Isolation and Expansion of Cardiosphere‐Derived Stem Cells

Jun‐Jie Tan1, Carolyn A. Carr1, Daniel J. Stuckey1, Georgina M. Ellison2, Elisa Messina3, Alessandro Giacomello3, Kieran Clarke1

1 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom, 2 Stem Cell and Molecular Physiology Laboratory, Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, United Kingdom, 3 Department of Experimental Medicine, Cenci‐Bolognetti Foundation, Pasteur Institute, University of Rome ‘Sapienza,’ Rome, Italy
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2C.3
DOI:  10.1002/9780470151808.sc02c03s16
Online Posting Date:  February, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The isolation and in vitro expansion of stem cells from the adult heart may provide a cell therapy for regenerating damaged myocardium. Cardiac stem cells can be isolated via magnetic or fluorescent cell sorting using specific cell‐surface markers, including c‐kit, Sca‐1, and Isl‐1. Because these isolation methods yield relatively few cells, substantial in vitro expansion is required to generate sufficient cell numbers for therapy. An alternative method uses cells spontaneously shed from cultured heart explants, which are harvested, induced to form cardiospheres, and expanded as a monolayer for several passages. This method for generating therapeutically relevant numbers of cells in a shorter time period than cell surface marker–based isolations is ideally suited for autologous cardiac stem cell therapy after myocardial infarction. Curr. Protoc. Stem Cell Biol. 16:2C.3.1‐2C.3.12. © 2011 by John Wiley & Sons, Inc.

Keywords: cardiac stem cells; cardiospheres; cardiosphere‐derived cells; myocardial regeneration therapy

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Neonatal Rat Heart Excision, Processing, and Explant Culture
  • Basic Protocol 2: Cardiosphere Culture
  • Basic Protocol 3: Culture and Expansion of Cardiosphere‐Derived Cells
  • Support Protocol 1: Characterization of Cardiosphere‐Derived Cells by Immunofluorescent Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Neonatal Rat Heart Excision, Processing, and Explant Culture

  Materials
  • 1 mg/ml fibronectin stock solution from bovine plasma (Sigma)
  • Dulbecco's phosphate‐buffered saline without CaCl 2 and MgCl 2 (DPBS, Sigma)
  • Complete explant medium (CEM, see recipe)
  • 0.05% trypsin/EDTA (Invitrogen), 37°C
  • Sprague‐Dawley neonatal rat
  • 60‐mm sterile tissue culture–treated petri dishes
  • Dumont no.7 forceps, sterilized
  • Fine iris scissors, sterilized
  • Inverted light microscope

Basic Protocol 2: Cardiosphere Culture

  Materials
  • Explant‐derived cells with 80% to 90% confluency ( protocol 1)
  • 2 mg/ml poly‐D‐lysine (Sigma, cat. no. P7280) stock solution in DPBS (store in 100‐µl aliquots at –20°C)
  • Dulbecco's phosphate‐buffered saline without CaCl 2 and MgCl 2 (DPBS, Sigma)
  • Versene (Invitrogen)
  • 0.05% trypsin/EDTA (Invitrogen)
  • Complete explant medium (CEM, see recipe)
  • Cardiosphere growth medium (CGM, see recipe)
  • Inverted light microscope
  • 24‐well tissue culture plates (see recipe)
  • 50‐ml conical centrifuge tube
  • Benchtop centrifuge: e.g., ALC PK121R multispeed centrifuge
  • Hemacytometer (also see unit 1.3)
  • Additional reagents and equipment for counting cells using a hemacytometer (unit 1.3)

Basic Protocol 3: Culture and Expansion of Cardiosphere‐Derived Cells

  Materials
  • 1 mg/ml fibronectin stock solution from bovine plasma (Sigma)
  • Dulbecco's phosphate‐buffered saline without CaCl 2 and MgCl 2 (DPBS, Sigma)
  • Cardiospheres in 24‐well plate ( protocol 2)
  • Complete explant medium (CEM)
  • Trypsin, 0.05% with sodium EDTA, liquid
  • Cardiosphere growth medium (see recipe)
  • 25‐cm2 cell culture flasks
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • 1‐ml (P‐1000) pipet tip
  • Hemacytometer inverted light microscope

Support Protocol 1: Characterization of Cardiosphere‐Derived Cells by Immunofluorescent Staining

  Materials
  • Fibronectin (Sigma)
  • Rat cardiosphere‐derived cells (CDCs; protocol 3)
  • Dulbecco's phosphate‐buffered saline without CaCl 2 and MgCl 2 (DPBS, Sigma)
  • 4% (w/v) paraformaldehyde
  • Washing solution: DPBS without CaCl 2 and MgCl 2, containing 0.1% (v/v) Tween 20
  • Permeabilization solution: DPBS without CaCl 2 and MgCl 2, containing 0.1% (v/v) Tween 20 and 0.1% (v/v) Triton X‐100
  • Blocking solution: DPBS without CaCl 2 and MgCl 2, containing 0.1% (v/v) Tween 20 and 0.1% (v/v) Triton X‐100
  • Primary and secondary antibodies (see Table 2.3.1 for suppliers and dilution factors)
  • 1 µg/ml DAPI (Sigma)
  • Vectashield mounting medium (Vector Laboratories)
  • 4‐well chamber slides (Sigma)
  • Platform shaker
  • Humidified chamber: e.g., Tupperware box with lid containing moistened paper towels
  • 22 × 50 mm coverslips
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Barile, L., Messina, E., Giacomello, A., and Marban, E. 2007. Endogenous cardiac stem cells. Prog. Cardiovasc. Dis. 50:31‐48.
   Beltrami, A.P., Barlucchi, L., Torella, D., Baker, M., Limana, F., Chimenti, S., Kasahara, H., Rota, M., Musso, E., Urbanek, K., Leri, A., Kajstura, J., Nadal‐Ginard, B., and Anversa, P. 2003. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 114:763‐776.
   Cowie, M.R., Wood, D.A., Coats, A.J.S., Thompson, S.G., Suresh, V., Poole‐Wilson, P.A., and Sutton, G.C. 2000. Survival of patients with a new diagnosis of heart failure: A population based study. Heart 83:505‐510.
   Davis, D.R., Ruckdeschel Smith, R., and Marban, E. 2010. Human cardiospheres are a source of stem cells with cardiomyogenic potential. Stem Cells 28:903‐904.
   Itzhaki‐Alfia, A., Leor, J., Raanani, E., Sternik, L., Spiegelstein, D., Netser, S., Holbova, R., Pevsner‐Fischer, M., Lavee, J., and Barbash, I.M. 2009. Patient characteristics and cell source determine the number of isolated human cardiac progenitor cells. Circulation 120:2559‐2566.
   Lipinski, M.J., Biondi‐Zoccai, G.G., Abbate, A., Khianey, R., Sheiban, I., Bartunek, J., Vanderheyden, M., Kim, H.S., Kang, H.J., Strauer, B.E., and Vetrovec, G.W. 2007. Impact of intracoronary cell therapy on left ventricular function in the setting of acute myocardial infarction: A collaborative systematic review and meta‐analysis of controlled clinical trials. J. Am. Coll. Cardiol. 50:1761‐1767.
   Matsuura, K., Nagai, T., Nishigaki, N., Oyama, T., Nishi, J., Wada, H., Sano, M., Toko, H., Akazawa, H., Sato, T., Nakaya, H., Kasanuki, H., and Komuro, I. 2004. Adult cardiac Sca‐1‐positive cells differentiate into beating cardiomyocytes. J. Biol. Chem. 279:11384‐11391.
   Menasche, P. 2007. Skeletal myoblasts as a therapeutic agent. Progr. Cardiovasc. Dis. 50:7‐17.
   Messina, E., De Angelis, L., Frati, G., Morrone, S., Chimenti, S., Fiordaliso, F., Salio, M., Battaglia, M., Latronico, M.V., Coletta, M., Vivarelli, E., Frati, L., Cossu, G., and Giacomello, A. 2004. Isolation and expansion of adult cardiac stem cells from human and murine heart. Circulation Res. 95:911‐921.
   Murry, C.E. and Keller, G. 2008. Differentiation of embryonic stem cells to clinically relevant populations: Lessons from embryonic development. Cell 132:661‐680.
   Oh, H., Bradfute, S.B., Gallardo, T.D., Nakamura, T., Gaussin, V., Mishina, Y., Pocius, J., Michael, L.H., Behringer, R.R., Garry, D.J., Entman, M.L., and Schneider, M.D. 2003. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc. Natl. Acad. Sci. U.S.A. 100:12313‐12318.
   Orlic, D. 2003. Adult bone marrow stem cells regenerate myocardium in ischemic heart disease. Ann. New York Acad. Sci. :152‐157.
   Schachinger, V., Erbs, S., Elsasser, A., Haberbosch, W., Hambrecht, R., Holschermann, H., Yu, J., Corti, R., Mathey, D.G., Hamm, C.W., Süselbeck, T., Assmus, B., Tonn, T. Dimmeler, S., and Zeiher, A.M. 2006. Intracoronary bone marrow‐derived progenitor cells in acute myocardial infarction. N. Engl. J. Med. 355:1210‐1221.
   Smith, R.R., Barile, L., Cho, H.C., Leppo, M.K., Hare, J.M., Messina, E., Giacomello, A., Abraham, M.R., and Marban, E. 2007. Regenerative potential of cardiosphere‐derived cells expanded from percutaneous endomyocardial biopsy specimens. Circulation 115:896‐908.
   Tang, Y.L., Zhu, W., Cheng, M., Chen, L., Zhang, J., Sun, T., Kishore, R., Phillips, M.I., Losordo, D.W., and Qin, G. 2009. Hypoxic preconditioning enhances the benefit of cardiac progenitor cell therapy for treatment of myocardial infarction by inducing CXCR4 expression. Circulation Res. 104:1209‐1216.
   Tateishi, K., Ashihara, E., Honsho, S., Takehara, N., Nomura, T., Takahashi, T., Ueyama, T., Yamagishi, M., Yaku, H., Matsubara, H., and Oh, H. 2007. Human cardiac stem cells exhibit mesenchymal features and are maintained through Akt/GSK‐3beta signaling. Biochem. Biophys. Res. Commun. 352:635‐641.
   Torella, D., Ellison, G.M., Mendez‐Ferrer, S., Ibanez, B., and Nadal‐Ginard, B. 2006. Resident human cardiac stem cells: Role in cardiac cellular homeostasis and potential for myocardial regeneration. Nat. Clin. Practice 3:S8‐S13.
Internet Resources
   http://www.heartstats.org/datapage.asp?id=752
  Online version of Cowie et al. (); also see Literature Cited.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library