Differentiation of Mouse Embryonic Stem Cells into Cardiomyocytes via the Hanging‐Drop and Mass Culture Methods

Christopher J. Fuegemann1, Ajoy K. Samraj2, Stuart Walsh2, Bernd K. Fleischmann1, Stefan Jovinge3, Martin Breitbach1

1 Institute of Physiology I, Life & Brain Center, University of Bonn, Bonn, Germany, 2 Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund, Sweden, 3 Department of Cardiology, Lund University Hospital, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund, Sweden
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.11
DOI:  10.1002/9780470151808.sc01f11s15
Online Posting Date:  December, 2010
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Abstract

Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial‐, ventricular‐, nodal‐like cells). The duration of cell culture determines whether preferentially early– or late–developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes. Curr. Protoc. Stem Cell Biol. 15:1F.11.1‐1F.11.13. © 2010 by John Wiley & Sons, Inc.

Keywords: mouse embryonic stem cells (mESCs); differentiation; hanging drops; mass culture; embryoid bodies; cardiomyocytes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Vitro Differentiation of mESCs Via Hanging Drops
  • Basic Protocol 2: In Vitro Differentiation of mESCs Via Mass Culture
  • Support Protocol 1: Freezing mESCs
  • Support Protocol 2: Thawing mESCs
  • Support Protocol 3: Culturing mESCs
  • Support Protocol 4: Depletion of Feeder Cells (“Pre‐Plating”)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Vitro Differentiation of mESCs Via Hanging Drops

  Materials
  • PBS without Mg2+ or Ca2+ (CMF‐PBS; Invitrogen, cat. no. 18912‐014), sterile
  • Pluripotent mESCs ( protocol 5)
  • mESC differentiation medium (see recipe)
  • Bacterial dishes or ultra‐low‐attachment dishes (Greiner, cat. no. 633180), 100‐mm diameter
  • Omni Trays (optional; Nunc, cat. no. 242811)
  • Horizontal shaker
  • 100‐mm diameter tissue culture dishes (BD Falcon, cat. no. 35‐3003), gelatin‐coated (see recipe) or noncoated
  • Additional reagents and equipment for culturing mESCs, including trypsinization ( protocol 5), for feeder cell depletion ( protocol 6), and for determination of viable cell number (unit 1.3)

Basic Protocol 2: In Vitro Differentiation of mESCs Via Mass Culture

  Materials
  • Pluripotent mESCs ( protocol 5)
  • PBS without Mg2+ or Ca2+ (CMF‐PBS; Invitrogen, cat. no. 18912‐014), sterile
  • mESC differentiation medium (recipe see below)
  • Bacterial dishes or ultra‐low‐attachment dishes (Greiner, cat. no. 633180), 100‐mm diameter
  • Omni Trays (optional; Nunc, cat. no. 242811)
  • Horizontal shaker: Gesellschaft für Labortechnik mbH (GFL, http://www.gfl.de/)
  • 50‐ml conical tubes
  • Microscope: 5×, 10×, and 20× oculars
  • 100‐mm diameter tissue culture dishes (BD Falcon, cat. no. 35‐3003), gelatin‐coated (see recipe) or noncoated
  • Additional reagents and equipment for culturing mESCs, including trypsinization ( protocol 5), for feeder cell depletion ( protocol 6), and for determination of viable cell number (unit 1.3)

Support Protocol 1: Freezing mESCs

  Materials
  • Cultures of mESC
  • mESC culture medium (see recipe)
  • Freezing medium: 80% (v/v) FBS/20% (v/v) DMSO, ice cold
  • Liquid N 2
  • Cryotubes (Nunc, cat. no. 375418)
  • 15‐ and 50‐ml conical tubes
  • Freezing container (Nalgene 5100‐0001)
  • Liquid nitrogen container
  • Additional reagents and equipment for trypsinizing mESCs ( protocol 4) and determining viable cell number (unit 1.3)

Support Protocol 2: Thawing mESCs

  Materials
  • Pluripotent mouse embryonic stem cells (mESCs; protocol 5), frozen ( protocol 3)
  • mESC culture medium (see recipe)
  • MEFs, mitotically inactivated (γ irradiated or mitomycin C treated; unit 1.3)
  • 15‐ and 50‐ml conical centrifuge tubes
  • Centrifuge
  • 25‐cm2 tissue culture flasks
  • Additional reagents and equipment for counting viable cells (unit 1.3)

Support Protocol 3: Culturing mESCs

  Materials
  • Cultures of mESC in 25‐cm2 or 75‐cm2 flasks
  • Phosphate‐buffered saline, Ca2+ and Mg2+ free (CMF‐PBS; Invitrogen, cat. no. 18912‐014), sterile
  • Trypsin‐EDTA (Invitrogen, cat. no. 25300)
  • mESC culture medium (see recipe)
  • Mitotically inactivated MEF feeder cells (unit 1.3)
  • 15‐ and 50‐ ml conical tubes (e.g., BD Falcon)
  • Centrifuge
  • 25‐cm2 tissue culture flasks
  • Additional reagents and equipment for preparing mitotically inactivated MEF feeder layers (unit 1.3)

Support Protocol 4: Depletion of Feeder Cells (“Pre‐Plating”)

  Materials
  • Culture of mESCs
  • mESC culture medium (see recipe)
  • 100‐mm gelatin‐coated tissue culture dishes (see recipe)
  • 15‐ml conical centrifuge tubes
  • Additional reagents and equipment for culturing mESCs, including trypsinization ( protocol 5)
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Figures

Videos

Literature Cited

Literature Cited
   Boheler, K.R., Czyz, J., Tweedie, D., Yang, H.T., Anisimov, S.V., and Wobus, A.M. 2002. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ. Res. 91:189‐201.
   Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W., and 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.
   Hescheler, J., Fleischmann, B.K., Lentini, S., Maltsev, V.A., Rohwedel, J., Wobus, A.M., and Addicks, K. 1997. Embryonic stem cells: A model to study structural and functional properties in cardiomyogenesis. Cardiovasc. Res. 36:149‐162.
   Kattman, S.J., Huber, T.L., and Keller, G.M. 2006. Multipotent flk‐1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages. Dev. Cell 11:723‐732.
   Klug, M.G., Soonpaa, M.H., Koh, G.Y., and Field, L.J. 1996. Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J. Clin. Invest. 98:216‐224.
   Kolossov, E., Bostani, T., Roell, W., Breitbach, M., Pillekamp, F., Nygren, J.M., Sasse, P., Rubenchik, O., Fries, J.W., Wenzel, D., Geisen, C., Xia, Y., Lu, Z., Duan, Y., Kettenhofen, R., Jovinge, S., Bloch, W., Bohlen, H., Welz, A., Hescheler, J., Jacobsen, S.E., and Fleischmann, B.K. 2006. Engraftment of engineered ES cell‐derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium. J. Exp. Med. 203:2315‐2327.
   Kuzmenkin, A., Liang, H., Xu, G., Pfannkuche, K., Eichhorn, H., Fatima, A., Luo, H., Saric, T., Wernig, M., Jaenisch, R., and Hescheler, J. 2009. Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro. FASEB J. 23:4168‐4180.
   Laflamme, M.A., Chen, K.Y., Naumova, A.V., Muskheli, V., Fugate, J.A., Dupras, S.K., Reinecke, H., Xu, C., Hassanipour, M., Police, S., O'Sullivan, C., Collins, L., Chen, Y., Minami, E., Gill, E.A., Ueno, S., Yuan, C., Gold, J., and Murry, C.E. 2007. Cardiomyocytes derived from human embryonic stem cells in pro‐survival factors enhance function of infarcted rat hearts. Nat. Biotechnol. 25:1015‐1024.
   Nagy, A., Rossant, J., Nagy, R., Bramow‐Newerly, W., and Roder, J.C. 1993. Derivation of completely cell culture‐derived mice from early‐passage embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 90:8424‐8428.
   Puceat, M. 2008. Protocols for cardiac differentiation of embryonic stem cells. Methods 45:168‐171.
   Robbins, J., Gulick, J., Sanchez, A., Howles, P., and Doetschman, T. 1990. Mouse embryonic stem cells express the cardiac myosin heavy chain genes during development in vitro. J. Biol. Chem. 265:11905‐11909.
   Sachinidis, A., Fleischmann, B.K., Kolossov, E., Wartenberg, M., Sauer, H., and Hescheler, J. 2003. Cardiac specific differentiation of mouse embryonic stem cells. Cardiovasc. Res. 58:278‐291.
   Sartiani, L., Bettiol, E., Stillitano, F., Mugelli, A., Cerbai, E., and Jaconi, M.E. 2007. Developmental changes in cardiomyocytes differentiated from human embryonic stem cells: A molecular and electrophysiological approach. Stem Cells 25:1136‐1144.
   Singla, D.K. and Sobel, B.E. 2005. Enhancement by growth factors of cardiac myocyte differentiation from embryonic stem cells: A promising foundation for cardiac regeneration. Biochem. Biophys. Res. Commun. 335:637‐642.
   Snir, M., Kehat. I., Gepstein. A., Coleman. R., Itskovitz‐Eldor. J., Livne, E., and Gepstein, L. 2003. Assessment of the ultrastructural and proliferative properties of human embryonic stem cell‐derived cardiomyocytes. Am J. Physiol. Heart Circ. Physiol. 285:H2355‐H2363.
   Wobus, A.M., Wallukat, G., and Hescheler, J. 1991. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation 48:173‐182.
   Wobus, A.M., Kaomei, G., Shan, J., Wellner, M.C., Rohwedel, J., Ji, G., Fleischmann, B., Katus, H.A., Hescheler, J., and Franz, W.M. 1997. Retinoic acid accelerates embryonic stem cell‐derived cardiac differentiation and enhances development of ventricular cardiomyocytes. J. Mol. Cell Cardiol. 29:1525‐1539.
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