances in stem cell biology have allowed for the mass production of patient-specific human cardiomyocytes from human-induced pluripotent stem cells. These cells can be produced via chemically defined differentiation of human pluripotent stem cells in a matter of weeks and could theoretically be utilized directly for therapeutic purposes to replace damaged myocardium. However, stem cell-derived cardiomyocytes, like their adult counterparts, are post-mitotic and incapable of large-scale expansion after reaching a certain stage of in vitro differentiation. Due to this shared characteristic, these stem cell-derived cardiomyocytes may provide a platform for studying genes, pathways, and small molecules that induce cell cycle reentry and proliferation of human cardiomyocytes. Ultimately, the discovery of novel mechanisms or pathways to induce human cardiomyocyte proliferation should improve our ability to regenerate adult cardiomyocytes and help restore cardiac function following injury.

  View details for DOI 10.1007/s11936-015-0404-z

  View details for PubMedID 26324824

• Members Only: Hypoxia-Induced Cell-Cycle Activation in Cardiomyocytes. Cell metabolism Sharma, A., Wu, S. M. 2015; 22 (3): 365-366

  Abstract

  A low level of cardiomyocyte turnover occurs in the adult mammalian heart, but the source of these new cells remains unknown. Kimura et al., 2015 utilized a novel hypoxia-induced fate mapping system to identify a rare population of adult cardiomyocytes undergoing cell-cycle entry and expansion in healthy adult hearts and following ischemic injury.

  View details for DOI 10.1016/j.cmet.2015.08.004

  View details for PubMedID 26331604

• Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes PLOS ONE Tarnawski, L., Xian, X., Monnerat, G., Macaulay, I. C., Malan, D., Borgman, A., Wu, S. M., Fleischmann, B. K., Jovinge, S. 2015; 10 (8)

  Abstract

  In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.

  View details for DOI 10.1371/journal.pone.0135880

  View details for Web of Science ID 000360435500010

• Fetal Mammalian Heart Generates a Robust Compensatory Response to Cell Loss. Circulation Sturzu, A. C., Rajarajan, K., Passer, D., Plonowska, K., Riley, A., Tan, T. C., Sharma, A., Xu, A. F., Engels, M. C., Feistritzer, R., Li, G., Selig, M. K., Geissler, R., Robertson, K. D., Scherrer-Crosbie, M., Domian, I. J., Wu, S. M. 2015; 132 (2): 109-121

  Abstract

  -Heart development is tightly regulated by signaling events acting upon a defined number of progenitor and differentiated cardiac cells. While loss-of-function of these signaling pathways leads to congenital malformation, the consequences of cardiac progenitor cell (CPC) or embryonic cardiomyocyte loss are less clear. In this study, we tested the hypothesis that embryonic mouse hearts exhibit a robust mechanism for regeneration following extensive cell loss.-By combining a conditional cell ablation approach with a novel blastocyst complementation strategy, we generated murine embryos that exhibit a full spectrum of CPC or cardiomyocyte ablation. Remarkably, ablation of up to 60% of CPCs at embryonic day 7.5 was well-tolerated and permitted embryo survival. Ablation of embryonic cardiomyocytes to a similar degree (50-60%) at embryonic day 9.0 could be fully rescued by residual myocytes with no obvious adult cardiac functional deficit. In both ablation models, an increase in cardiomyocyte proliferation rate was detected and accounted for at least some of the rapid recovery of myocardial cellularity and heart size.-Our study defines the threshold for cell loss in the embryonic mammalian heart and reveals a robust cardiomyocyte compensatory response that sustains normal fetal development.

  View details for DOI 10.1161/CIRCULATIONAHA.114.011490

  View details for PubMedID 25995316

• Identification of cardiovascular lineage descendants at single-cell resolution. Development Li, G., Plonowska, K., Kuppusamy, R., Sturzu, A., Wu, S. M. 2015; 142 (5): 846-857

  Abstract

  The transcriptional profiles of cardiac cells derived from murine embryos and from mouse embryonic stem cells (mESCs) have primarily been studied within a cell population. However, the characterization of gene expression in these cells at a single-cell level might demonstrate unique variations that cannot be appreciated within a cell pool. In this study, we aimed to establish a single-cell quantitative PCR platform and perform side-by-side comparison between cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) derived from mESCs and mouse embryos. We first generated a reference map for cardiovascular single cells through quantifying lineage-defining genes for CPCs, CMs, smooth muscle cells (SMCs), endothelial cells (EDCs), fibroblasts and mESCs. This panel was then applied against single embryonic day 10.5 heart cells to demonstrate its ability to identify each endocardial cell and chamber-specific CM. In addition, we compared the gene expression profile of embryo- and mESC-derived CPCs and CMs at different developmental stages and showed that mESC-derived CMs are phenotypically similar to embryo-derived CMs up to the neonatal stage. Furthermore, we showed that single-cell expression assays coupled with time-lapse microscopy can resolve the identity and the lineage relationships between progenies of single cultured CPCs. With this approach, we found that mESC-derived Nkx2-5(+) CPCs preferentially become SMCs or CMs, whereas single embryo-derived Nkx2-5(+) CPCs represent two phenotypically distinct subpopulations that can become either EDCs or CMs. These results demonstrate that multiplex gene expression analysis in single cells is a powerful tool for examining the unique behaviors of individual embryo- or mESC-derived cardiac cells.

  View details for DOI 10.1242/dev.116897

  View details for PubMedID 25633351

• Small RNAs make big impact in cardiac repair. Circulation research Krane, M., Deutsch, M., Doppler, S., Lange, R., Wu, S. M. 2015; 116 (3): 393-395

  View details for DOI 10.1161/CIRCRESAHA.114.305676

  View details for PubMedID 25634967

  View details for PubMedCentralID PMC4313563

• 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

• Pharmacological inhibition of TGFß receptor improves Nkx2.5 cardiomyoblast-mediated regeneration. Cardiovascular research Chen, W., Liu, Y., Ho, Y., Wu, S. M. 2015; 105 (1): 44-54

  Abstract

  Our previous study found that A83-01, a small molecule type 1 TGFβ receptor inhibitor, could induce proliferation of postnatal Nkx2.5(+) cardiomyoblasts in vitro and enhance their cardiomyogenic differentiation. The present study addresses whether A83-01 treatment in vivo could increase cardiomyogenesis and improve cardiac function after myocardial infarction through an Nkx2.5(+) cardiomyoblast-dependent process.To determine the effect of A83-01 on the number of Nkx2.5(+) cardiomyoblasts in the heart after myocardial injury, we treated transgenic Nkx2.5 enhancer-GFP reporter mice for 7 days with either A83-01 or DMSO and measured the number of GFP(+) cardiomyoblasts in the heart at 1 week after injury by flow cytometry. To determine the degree of new cardiomyocyte formation after myocardial injury and the effect of A83-01 in this process, we employed inducible Nkx2.5 enhancer-Cre transgenic mice to lineage label postnatal Nkx2.5(+) cardiomyoblasts and their differentiated progenies after myocardial injury. We also examined the cardiac function of each animal by intracardiac haemodynamic measurements. We found that A83-01 treatment significantly increased the number of Nkx2.5(+) cardiomyoblasts at baseline and after myocardial injury, resulting in an increase in newly formed cardiomyocytes. Finally, we showed that A83-01 treatment significantly improved ventricular elastance and stroke work, leading to improved contractility after injury.Pharmacological inhibition of TGFβ signalling improved cardiac function in injured mice and promoted the expansion and cardiomyogenic differentiation of Nkx2.5(+) cardiomyoblasts. Direct modulation of resident cardiomyoblasts in vivo may be a promising strategy to enhance therapeutic cardiac regeneration.

  View details for DOI 10.1093/cvr/cvu229

  View details for PubMedID 25362681

• Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation. Journal of visualized experiments : JoVE Sharma, A., Li, G., Rajarajan, K., Hamaguchi, R., Burridge, P. W., Wu, S. M. 2015

  Abstract

  Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/β-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

  View details for DOI 10.3791/52628

  View details for PubMedID 25867738

• Comparing mouse and human pluripotentstem cell derived cardiac cells: both systemshave advantages for pharmacological and toxicological screening. Journal of pharmacological and toxicological methods Lagerqvist, E. L., Finnin, B. A., Elliott, D. n., Anderson, D. n., Wu, S. M., Pouton, C. W., Haynes, J. M. 2015

  Abstract

  Pluripotent stem cells offer an unparalleled opportunity to investigate cardiac physiology, pharmacology, toxicology and pathophysiology. In this paper we describe the use of both mouse (Nkx2-5(eGFP/w)) and human (NKX2-5(eGFP/w)) pluripotent stem cell reporter lines, differentiated toward cardiac lineage, for live single cell high acquisition rate calcium imaging. We also assess the potential of NKX2-5(eGFP/w) cardiac lineage cells for use toxicological screening as well as establish their sensitivity to a shift between low and high oxygen environments. Differentiated mouse Nkx2-5(eGFP/w) cells demonstrated a wide range of spontaneous oscillation rates that could be reduced by ryanodine (10 μM), thapsigargin (1 μM) and ZD7288 (10μM). In contrast human NKX2-5(eGFP/w) cell activity was only reduced by thapsigargin (1 μM). Human cells were also sensitive to the addition of trastuzumab and doxorubicin as well as a low oxygen environment. We suggest that the human NKX2-5(eGFP/w) cells are less suitable for studies of compounds affecting cardiac pacemaker activity than mouse Nkx2-5(eGFP/w) cells, but are very suitable for cardiac toxicity studies.

  View details for PubMedID 25957031

• Patching up broken hearts: cardiac cell therapy gets a bioengineered boost. Cell stem cell Serpooshan, V., Wu, S. M. 2014; 15 (6): 671-673

  Abstract

  Preclinical and clinical studies for cardiac cell therapy have only seen moderate success due to poor engraftment and survival of transplanted cells. In this issue of Cell Stem Cell, Ye et al. (2014) employ a growth-factor-loaded fibrin patch and show improved cardiovascular cell survival after cell transplantation into a porcine model of ischemia reperfusion.

  View details for DOI 10.1016/j.stem.2014.11.008

  View details for PubMedID 25479741

• Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus b3-induced myocarditis and antiviral drug screening platform. Circulation research Sharma, A., Marceau, C., Hamaguchi, R., Burridge, P. W., Rajarajan, K., Churko, J. M., Wu, H., Sallam, K. I., Matsa, E., Sturzu, A. C., Che, Y., Ebert, A., Diecke, S., Liang, P., Red-Horse, K., Carette, J. E., Wu, S. M., Wu, J. C. 2014; 115 (6): 556-566

  Abstract

  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.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.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 interferonβ1, 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 interferonβ1 treatment.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.

  View details for DOI 10.1161/CIRCRESAHA.115.303810

  View details for PubMedID 25015077

  View details for PubMedCentralID PMC4149868

• Somatic Cell Reprogramming into Cardiovascular Lineages JOURNAL OF CARDIOVASCULAR PHARMACOLOGY AND THERAPEUTICS Chen, J. X., Plonowska, K., Wu, S. M. 2014; 19 (4): 340-349

  Abstract

  Ischemic cardiac disease is the leading cause of death in the developed world. The inability of the adult mammalian heart to adequately repair itself has motivated stem cell researchers to explore various strategies to regenerate cardiomyocytes after myocardial infarction. Over the past century, progressive gains in our knowledge about the cellular mechanisms governing fate determination have led to recent advances in cellular reprogramming. The identification of specific factors capable of inducing pluripotent phenotype in somatic cells as well as factors that can directly reprogram somatic cells into cardiomyocytes suggests the potential for these approaches to translate into clinical therapies in the future. Although conceptually appealing, the field of cell lineage reprogramming is in its infancy, and further research will be needed to improve the efficiency of the reprogramming process and the fidelity of the reprogrammed cells to their in vivo counterpart.

  View details for DOI 10.1177/1074248414527641

  View details for Web of Science ID 000338394200003

• Insulin-like growth factor promotes cardiac lineage induction in vitro by selective expansion of early mesoderm. Stem cells Engels, M. C., Rajarajan, K., Feistritzer, R., Sharma, A., Nielsen, U. B., Schalij, M. J., de Vries, A. A., Pijnappels, D. A., Wu, S. M. 2014; 32 (6): 1493-1502

  Abstract

  A thorough understanding of the developmental signals that direct pluripotent stem cells (PSCs) towards a cardiac fate is essential for translational applications in disease modeling and therapy. We screened a panel of 44 cytokines/signaling molecules for their ability to enhance Nkx2.5(+) cardiac progenitor cell (CPC) formation during in vitro embryonic stem cell (ESC) differentiation. Treatment of murine ESCs with insulin or insulin-like growth factors (IGF1/2) during early differentiation increased mesodermal cell proliferation and, consequently, CPC formation. Furthermore, we show that downstream mediators of IGF signaling (e.g. phospho-Akt and mTOR) are required for this effect. These data support a novel role for IGF family ligands to expand the developing mesoderm and promote cardiac differentiation. Insulin or IGF treatment could provide an effective strategy to increase the PSC-based generation of CPCs and cardiomyocytes for applications in regenerative medicine. Stem Cells 2014.

  View details for DOI 10.1002/stem.1660

  View details for PubMedID 24496962

• Telocytes in human heart valves JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Yang, Y., Sun, W., Wu, S. M., Xiao, J., Kong, X. 2014; 18 (5): 759-765

  Abstract

  Valve interstitial cells (VICs) are responsible for maintaining the structural integrity and dynamic behaviour of the valve. Telocytes (TCs), a peculiar type of interstitial cells, have been recently identified by Popescu's group in epicardium, myocardium and endocardium (visit www.telocytes.com). The presence of TCs has been identified in atria, ventricles and many other tissues and organ, but not yet in heart valves. We used transmission electron microscopy and immunofluorescence methods (double labelling for CD34 and c-kit, or vimentin, or PDGF Receptor-β) to provide evidence for the existence of TCs in human heart valves, including mitral valve, tricuspid valve and aortic valve. TCs are found in both apex and base of heart valves, with a similar density of 27-28 cells/mm(2) in mitral valve, tricuspid valve and aortic valve. Since TCs are known for the participation in regeneration or repair biological processes, it remains to be determined how TCs contributes to the valve attempts to re-establish normal structure and function following injury, especially a complex junction was found between TCs and a putative stem (progenitor) cell.

  View details for DOI 10.1111/jcmm.12285

  View details for Web of Science ID 000335861500002

  View details for PubMedID 24674389

• Myeloid zinc finger 1 (mzf1) differentially modulates murine cardiogenesis by interacting with an nkx2.5 cardiac enhancer. PloS one Doppler, S. A., Werner, A., Barz, M., Lahm, H., Deutsch, M., Dreßen, M., Schiemann, M., Voss, B., Gregoire, S., Kuppusamy, R., Wu, S. M., Lange, R., Krane, M. 2014; 9 (12): e113775

  Abstract

  Vertebrate heart development is strictly regulated by temporal and spatial expression of growth and transcription factors (TFs). We analyzed nine TFs, selected by in silico analysis of an Nkx2.5 enhancer, for their ability to transactivate the respective enhancer element that drives, specifically, expression of genes in cardiac progenitor cells (CPCs). Mzf1 showed significant activity in reporter assays and bound directly to the Nkx2.5 cardiac enhancer (Nkx2.5 CE) during murine ES cell differentiation. While Mzf1 is established as a hematopoietic TF, its ability to regulate cardiogenesis is completely unknown. Mzf1 expression was significantly enriched in CPCs from in vitro differentiated ES cells and in mouse embryonic hearts. To examine the effect of Mzf1 overexpression on CPC formation, we generated a double transgenic, inducible, tetOMzf1-Nkx2.5 CE eGFP ES line. During in vitro differentiation an early and continuous Mzf1 overexpression inhibited CPC formation and cardiac gene expression. A late Mzf1 overexpression, coincident with a second physiological peak of Mzf1 expression, resulted in enhanced cardiogenesis. These findings implicate a novel, temporal-specific role of Mzf1 in embryonic heart development. Thereby we add another piece of puzzle in understanding the complex mechanisms of vertebrate cardiac development and progenitor cell differentiation. Consequently, this knowledge will be of critical importance to guide efficient cardiac regenerative strategies and to gain further insights into the molecular basis of congenital heart malformations.

  View details for DOI 10.1371/journal.pone.0113775

  View details for PubMedID 25436607

  View details for PubMedCentralID PMC4249966

• Patching Up Broken Hearts: Cardiac Cell Therapy Gets a Bioengineered Boost Cell Stem Cell Serpooshan, V., Wu, S. M. 2014; 15 (6): 671–673

  Abstract

  Preclinical and clinical studies for cardiac cell therapy have only seen moderate success due to poor engraftment and survival of transplanted cells. In this issue of Cell Stem Cell, Ye et al. (2014) employ a growth-factor-loaded fibrin patch and show improved cardiovascular cell survival after cell transplantation into a porcine model of ischemia reperfusion.

  View details for DOI 10.1016/j.stem.2014.11.008

• Induced pluripotent stem cell-derived cardiomyocytes for cardiovascular disease modeling and drug screening STEM CELL RESEARCH & THERAPY Sharma, A., Wu, J. C., Wu, S. M. 2013; 4

  View details for DOI 10.1186/scrt380

  View details for Web of Science ID 000329186900001

  View details for PubMedID 24476344

• Screening drug-induced arrhythmia events using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays. Circulation Navarrete, E. G., Liang, P., Lan, F., Sanchez-Freire, V., Simmons, C., Gong, T., Sharma, A., Burridge, P. W., Patlolla, B., Lee, A. S., Wu, H., Beygui, R. E., Wu, S. M., Robbins, R. C., Bers, D. M., Wu, J. C. 2013; 128 (11): S3-13

  Abstract

  Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls.We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

  View details for DOI 10.1161/CIRCULATIONAHA.112.000570

  View details for PubMedID 24030418

  View details for PubMedCentralID PMC3855862

• Meta-Analysis of Stem Cell Therapy in Chronic Ischemic Cardiomyopathy AMERICAN JOURNAL OF CARDIOLOGY Kandala, J., Upadhyay, G. A., Pokushalov, E., Wu, S., Drachman, D. E., Singh, J. P. 2013; 112 (2): 217-225

  Abstract

  Studies investigating bone marrow stem cell therapy (BMSCT) in patients with chronic ischemic cardiomyopathy have yielded mixed results. A meta-analysis of randomized controlled trials of BMSCT in patients with chronic ischemic cardiomyopathy was undertaken to assess its efficacy and the best route of administration. The MEDLINE, Embase, Cumulative Index to Nursing & Allied Health Literature, and Cochrane Library databases were searched through April 2012 for randomized controlled trials that investigated the impact of BMSCT and its route of administration on left ventricular (LV) function in patients with chronic ischemic cardiomyopathy and systolic dysfunction. Of the 226 reports identified, 10 randomized controlled trials including 519 patients (average LV ejection fraction [LVEF] at baseline 32 ± 7%) were included in the analysis. On the basis of a random-effects model, BMSCT improved the LVEF at 6 months by 4.48% (95% confidence interval [CI] 2.43% to 6.53%, p = 0.0001). A greater improvement in the LVEF was seen with intramyocardial injection compared with intracoronary infusion (5.13% [95% CI 3.17% to 7.10%], p <0.00001, vs 2.32% [95% CI -2.06% to 6.70%], p = 0.30). Overall, there were reductions in LV end-systolic volume of -20.64 ml (95% CI -33.21 to -8.07, p = 0.001) and LV end-diastolic volume of -16.71 ml (95% CI -31.36 to -2.06, p = 0.03). In conclusion, stem cell therapy may improve LVEF and favorably remodel the heart in patients with chronic ischemic cardiomyopathy. On the basis of current limited data, intramyocardial injection may be superior to intracoronary infusion in patients with LV systolic dysfunction.

  View details for DOI 10.1016/j.amjcard.2013.03.021

  View details for Web of Science ID 000322206500013

  View details for PubMedID 23623290

• A83-01, a TGF beta RI inhibitor, can proliferate adult cardiac progenitor cells and improve cardiac contractility of myocardial infarcted mice Chen, W., Liu, Y., Ho, Y., Wu, S. M. ACTA PHARMACOLOGICA SINICA. 2013: 57–57

  View details for Web of Science ID 000322051500249

• Autophagy - the friendly fire in endothelial cell regeneration. Focus on "Autophagy in endothelial progenitor cells is cytoprotective in hypoxic conditions" AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Sharma, A., Wu, S. M. 2013; 304 (7): C614-C616

  View details for DOI 10.1152/ajpcell.00046.2013

  View details for Web of Science ID 000316995700003

  View details for PubMedID 23407881

  View details for PubMedCentralID PMC3625717

• At a Crossroad Cell Therapy for Cardiac Repair CIRCULATION RESEARCH Deutsch, M., Sturzu, A., Wu, S. M. 2013; 112 (6): 884-890

  View details for DOI 10.1161/CIRCRESAHA.112.275974

  View details for Web of Science ID 000316189900005

  View details for PubMedID 23493304

  View details for PubMedCentralID PMC3614339

• Essential and Unexpected Role of Yin Yang 1 to Promote Mesodermal Cardiac Differentiation CIRCULATION RESEARCH Gregoire, S., Karra, R., Passer, D., Deutsch, M., Krane, M., Feistritzer, R., Sturzu, A., Domian, I., Saga, Y., Wu, S. M. 2013; 112 (6): 900-U104

  Abstract

  Cardiogenesis is regulated by a complex interplay between transcription factors. However, little is known about how these interactions regulate the transition from mesodermal precursors to cardiac progenitor cells (CPCs). Objective: To identify novel regulators of mesodermal cardiac lineage commitment.We performed a bioinformatic-based transcription factor binding site analysis on upstream promoter regions of genes that are enriched in embryonic stem cell-derived CPCs. From 32 candidate transcription factors screened, we found that Yin Yang 1 (YY1), a repressor of sarcomeric gene expression, is present in CPCs in vivo. Interestingly, we uncovered the ability of YY1 to transcriptionally activate Nkx2.5, a key marker of early cardiogenic commitment. YY1 regulates Nkx2.5 expression via a 2.1-kb cardiac-specific enhancer as demonstrated by in vitro luciferase-based assays, in vivo chromatin immunoprecipitation, and genome-wide sequencing analysis. Furthermore, the ability of YY1 to activate Nkx2.5 expression depends on its cooperative interaction with Gata4 at a nearby chromatin. Cardiac mesoderm-specific loss-of-function of YY1 resulted in early embryonic lethality. This was corroborated in vitro by embryonic stem cell-based assays in which we showed that the overexpression of YY1 enhanced the cardiogenic differentiation of embryonic stem cells into CPCs.These results demonstrate an essential and unexpected role for YY1 to promote cardiogenesis as a transcriptional activator of Nkx2.5 and other CPC-enriched genes.

  View details for DOI 10.1161/CIRCRESAHA.113.259259

  View details for Web of Science ID 000316189900007

  View details for PubMedID 23307821

  View details for PubMedCentralID PMC3629954

• Of fish and men: clonal lineage analysis identifies divergence in myocardial development. Circulation research Sharma, A., Wu, S. M. 2013; 112 (4): 583-585

  View details for DOI 10.1161/CIRCRESAHA.113.300964

  View details for PubMedID 23410876

  View details for PubMedCentralID PMC3600367

• iPS Cell Modeling of Cardiometabolic Diseases JOURNAL OF CARDIOVASCULAR TRANSLATIONAL RESEARCH Nakamura, K., Hirano, K., Wu, S. M. 2013; 6 (1): 46-53

  Abstract

  Cardiometabolic diseases encompass simple monogenic enzyme deficiencies with well-established pathogenesis and clinical outcomes to complex polygenic diseases such as the cardiometabolic syndrome. The limited availability of relevant human cell types such as cardiomyocytes has hampered our ability to adequately model and study pathways or drugs relevant to these diseases in the heart. The recent discovery of induced pluripotent stem (iPS) cell technology now offers a powerful opportunity to establish translational platforms for cardiac disease modeling, drug discovery, and pre-clinical testing. In this article, we discuss the excitement and challenges of modeling cardiometabolic diseases using iPS cell and their potential to revolutionize translational research.

  View details for DOI 10.1007/s12265-012-9413-4

  View details for Web of Science ID 000313657700006

  View details for PubMedID 23070616

  View details for PubMedCentralID PMC3547131

• Early cardiac development: a view from stem cells to embryos CARDIOVASCULAR RESEARCH van Vliet, P., Wu, S. M., Zaffran, S., Puceat, M. 2012; 96 (3): 352-362

  Abstract

  From the 1920s, early cardiac development has been studied in chick and, later, in mouse embryos in order to understand the first cell fate decisions that drive specification and determination of the endocardium, myocardium, and epicardium. More recently, mouse and human embryonic stem cells (ESCs) have demonstrated faithful recapitulation of early cardiogenesis and have contributed significantly to this research over the past few decades. Derived almost 15 years ago, human ESCs have provided a unique developmental model for understanding the genetic and epigenetic regulation of early human cardiogenesis. Here, we review the biological concepts underlying cell fate decisions during early cardiogenesis in model organisms and ESCs. We draw upon both pioneering and recent studies and highlight the continued role for in vitro stem cells in cardiac developmental biology.

  View details for DOI 10.1093/cvr/cvs270

  View details for Web of Science ID 000311306800006

  View details for PubMedID 22893679

  View details for PubMedCentralID PMC3500045

• Reprogramming the Beat Kicking It Up a Notch CIRCULATION Wu, S. M., Milan, D. J. 2012; 126 (9): 1009-1011

  View details for DOI 10.1161/CIRCULATIONAHA.112.126482

  View details for Web of Science ID 000308355100008

  View details for PubMedID 22837161

• Inefficient Reprogramming of Fibroblasts into Cardiomyocytes Using Gata4, Mef2c, and Tbx5 CIRCULATION RESEARCH Chen, J. X., Krane, M., Deutsch, M., Wang, L., Rav-Acha, M., Gregoire, S., Engels, M. C., Rajarajan, K., Karra, R., Abel, E. D., Wu, J. C., Milan, D., Wu, S. M. 2012; 111 (1): 50-55

  Abstract

  Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown.To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT).We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes.Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy.

  View details for DOI 10.1161/CIRCRESAHA.112.270264

  View details for Web of Science ID 000306061700012

  View details for PubMedID 22581928

  View details for PubMedCentralID PMC3390172

• Small molecule regulators of postnatal Nkx2.5 cardiomyoblast proliferation and differentiation JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Chen, W., Wu, S. M. 2012; 16 (5): 961-965

  Abstract

  While recent data have supported the capacity for a neonatal heart to undergo cardiomyogenesis, it is unclear whether these new cardiomyocytes arise from an immature cardiomyoblast population or from the division of mature cardiomyocytes. By following the expression of enhanced Green Fluorescent Protein (eGFP) in an Nkx2.5 enhancer-eGFP transgenic mice, we have identified a population of immature cells that can undergo cardiomyogenic as well as smooth muscle cell differentiation in the neonatal heart. Here, we examined growth factors and small molecule regulators that potentially regulate the proliferation and cardiomyogenic versus smooth muscle cell differentiation of neonatal Nkx2.5-GFP (+) cells in vitro. We found that A83-01 (A83), an inhibitor of TGF-βRI, was able to induce an expansion of neonatal Nkx2.5-eGFP (+) cells. In addition, the ability of A83 to expand eGFP (+) cells in culture was dependent on signalling from the mitogen-activated protein kinase kinase (MEK) as treatment with a MEK inhibitor, PD0325901, abolished this effect. On the other hand, activation of neonatal Nkx2.5-eGFP (+) cells with TGF-β1, but not activin A nor BMP2, led to smooth muscle cell differentiation, an effect that can be reversed by treatment with A83. In summary, small molecule inhibition of TGF-β signalling may be a promising strategy to induce the expansion of a rare population of postnatal cardiomyoblasts.

  View details for DOI 10.1111/j.1582-4934.2011.01513.x

  View details for Web of Science ID 000303239500002

  View details for PubMedID 22212626

  View details for PubMedCentralID PMC3325363

• Putting the Pieces Together: Stem Cells and The Quest to Heal A Broken Heart CardioSource World News Wu, S. M., Singh, J. P. 2012; 12: 22-27
• Induced pluripotent stem cell modeling of complex genetic diseases. Drug discovery today. Disease models Hinson, J. T., Nakamura, K., Wu, S. M. 2012; 9 (4): e147-e152

  Abstract

  The study of complex disease genetics by genome-wide association studies (GWAS) has led to hundreds of genomic loci associated with disease traits in humans. However, the functional consequences of most loci are largely undefined. We discuss here the potential for human induced pluripotent stem (iPS) cells to bridge the gap between genetic variant and mechanisms of complex disease. We also highlight specific diseases and the roadblocks that must be overcome before iPS cell technology can be widely adopted for complex disease modeling.

  View details for DOI 10.1016/j.ddmod.2012.04.002

  View details for PubMedID 23690830

  View details for PubMedCentralID PMC3653342

• A Brief Primer on the Development of the Heart Heart Failure, 2nd Ed. Gregoire S, Wu SM 2012: Chapter 1
• Epigenetic mechanisms in cardiac development and disease ACTA BIOCHIMICA ET BIOPHYSICA SINICA Vallaster, M., Vallaster, C. D., Wu, S. M. 2012; 44 (1): 92-102

  Abstract

  During mammalian development, cardiac specification and ultimately lineage commitment to a specific cardiac cell type is accomplished by the action of specific transcription factors (TFs) and their meticulous control on an epigenetic level. In this review, we detail how cardiac-specific TFs function in concert with nucleosome remodeling and histone-modifying enzymes to regulate a diverse network of genes required for processes such as cell growth and proliferation, or epithelial to mesenchymal transition (EMT), for instance. We provide examples of how several cardiac TFs, such as Nkx2.5, WHSC1, Tbx5, and Tbx1, which are associated with developmental and congenital heart defects, are required for the recruitment of histone modifiers, such as Jarid2, p300, and Ash2l, and components of ATP-dependent remodeling enzymes like Brg1, Baf60c, and Baf180. Binding of these TFs to their respective sites at cardiac genes coincides with a distinct pattern of histone marks, indicating that the precise regulation of cardiac gene networks is orchestrated by interactions between TFs and epigenetic modifiers. Furthermore, we speculate that an epigenetic signature, comprised of TF occupancy, histone modifications, and overall chromatin organization, is an underlying mechanism that governs cardiac morphogenesis and disease.

  View details for DOI 10.1093/abbs/gmr090

  View details for Web of Science ID 000298386700010

  View details for PubMedID 22194017

  View details for PubMedCentralID PMC3244653

• Reprogramming of mouse, rat, pig, and human fibroblasts into iPS cells. Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] Rajarajan, K., Engels, M. C., Wu, S. M. 2012; Chapter 23: Unit 23 15-?

  Abstract

  The induction of pluripotency in somatic cells by transcription-factor overexpression has been widely regarded as one of the major breakthroughs in stem cell biology within this decade. The generation of these induced pluripotent stem cells (iPSCs) has enabled investigators to develop in vitro disease models for biological discovery and drug screening, and in the future, patient-specific therapy for tissue or organ regeneration. While new technologies for reprogramming are continually being discovered, the availability of iPSCs from different species is also increasing rapidly. Comparison of iPSCs across species may provide new insights into key aspects of pluripotency and early embryonic development. iPSCs from large animals may enable the generation of genetically modified large animal models or potentially transplantable donor tissues or organs. This unit describes the procedure for the generation of iPSCs from mouse, rat, pig and human fibroblasts.

  View details for DOI 10.1002/0471142727.mb2315s97

  View details for PubMedID 22237859

• Developmental and Regenerative Biology of Multipotent Cardiovascular Progenitor Cells CIRCULATION RESEARCH Sturzu, A. C., Wu, S. M. 2011; 108 (3): 353-364

  Abstract

  Our limited ability to improve the survival of patients with heart failure is attributable, in part, to the inability of the mammalian heart to meaningfully regenerate itself. The recent identification of distinct families of multipotent cardiovascular progenitor cells from endogenous, as well as exogenous, sources, such as embryonic and induced pluripotent stem cells, has raised much hope that therapeutic manipulation of these cells may lead to regression of many forms of cardiovascular disease. Although the exact source and cell type remains to be clarified, our greater understanding of the scientific underpinning behind developmental cardiovascular progenitor cell biology has helped to clarify the origin and properties of diverse cells with putative cardiogenic potential. In this review, we highlight recent advances in the understanding of cardiovascular progenitor cell biology from embryogenesis to adulthood and their implications for therapeutic cardiac regeneration. We believe that a detailed understanding of cardiogenesis will inform future applications of cardiovascular progenitor cells in heart failure therapy and regenerative medicine.

  View details for DOI 10.1161/CIRCRESAHA.110.227066

  View details for Web of Science ID 000286930500012

  View details for PubMedID 21293007

  View details for PubMedCentralID PMC3073355

• Regenerative strategies for cardiac disease In: Stem Cells and Regenerative Medicine. Humana Press. Huang X, Oh JB, Wu SM 2011; 1: 579-593
• Origin of Cardiac Progenitor Cells in the Developing and Postnatal Heart JOURNAL OF CELLULAR PHYSIOLOGY Kuhn, E. N., Wu, S. M. 2010; 225 (2): 321-325

  Abstract

  The mammalian heart lacks the capacity to replace the large numbers of cardiomyocytes lost due to cardiac injury. Several different cell-based routes to myocardial regeneration have been explored, including transplantation of cardiac progenitors and cardiomyocytes into injured myocardium. As seen with cell-based therapies in other solid organ systems, inherent limitations, such as host immune response, cell death and long-term graft instability have hampered meaningful cardiac regeneration. An understanding of the cell biology of cardiac progenitors, including their developmental origin, lineage markers, renewal pathways, differentiation triggers, microenvironmental niche, and mechanisms of homing and migration to the site of injury, will enable further refinement of therapeutic strategies to enhance clinically meaningful cardiac repair.

  View details for DOI 10.1002/jcp.22281

  View details for Web of Science ID 000283003400007

  View details for PubMedID 20568226

  View details for PubMedCentralID PMC3620291

• Isolation and functional characterization of pluripotent stem cell-derived cardiac progenitor cells. Current protocols in stem cell biology Huang, X., Wu, S. M. 2010; Chapter 1: Unit 1F 10-?

  Abstract

  The use of transgenic markers in pluripotent stem cells allows the facile isolation of transient cell populations that appear at certain phases of embryonic development. Here, we describe a procedure for deriving cardiac progenitors from mouse pluripotent stem cells carrying a GFP reporter under the control of an Nkx2.5 enhancer sequence. The cells are propagated under standard conditions and are differentiated using the hanging-droplet method with medium optimized for commitment to the cardiac lineage. Cardiac progenitors are isolated from the differentiation culture using fluorescence-activated cell sorting (FACS) and can be cultured further for functional characterization and experimentation. The protocols described here can be applied to both embryonic and induced pluripotent stem cells and can easily be adapted to transgenic lines carrying other cardiac cell lineage reporters.

  View details for DOI 10.1002/9780470151808.sc01f10s14

  View details for PubMedID 20814937

  View details for PubMedCentralID PMC2947085

• Promises and pitfalls in cell replacement therapy for heart failure. Drug discovery today. Disease mechanisms Krane, M., Wernet, O., Wu, S. M. 2010; 7 (2): e109-e115

  Abstract

  Symptomatic heart failure is a complex clinical syndrome with a poor prognosis. Many efforts have been made to develop new therapeutic strategies to improve prognosis associated with heart failure. In this context, different stem cell populations for cardiac regenerative therapy have been examined recently. Here we discuss the potential strategies for using stem cells in cardiac regenerative therapy and the barriers that remain before an effective cell-based cardiac regenerative therapy can be employed clinically.

  View details for PubMedID 21180399

• Cardiac progenitor cells: from embryonic to the aging heart. Aging Health Liu, Y-H., Kuhn, E.B., Wu, S.M. 2010; 6 (6): 679-686
• The integrative aspects of cardiac physiology and their implications for cell-based therapy Ananlysis of Cardiac Development: From Embryo to Old Age Pijnappels, D. A., Gregoire, S., Wu, S. M. Annals of the New York Academy of Sciences. 2010; 1188: 7–14

  View details for DOI 10.1111/j.1749-6632.2009.05077.x

• Myocardial Injury Induces the Expansion and Cardiomyogenic Differentiation of Postnatal Nkx2.5 Progenitor Cells via Inflammatory Signals 82nd National Conference and Exhibitions and Annual Scientific Session of the American-Heart-Association Liu, Y., Rawnsley, D., Zeng, M., Yu, E., Pijnappels, D., Thibault, H., Scherrer-Crosbie, M., Wu, S. M. LIPPINCOTT WILLIAMS & WILKINS. 2009: S756–S756

  View details for Web of Science ID 000271831502235

• VISIONS: the art of science. Molecular reproduction and development Wu, S. M. 2009; 76 (6): 525-?

  View details for DOI 10.1002/mrd.21022

  View details for PubMedID 19358263

• Committed Ventricular Progenitors in the Islet-1 Lineage Expand and Assemble Into Functional Ventricular Heart Muscle 58th Annual Scientific Session of the American-College-of-Cardiology van der Meer, P., Domian, I. J., Chiravuri, M., Feinberg, A. F., Wu, S. M., Parker, K. K., Chien, K. R. ELSEVIER SCIENCE INC. 2009: A468–A468

  View details for Web of Science ID 000263864201940

• Platypnea-orthodeoxia syndrome in two previously healthy adults: a case-based review Clinical Medicine Insights: Cardiology Ptaszek LM, Saldana F, Palacios IF, Wu SM 2009; 3: 37-43
• Derivation and Functional Characterization of Nkx2.5+Cardiac Progenitor Cells from Mouse Induced Pluripotent Stem Cells 81st Annual Scientific Session of the American-Heart-Association Pijnappels, D. A., Stadtfeld, M., Zeng, M., Yu, E., Fujiwara, Y., Wang, G., Orkin, S. H., Jackson-Grusby, L., Hochedlinger, K., Wu, S. M. LIPPINCOTT WILLIAMS & WILKINS. 2008: S428–S428

  View details for Web of Science ID 000262104500714

• Mesp1 at the heart of mesoderm lineage specification CELL STEM CELL Wu, S. M. 2008; 3 (1): 1-2

  Abstract

  Stem cell-based cardiac regeneration requires a detailed understanding of the factors that induce cardiac lineage commitment. In this issue of Cell Stem Cell, Lindsley et al. (2008) and Bondue et al. (2008) use embryonic stem cells to identify a key role for Mesp1 in this process.

  View details for DOI 10.1016/j.stem.2008.06.017

  View details for Web of Science ID 000257622300001

  View details for PubMedID 18593549

• Multipotent stem cells in cardiac regenerative therapy REGENERATIVE MEDICINE Karra, R., Wu, S. M. 2008; 3 (2): 189-198

  Abstract

  The potential for stem cells to ameliorate or cure heart diseases has galvanized a cadre of cardiovascular translational and clinical scientists to take a 'first-in-man' approach using autologous stem cells from a variety of tissues. However, recent clinical trial data show that when these cells are given by intracoronary infusion or direct myocardial injection, limited improvement in heart function occurs with no evidence of cardiomyogenesis. These studies illustrate the great need to understand the logic of cell-lineage commitment and the principles of cardiac differentiation. Recent identification of stem/progenitor cells of embryological origin with intrinsic competence to differentiate into multiple lineages within the heart offers new possibilities for cardiac regeneration. When combined with developments in nuclear reprogramming and provided that tumor risks and other challenges of embryonic cell transplantation can be overcome, the prospect of achieving autologous, cardiomyogenic, stem cell-based therapy may be within reach.

  View details for DOI 10.2217/17460751.3.2.189

  View details for Web of Science ID 000257995000013

  View details for PubMedID 18307403

  View details for PubMedCentralID PMC2519870

• Cardiovascular Stem Cells in Regenerative Medicine: Ready for Prime Time? Drug discovery today. Therapeutic strategies Liu, Y., Karra, R., Wu, S. M. 2008; 5 (4): 201-207

  Abstract

  Restoration of cardiovascular function is the ultimate goal of stem cell-based therapy. In principle, cardiovascular stem cells can improve cardiac function via de novo cardiomyogenesis, enhanced myocardial neovascularization, and prevention of post-infarct remodeling. Stem cell transplantation to improve cardiac function has received mixed results in human clinical trials. These early data suggest that a critical reassessment of the scientific basis to stem cell-based therapy is needed in order to bring this highly promising treatment modality to mainstream clinical care.

  View details for PubMedID 20054428

• alpha(2)-Macraglobulin from rheumatoid arthritis synovial fluid: Functional analysis defines a role for oxidation in inflammation ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Wu, S. M., Pizzo, S. V. 2001; 391 (1): 119-126

  Abstract

  A hallmark of inflammation is the release of oxidants, proteinases, and cytokines, all important mediators of the inflammatory cascade. alpha(2)-Macroglobulin (alpha(2)M) is a high-affinity, broad-specificity proteinase inhibitor that also binds and regulates the biological activities of a number of cytokines. We demonstrated recently that hypochlorite-oxidized alpha(2)M has decreased ability to inhibit proteinases and regulate cytokines in vitro. The role of oxidation in regulating alpha(2)M functions in vivo is largely unknown. To determine the extent and biological consequence of in vivo alpha(2)M oxidation, we measured the degree of oxidative alpha(2)M modification from rheumatoid arthritis (RA) synovial fluid and compared this with osteoarthritis (OA) as noninflammatory controls. We found that RA synovial fluid alpha(2)M is significantly more oxidized than that from OA. RA synovial fluid also contains a twofold higher median alpha(2)M level than OA, while having only half the alpha(2)M-proteinase inhibitory activity. Detailed biochemical analysis demonstrates proteolytically degraded alpha(2)M in RA greater than in OA synovial fluid. Additionally, the hypochlorite-mediated oxidation product, chlorotyrosine, is present in RA more than in OA or plasma alpha(2)M samples. Taken together, these findings confirm a role for oxidative regulation of inflammation by altering the functions of extracellular mediators such as alpha(2)M.

  View details for Web of Science ID 000169700200015

  View details for PubMedID 11414692

• Differential regulation of the fibroblast growth factor (FGF) family by alpha(2)-macroglobulin: evidence for selective modulation of FGF-2-induced angiogenesis BLOOD Asplin, I. R., Wu, S. M., Mathew, S., Bhattacharjee, G., Pizzo, S. V. 2001; 97 (11): 3450-3457

  Abstract

  The fibroblast growth factor (FGF) family has an important role in processes such as angiogenesis, wound healing, and development in which precise control of proteinase activity is important. The human plasma proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M) regulates cellular growth by binding and modulating the activity of many cytokines and growth factors. These studies investigate the ability of native and activated alpha(2)M (alpha(2)M*) to bind to members of the FGF family. Both alpha(2)M and alpha(2)M* bind specifically and saturably to FGF-1, -2, -4, and -6, although the binding to alpha(2)M* is of significantly higher affinity. Neither alpha(2)M nor alpha(2)M* bind to FGF-5, -7, -9, or -10. FGF-2 was chosen for more extensive study in view of its important role in angiogenesis. It was demonstrated that FGF-2 binds to the previously identified TGF-beta binding site. The alpha(2)M* inhibits FGF-2-dependent fetal bovine heart endothelial cell proliferation in a dose-dependent manner. Unexpectedly, alpha(2)M* does not affect FGF-2-induced vascular tubule formation on Matrigel basement membrane matrix or collagen gels. Further studies demonstrate that FGF-2 partitions between fluid-phase alpha(2)M* and solid-phase Matrigel or collagen. These studies suggest that the ability of alpha(2)M* to modulate the activity of FGF-2 is dependent on an interplay with extracellular matrix components. (Blood. 2001;97:3450-3457)

  View details for Web of Science ID 000168927900019

  View details for PubMedID 11369636

• The conformation-dependent interaction of alpha(2)-macroglobulin with vascular endothelial growth factor - A novel mechanism of alpha(2)-macroglobulin/growth factor binding JOURNAL OF BIOLOGICAL CHEMISTRY Bhattacharjee, G., Asplin, I. R., Wu, S. M., Gawdi, G., Pizzo, S. V. 2000; 275 (35): 26806-26811

  Abstract

  alpha(2)-Macroglobulin (alpha(2)M) is a highly conserved proteinase inhibitor present in human plasma at high concentration (2-4 mg/ml). alpha(2)M exists in two conformations, a native form and an activated, receptor-recognized form. While alpha(2)M binds to numerous cytokines and growth factors, in most cases, the nature of the alpha(2)M interaction with these factors is poorly understood. We examined in detail the interaction between alpha(2)M and vascular endothelial growth factor (VEGF) and found a novel and unexpected mechanism of interaction as demonstrated by the following observations: 1) the binding of VEGF to alpha(2)M occurs at a site distinct from the recently characterized growth factor binding site; 2) VEGF binds different forms of alpha(2)M with distinct spatial arrangement, namely to the interior of methylamine or ammonia-treated alpha(2)M and to the exterior of native and proteinase-converted alpha(2)M; and 3) VEGF (molecular mass approximately 40 kDa) can access the interior of receptor-recognized alpha(2)M in the absence of a proteinase trapped within the molecule. VEGF bound to receptor-recognized forms of alpha(2)M is internalized and degraded by macrophages via the alpha(2)M receptor, the low density lipoprotein receptor-related protein. Oxidation of both native and receptor-recognized alpha(2)M results in significant inhibition of VEGF binding. We also examined the biological significance of this interaction by studying the effect of alpha(2)M on VEGF-induced cell proliferation and VEGF-induced up-regulation of intracellular Ca(2+) levels. We demonstrate that under physiological conditions, alpha(2)M does not impact the ability of VEGF to induce cell proliferation or up-regulate Ca(2+).

  View details for Web of Science ID 000089144800021

  View details for PubMedID 10862607

• a-Macroglobulins/Kunins In: Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 4th Ed. Pizzo SV, Wu SM 2000: 367-379
• Mechanism of hypochlorite-mediated inactivation of proteinase inhibition by alpha(2)-Macroglobulin BIOCHEMISTRY Wu, S. M., Pizzo, S. V. 1999; 38 (42): 13983-13990

  Abstract

  The proteinase-proteinase inhibitor balance plays an important role in mediating inflammation-associated tissue destruction. alpha 2-Macroglobulin (alpha 2M) is a high-affinity, broad-spectrum proteinase inhibitor found abundantly in plasma and interstitial fluids. Increased levels of alpha 2M and proteinase-alpha 2M complexes can be demonstrated in patients with sepsis, emphysema, peridontitis, rheumatoid arthritis, and other inflammatory diseases. Despite these increased levels, proteolysis remains a significant problem. We hypothesized that a mechanism for inactivating alpha 2M-mediated proteinase inhibition must exist and recently demonstrated that alpha 2M isolated from human rheumatoid arthritis synovial fluid is oxidized and has decreased functional activity. The oxidant responsible for alpha 2M inactivation and the mechanism of such destruction were not studied. We now report that while hypochlorite and hydroxyl radical both modify amino acid residues on alpha 2M, only hypochlorite can abolish the ability of alpha 2M to inhibit proteinases. Hydrogen peroxide, on the other hand, has no effect on alpha 2M structure or function. Protein unfolding with increased susceptibility to proteolytic cleavage appears to be involved in alpha 2M inactivation by oxidation. The in vivo relevance of this mechanism is supported by the presence of multiple cleavage fragments of alpha 2M in synovial fluid from patients with rheumatoid arthritis, where significant tissue destruction occurs, but not in patients with osteoarthritis. These results provide strong evidence that hypochlorite oxidation contributes to enhanced tissue destruction during inflammation by inactivating alpha 2M.

  View details for Web of Science ID 000083288400025

  View details for PubMedID 10529245

• Oxidized alpha(2)-macroglobulin (alpha(2)M) differentially regulates receptor binding by cytokines growth factors: Implications for tissue injury and repair mechanisms in inflammation JOURNAL OF IMMUNOLOGY Wu, S. M., Patel, D. D., Pizzo, S. V. 1998; 161 (8): 4356-4365

  Abstract

  Alpha2M binds specifically to TNF-alpha, IL-1beta, IL-2, IL-6, IL-8, basic fibroblast growth factor (bFGF), beta-nerve growth factor (beta-NGF), platelet-derived growth factor (PDGF), and TGF-beta. Since many of these cytokines are released along with neutrophil-derived oxidants during acute inflammation, we hypothesize that oxidation alters the ability of alpha2M to bind to these cytokines, resulting in differentially regulated cytokine functions. Using hypochlorite, a neutrophil-derived oxidant, we show that oxidized alpha2M exhibits increased binding to TNF-alpha, IL-2, and IL-6 and decreased binding to beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Hypochlorite oxidation of methylamine-treated alpha2M (alpha2M*), an analogue of the proteinase/alpha2M complex, also results in decreased binding to bFGF, beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Concomitantly, we observed decreased ability to inhibit TGF-beta binding and regulation of cells by oxidized alpha2M and alpha2M*. We then isolated alpha2M from human rheumatoid arthritis synovial fluid and showed that the protein is extensively oxidized and has significantly decreased ability to bind to TGF-beta compared with alpha2M derived from plasma and osteoarthritis synovial fluid. We, therefore, propose that oxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-alpha, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, beta-NGF, PDGF, and TGF-beta from binding to alpha2M.

  View details for Web of Science ID 000076343300073

  View details for PubMedID 9780213

• The binding of receptor-recognized alpha(2)-macroglobulin to the low density lipoprotein receptor-related protein and the alpha(2)M signaling receptor is decoupled by oxidation JOURNAL OF BIOLOGICAL CHEMISTRY Wu, S. M., BOYER, C. M., Pizzo, S. V. 1997; 272 (33): 20627-20635

  Abstract

  Receptor-recognized forms of alpha2-macroglobulin (alpha2M*) bind to two classes of cellular receptors, a high affinity site comprising approximately 1500 sites/cell and a lower affinity site comprising about 60,000 sites/cell. The latter class has been identified as the so-called low density lipoprotein receptor-related protein (LRP). Ligation of receptors distinct from LRP activates cell signaling pathways. Strong circumstantial evidence suggests that the high affinity binding sites are responsible for cell signaling induced by alpha2M*. Using sodium hypochlorite, a powerful oxidant produced by the H2O2-myeloperoxidase-Cl- system, we now demonstrate that binding to the high affinity sites correlates directly with activation of the signaling cascade. Oxidation of alpha2M* using 200 microM hypochlorite completely abolishes its binding to LRP without affecting its ability to activate the macrophage signaling cascade. Scatchard analysis shows binding to a single class of high affinity sites (Kd - 71 +/- 12 pM). Surprisingly, oxidation of native alpha2-macroglobulin (alpha2M) with 125 microM hypochlorite results in the exposure of its receptor-binding site to LRP, but the ligand is unable to induce cell signaling. Scatchard analysis shows binding to a single class of lower affinity sites (Kd - 0.7 +/- 0.15 nM). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of alpha2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding Kd, supporting our earlier finding that the oxidation-sensitive site is predominantly outside of RBF. Attempts to understand the mechanism responsible for the selective exposure of LRP-binding sites in oxidized native alpha2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (Kd - 71 pM) are the alpha2M* signaling receptor.

  View details for Web of Science ID A1997XR22100048

  View details for PubMedID 9252378

• Crashing the Boards: A User Friendly Study Guide for the USMLE Step 1 Lippincott-Raven Yeh B, Paydafar JA, Flynn M, Biswas SS, Bulsara KR, Liao L, Wu SM 1997; 1
• Low-density lipoprotein receptor-related protein alpha(2)-macroglobulin receptor on murine peritoneal macrophages mediates the binding and catabolism of low-density lipoprotein ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Wu, S. M., Pizzo, S. V. 1996; 326 (1): 39-47

  Abstract

  Low-density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor is a member of the low-density lipoprotein receptor family. It is known to bind a wide variety of unrelated ligands including alpha 2-macroglobulin-proteinase complexes, tissue plasminogen activator, apolipoprotein E-enriched very low density lipoprotein, lipoprotein lipase, and Pseudomonas exotoxin A. Receptor-associated protein (RAP), a protein which copurifies with LRP, can inhibit the binding and internalization of all known ligands to LRP. Recent studies have shown that some ligands can bind to more than one receptor in this family. However, the ability of low-density lipoprotein (LDL) to bind to LRP in addition to the LDL receptor has not been demonstrated consistently. In this study we demonstrate that LDL binds with high affinity to macrophage cell surface receptors at 4 degrees C (Kd = 1.8 nM) and competes for the binding of a receptor-recognized form of alpha 2-macroglobulin (alpha 2M*) (Ki = 3 nM). alpha 2M* and RAP can inhibit the binding of LDL to macrophages completely (96 and 100% inhibition, respectively), after cell surface heparin has been removed by treatment with heparinase. Using a solid-phase assay, we show that LDL binds specifically, saturably, and with high affinity to purified LRP (Kd = 5 nM). LDL can also completely inhibit the binding of alpha 2M* to purified LRP. These results indicate that LDL binds directly to LRP. The ability of LDL to cross-compete with alpha 2M* for binding to LRP suggests that LDL binds to a similar or overlapping site as alpha 2M*. In addition, the ability of alpha 2M* to inhibit most of the receptor-mediated binding of LDL to macrophages suggests that LDL receptors on murine peritoneal macrophages are predominantly LRP.

  View details for Web of Science ID A1996TT88500006

  View details for PubMedID 8579370