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Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells

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Abstract

In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (CMs). Protocols for cardiac differentiation of hESCs and hiPSCs include formation of the three-dimensional cell aggregates called embryoid bodies (EBs). The traditional suspension method for EB formation from clumps of cells results in an EB population heterogeneous in size and shape. In this study we show that forced aggregation of a defined number of single cells on AggreWell plates gives a high number of homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors in the media. For cardiac differentiation, we used three hESC lines and one hiPSC line. Our contracting EBs and the resulting CMs express cardiac markers, namely myosin heavy chain α and β, cardiac ryanodine receptor/calcium release channel, and cardiac troponin T, shown by real-time polymerase chain reaction and immunocytochemistry. Using Ca2+ imaging and atomic force microscopy, we demonstrate the functionality of RyR2 to release Ca2+ from the sarcoplasmic reticulum as well as reliability in contractile and beating properties of hESC-EBs and hiPSC-EBs upon the stimulation or inhibition of the β-adrenergic pathway.

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References

  1. Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 87:27–45

    CAS  PubMed  Google Scholar 

  2. Doi K, Itoh H, Nakagawa O, Igaki T, Yamashita J, Chun TH, Inoue M, Masatsugu K, Nakao K (1997) Expression of natriuretic peptide system during embryonic stem cell vasculogenesis. Heart Vessels Suppl 12:18–22

    Google Scholar 

  3. Burridge PW, Anderson D, Priddle H, Barbadillo Munoz MD, Chamberlain S, Allegrucci C, Young LE, Denning C (2007) Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability. Stem Cells 25:929–938

    Article  CAS  PubMed  Google Scholar 

  4. Burridge PW, Thompson S, Millrod MA, Weinberg S, Yuan X, Peters A, Mahairaki V, Koliatsos VE, Tung L, Zambidis ET (2011) A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS One 6:e18293

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Kattman SJ, Witty AD, Gagliardi M, Dubois NC, Niapour M, Hotta A, Ellis J, Keller G (2011) Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8:228–240

    Article  CAS  PubMed  Google Scholar 

  6. Xu XQ, Graichen R, Soo SY, Balakrishnan T, Rahmat SN, Sieh S, Tham SC, Freund C, Moore J, Mummery C, Colman A, Zweigerdt R, Davidson BP (2008) Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells. Differentiation 76:958–970

    CAS  PubMed  Google Scholar 

  7. Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM, Field LJ, Keller GM (2008) Human cardiovascular progenitor cells develop from a KDR + embryonic-stem-cell-derived population. Nature 453:524–528

    Article  CAS  PubMed  Google Scholar 

  8. Dvorak P, Dvorakova D, Koskova S, Vodinska M, Najvirtova M, Krekac D, Hampl A (2005) Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells. Stem Cells 23:1200–1211

    Article  CAS  PubMed  Google Scholar 

  9. Eiselleova L, Peterkova I, Neradil J, Slaninova I, Hampl A, Dvorak P (2008) Comparative study of mouse and human feeder cells for human embryonic stem cells. Int J Dev Biol 52:353–363

    Article  CAS  PubMed  Google Scholar 

  10. Armstrong L, Tilgner K, Saretzki G, Atkinson SP, Stojkovic M, Moreno R, Przyborski S, Lako M (2010) Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells. Stem Cells 28:661–673

    Article  CAS  PubMed  Google Scholar 

  11. Dubois NC, Craft AM, Sharma P, Elliott DA, Stanley EG, Elefanty AG, Gramolini A, Keller G (2011) SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat Biotechnol 29:1011–1018

    Article  CAS  PubMed  Google Scholar 

  12. Lee MY, Cagavi Bozkulak E, Schliffke S, Amos PJ, Ren Y, Ge X, Ehrlich BE, Qyang Y (2011) High density cultures of embryoid bodies enhanced cardiac differentiation of murine embryonic stem cells. Biochem Biophys Res Commun 416:51–57

    Article  CAS  PubMed  Google Scholar 

  13. Fujita E, Nakanishi T, Nishizawa T, Hagiwara N, Matsuoka R (2013) Mutations in the cardiac troponin T gene show various prognoses in Japanese patients with hypertrophic cardiomyopathy. Heart Vessels. doi:10.1007/s00380-013-0332-3

  14. Zwi-Dantsis L, Huber I, Habib M, Winterstern A, Gepstein A, Arbel G, Gepstein L (2013) Derivation and cardiomyocyte differentiation of induced pluripotent stem cells from heart failure patients. Eur Heart J 34:1575–1586

    Article  CAS  PubMed  Google Scholar 

  15. Liu J, Sun N, Bruce MA, Wu JC, Butte MJ (2012) Atomic force mechanobiology of pluripotent stem cell-derived cardiomyocytes. PLoS One 7:e37559

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Itzhaki I, Rapoport S, Huber I, Mizrahi I, Zwi-Dantsis L, Arbel G, Schiller J, Gepstein L (2011) Calcium handling in human induced pluripotent stem cell derived cardiomyocytes. PLoS One 6:e18037

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Lee YK, Ng KM, Lai WH, Chan YC, Lau YM, Lian Q, Tse HF, Siu CW (2011) Calcium homeostasis in human induced pluripotent stem cell-derived cardiomyocytes. Stem Cell Rev 7:976–986

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Liu B, Wohlfart B, Johansson BW (1990) Effects of low temperature on contraction in papillary muscles from rabbit, rat, and hedgehog. Cryobiology 27:539–546

    Article  CAS  PubMed  Google Scholar 

  19. Fu Y, Zhang GQ, Hao XM, Wu CH, Chai Z, Wang SQ (2005) Temperature dependence and thermodynamic properties of Ca2+ sparks in rat cardiomyocytes. Biophys J 89:2533–2541

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Wang SQ, Huang YH, Liu KS, Zhou ZQ (1997) Dependence of myocardial hypothermia tolerance on sources of activator calcium. Cryobiology 35:193–200

    Article  CAS  PubMed  Google Scholar 

  21. Degubareff T, Sleator W Jr (1965) Effects of caffeine on mammalian atrial muscle, and its interaction with adenosine and calcium. J Pharmacol Exp Ther 148:202–214

    CAS  PubMed  Google Scholar 

  22. Axelsson J, Thesleff S (1958) Activation of the contractile mechanism in striated muscle. Acta Physiol Scand 44:55–66

    Article  CAS  PubMed  Google Scholar 

  23. Weber A, Herz R (1968) The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum. J Gen Physiol 52:750–759

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Bellin M, Marchetto MC, Gage FH, Mummery CL (2012) Induced pluripotent stem cells: the new patient? Nat Rev Mol Cell Biol 13:713–726

    Article  PubMed  Google Scholar 

  25. Bratt-Leal AM, Carpenedo RL, McDevitt TC (2009) Engineering the embryoid body microenvironment to direct embryonic stem cell differentiation. Biotechnol Prog 25:43–51

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Mohr JC, Zhang J, Azarin SM, Soerens AG, de Pablo JJ, Thomson JA, Lyons GE, Palecek SP, Kamp TJ (2010) The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells. Biomaterials 31:1885–1893

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Bauwens CL, Peerani R, Niebruegge S, Woodhouse KA, Kumacheva E, Husain M, Zandstra PW (2008) Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells 26:2300–2310

    Article  PubMed  Google Scholar 

  28. Antonchuk J (2013) Formation of embryoid bodies from human pluripotent stem cells using AggreWell plates. Methods Mol Biol 946:523–533

    Article  CAS  PubMed  Google Scholar 

  29. Preda MB, Burlacu A, Simionescu M (2013) Defined-size embryoid bodies formed in the presence of serum replacement increases the efficiency of the cardiac differentiation of mouse embryonic stem cells. Tissue Cell 45:54–60

    Article  CAS  PubMed  Google Scholar 

  30. Ren Y, Lee MY, Schliffke S, Paavola J, Amos PJ, Ge X, Ye M, Zhu S, Senyei G, Lum L, Ehrlich BE, Qyang Y (2011) Small molecule Wnt inhibitors enhance the efficiency of BMP-4-directed cardiac differentiation of human pluripotent stem cells. J Mol Cell Cardiol 51:280–287

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Gutstein DE, Marks AR (1997) Role of inositol 1,4,5-trisphosphate receptors in regulating apoptotic signaling and heart failure. Heart Vessels Suppl 12:53–57

    Article  Google Scholar 

  32. Satin J, Itzhaki I, Rapoport S, Schroder EA, Izu L, Arbel G, Beyar R, Balke CW, Schiller J, Gepstein L (2008) Calcium handling in human embryonic stem cell-derived cardiomyocytes. Stem Cells 26:1961–1972

    Article  CAS  PubMed  Google Scholar 

  33. Shibata S, Hollander PB (1967) Effects of caffeine on the contractility and membrane potentials of rat atrium. Experientia 23:559

    Article  CAS  PubMed  Google Scholar 

  34. Shinohara T, Kim D, Joung B, Maruyama M, Vembaiyan K, Back TG, Wayne Chen SR, Chen PS, Lin SF (2013) Carvedilol analog modulates both basal and stimulated sinoatrial node automaticity. Heart Vessels doi. doi:10.1007/s00380-013-0378-2

    Google Scholar 

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Acknowledgments

We would like to thank Dr Livia Eiselleova, Stanislava Koskova, Professor Ales Hampl, Dana Stritecka, and Eva Peslova for their assistance, as well as Professor Majlinda Lako for kindly providing the clone 4 hiPSC. This work was supported by grants from the Ministry of Education, Youth, and Sports of the Czech Republic (CZ.1.07/2.3.00/20.0011 and MSM0021622430), project FNUSA-ICRC (no. CZ.1.05/1.1.00/02.0123) from the European Regional Development Fund, SoMoPro—Marie Curie Actions—South Moravian Region, and by the European Society of Cardiology (ESC) to Albano C. Meli. The research leading to these results obtained a financial contribution from the European Community within the Seventh Framework Program (FP/2007-2013) under Grant Agreement No. 229603. This work was supported by CEITEC—Central European Institute of Technology (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund. Albano C. Meli was supported by a French Muscular Dystrophy Association Research Grant (AFM). Ivana Acimovic was supported by a PLURICELL grant (CZ.1.07/2.3.00/20.0011).

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Authors and Affiliations

  1. Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

    Martin Pesl, Ivana Acimovic, Aleksandra Vilotic, Jan Vrbsky, Vladimir Rotrekl, Petr Dvorak & Albano C. Meli

  2. ICRC, St Anne’s University Hospital, Brno, Czech Republic

    Martin Pesl, Jan Vrbsky, Peter Kruzliak, Tomas Kara & Petr Dvorak

  3. CEITEC, Masaryk University, Brno, Czech Republic

    Jan Pribyl, Renata Hezova & Petr Skladal

  4. INSERM U1046, University of Montpellier I, University of Montpellier II, 371 Avenue du Doyen G. Giraud, CHU Arnaud de Villeneuve, Building INSERM Crastes de Paulet, 34295, Montpellier, France

    Jeremy Fauconnier, Alain Lacampagne & Albano C. Meli

  5. Department of Biochemistry, Faculty of Science, Brno, Czech Republic

    Petr Skladal

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  1. Martin Pesl
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  2. Ivana Acimovic
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Corresponding author

Correspondence to Albano C. Meli.

Additional information

M. Pesl and I. Acimovic contributed equally to this work.

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Pesl, M., Acimovic, I., Pribyl, J. et al. Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells. Heart Vessels 29, 834–846 (2014). https://doi.org/10.1007/s00380-013-0436-9

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  • DOI: https://doi.org/10.1007/s00380-013-0436-9

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