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Cryopreservation of Human Embryonic Stem Cells Derived-Cardiomyocytes Induced by BMP2 in Serum-Free Condition

Abstract

Although previous studies showed that cardiomyocytes (CMs) can be generated from human embryonic stem cells (hESCs), the protocols for cryopreservation of hESC-derived CMs is not available to date. Here, we report on the efficient generation of hESC-derived CMs by direct differentiation using BMP2 in a serum-free condition, along with successful cryopreservation of derived CMs using Rho-associated kinase (ROCK) inhibitor. To induce differentiation, hESCs were treated with activin A and BMP2 for 5 days. A mesodermal gene, Brachyury, was expressed from day 3, and cardiac-specific markers such as Nkx2.5 and cTnI were detected at day 14. Furthermore, these cardiac progenitors expressed ion channel-related transcripts such as HCN1 and HCN2 from day 10. Beating clusters were observed from 14 days of differentiation for up to 35 days. Using mass cryopreservation, we froze hESC-derived CMs at 2 stages, at day 12 and 16 (prebeating and postbeating), after treating with ROCK inhibitor, Y-27632. Postthaw survival of CMs was higher in day 12 group compared to day 16, and some cell clusters from day 12 group recovered their contraction. From transmission electron microscope (TEM) analysis, less ultrastructural alterations were observed in day 12 group. Our results provide an insight into the use of BMP2 for cardiac lineage differentiation in a serum-free condition and a possibility of long-term storage of hESC-derived CMs.

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References

  1. 1.

    Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282(5391):1145–1147.

  2. 2.

    Oh SK, Kim HS, Ahn HJ, et al. Derivation and characterization of new human embryonic stem cell lines: SNUhES1, SNUhES2, and SNUhES3. Stem Cells. 2005;23(2):211–219.

  3. 3.

    Sugi Y, Lough J. Activin-A and FGF-2 mimic the inductive effects of anterior endoderm on terminal cardiac myogenesis in vitro. Dev Biol. 1995;168(2):567–574.

  4. 4.

    Schultheiss TM, Burch JB, Lassar AB. A role for bone morphogenetic proteins in the induction of cardiac myogenesis. Genes Dev. 1997;11(4):451–462.

  5. 5.

    Laflamme MA, Chen KY, Naumova AV, et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol. 2007;25(9):1015–1024.

  6. 6.

    Zhang P, Li J, Tan Z, et al. Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells. Blood. 2008;111(4):1933–1941.

  7. 7.

    Yang L, Soonpaa MH, Adler ED, et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature. 2008;453(7194):524–528.

  8. 8.

    Tomescot A, Leschik J, Bellamy V, et al. Differentiation in vivo of cardiac committed human embryonic stem cells in postmyocardial infarcted rats. Stem Cells. 2007;25(9):2200–2205.

  9. 9.

    Pal R, Khanna A. Similar pattern in cardiac differentiation of human embryonic stem cell lines, BG01V and ReliCellhES1, under low serum concentration supplemented with bone morphogenetic protein-2. Differentiation. 2007;75(2):112–122.

  10. 10.

    Kim YY, Ku SY, Jang J, et al. Use of long-term cultured embryoid bodies may enhance cardiomyocyte differentiation by BMP2. Yonsei Med J. 2008;49(5):819–827.

  11. 11.

    Kehat I, Kenyagin-Karsenti D, Snir M, et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest. 2001;108(3):407–414.

  12. 12.

    Xu C, Police S, Rao N, Carpenter MK. Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res. 2002;91(6):501–508.

  13. 13.

    Itskovitz-Eldor J, Schuldiner M, Karsenti D, et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med. 2000;6(2):88–95.

  14. 14.

    Keller GM. In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol. 1995;7(6):862–869.

  15. 15.

    Li T, Zhou C, Liu C, Mai Q, Zhuang G. Bulk vitrification of human embryonic stem cells. Hum Reprod. 2008;23(2):358–364.

  16. 16.

    Meryman HT. Cryopreservation of living cells: principles and practice. Transfusion. 2007;47(5):935–945.

  17. 17.

    Li X, Meng G, Krawetz R, Liu S, Rancourt DE. The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation. Stem Cells Dev. 2008;17(6):1079–1085.

  18. 18.

    Watanabe K, Ueno M, Kamiya D, et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 2007;25(6):681–686.

  19. 19.

    Li X, Krawetz R, Liu S, Meng G, Rancourt DE. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum Reprod. 2009;24(3):580–589.

  20. 20.

    Mollamohammadi S, Taei A, Pakzad M, et al. A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells. Hum Reprod. 2009;24(10):2468–2476.

  21. 21.

    Martin-Ibanez R, Unger C, Stromberg A, Baker D, Canals JM, Hovatta O. Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor. Hum Reprod. 2008;23(12):2744–2754.

  22. 22.

    Norstrom A, Akesson K, Hardarson T, Hamberger L, Bjorquist P, Sartipy P. Molecular and pharmacological properties of human embryonic stem cell-derived cardiomyocytes. Exp Biol Med (Maywood). 2006;231(11):1753–1762.

  23. 23.

    Oh SK, Kim HS, Park YB, et al. Methods for expansion of human embryonic stem cells. Stem Cells. 2005;23(5):605–609.

  24. 24.

    He JQ, Ma Y, Lee Y, Thomson JA, Kamp TJ. Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization. Circ Res. 2003;93(1):32–39.

  25. 25.

    Kehat I, Khimovich L, Caspi O, et al. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol. 2004;22(10):1282–1289.

  26. 26.

    Jang J, Ku SY, Kim JE, et al. Notch inhibition promotes human embryonic stem cell-derived cardiac mesoderm differentiation. Stem Cells. 2008;26(11):2782–2790.

  27. 27.

    Heng BC, Ye CP, Liu H, et al. Loss of viability during freeze-thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to apoptosis rather than cellular necrosis. J Biomed Sci. 2006;13(3):433–445.

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Author information

Correspondence to Seung-Yup Ku MD, PhD or Young Min Choi MD, PhD.

Additional information

Authors’ Note

YYK, HJC and SKO carried out the in vitro analyses. HCL and SYM participated in the design of the in vitro studies. SYKandYMCconceived the study and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript. Supplemental figures and tables can be found at (http://rsx.sagepub.com/content/early/2011/01/06/1933719110385130/suppl/DC1).

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Kim, Y.Y., Ku, S., Liu, H. et al. Cryopreservation of Human Embryonic Stem Cells Derived-Cardiomyocytes Induced by BMP2 in Serum-Free Condition. Reprod. Sci. 18, 252–260 (2011). https://doi.org/10.1177/1933719110385130

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Keywords

  • human embryonic stem cells
  • cardiomyocytes
  • serum-free differentiation
  • BMP2
  • cryopreservation