Derivation of Epicardium‐Derived Progenitor Cells (EPDCs) from Adult Epicardium

Nicola Smart1, Paul R. Riley1

1 UCL Institute of Child Health, London, United Kingdom
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2C.2
DOI:  10.1002/9780470151808.sc02c02s8
Online Posting Date:  February, 2009
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The epicardium has, like the other cell lineages of the terminally differentiated adult heart, long been regarded as quiescent, incapable of migration or differentiation. In contrast, the embryonic epicardium possesses an innate ability to proliferate, migrate, and differentiate into a number of mature cardiovascular cell types, including vascular smooth muscle cells, fibroblasts, cardiomyocytes, and, arguably, some endothelial cells. In recapitulating its essential developmental role, we recognized the ability of the actin‐binding peptide thymosin β4 (Tβ4) to induce epicardium‐derived progenitor cell (EPDC) migration from adult heart and noted the derivation of cell types originating from embryonic epicardium. This protocol provides a means of enabling adult EPDC outgrowth and culture. We establish a model system in which to study the ability of factors to influence the migration of vascular precursors and their differentiation and to move towards screening of small molecules ex vivo prior to clinical trials of therapeutic cardiac repair. Curr. Protoc. Stem Cell Biol. 8:2C.2.1‐2C.2.9. © 2009 by John Wiley & Sons, Inc.

Keywords: epicardium; adult EPDCs; thymosin β4; adult heart

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Table of Contents

  • Introduction
  • Basic Protocol 1: Thymosin β4–Induced Outgrowth of Adult Epicardium‐Derived Cells (EPDCs)
  • Support Protocol 1: Characterization of EPDC Phenotypes by Immunofluorescence
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Thymosin β4–Induced Outgrowth of Adult Epicardium‐Derived Cells (EPDCs)

  • 0.1% gelatin solution (see recipe)
  • 8‐ to 12‐week‐old adult mice (C57Bl/6 strain used; other strains and ages not tested)
  • Dulbecco's phosphate‐buffered saline (DPBS; Invitrogen, cat. no. 14190)
  • EPDC culture medium (see recipe) supplemented with 100 ng/ml thymosin β4 (see recipe)
  • Tissue culture dishes or plates of desired size for culture (Table 2.2.1) and (optionally) glass coverslips of the appropriate size (also in Table 2.2.1)
  • Forceps (0.5‐mm approximate tip size), sterile
  • Dissection scissors, sterile
  • Sterile 60‐ or 100‐mm culture/bacteriological dish (not gelatin coated) for dissection
  • Scalpel blade
  • Humidified 37°C, 5% CO 2 incubator
    Table 2.0.1   MaterialsRecommended Parameters for EPDC Culture in Various Plate Formats

    TC plate format Culture volume (ml) a Coverslip size (mm) Amount tissue/well
    12‐well plate 0.8 13 1/8 heart
    6‐well plate 2.0 18 1/4 heart
    35‐mm dish 2.0 18 1/4 heart
    60‐mm dish 4.0 Not recommended 1/2 heart
    100‐mm dish 10.0 Not recommended 1 to 2 hearts

     aVolumes given apply to each well of multiwall plates.
  • Additional reagents and equipment for sacrifice of mice by cervical dislocation (Donovan and Brown, )

Support Protocol 1: Characterization of EPDC Phenotypes by Immunofluorescence

  • Heart explants cultured on glass coverslips ( protocol 1)
  • 4% (w/v) paraformaldehyde in PBS (freshly prepared)
  • Phosphate‐buffered saline (PBS; prepared according to manufacturer's instructions from PBS tablets; Sigma, cat. no. P‐4417)
  • Blocking solution containing 0.1% (v/v) Triton X‐100
  • Blocking solution (see recipe)
  • Primary antibodies of choice (refer to Table 2.2.2)
  • Appropriate fluorochrome‐conjugated secondary antibody (against Ig of species in which primary antibody was raised)
  • 5 µg/ml Hoechst 33342 in PBS
  • Suitable commercially available mounting medium or 50% (v/v) glycerol in PBS
  • Microscope slides
  • Fluorescence microscope with appropriate filters for fluorochrome used
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Literature Cited

Literature Cited
   Bock‐Marquette, I., Saxena, A., White, M.D., Dimaio, J.M., and Srivastava, D. 2004. Thymosin β4 activates integrin‐linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature 432:466‐472.
   Cai, C.L., Martin, J.C., Sun, Y., Cui, L., Wang, L., Ouyang, K., Yang, L., Bu, L., Liang, X., Zhang, X., Stallcup, W.B., Denton, C.P., McCulloch, A., Chen, J., and Evans, S.M. 2008. A myocardial lineage derives from Tbx18 epicardial cells. Nature 454:104‐108.
   Chen, T.H., Chang, T.C., Kang, J.O., Choudhary, B., Makita, T., Tran, C.M., Burch, J.B.E., Eid, H., and Sucov, H.M. 2002. Epicardial induction of fetal cardiomyocyte proliferation via a retinoic acid‐inducible trophic factor. Devel. Biol. 250:198‐207.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Perez‐Pomares, J.M., Mironov, V., Guadix, J., Macias, D., Markwald, R., and Munoz‐Chapuli, R. 2006. In vitro self‐assembly of proepicardial cell aggregates: An embryonic vasculogenic model for vascular tissue engineering. Anat. Rec. Part A 288A:700‐713.
   Smart, N. and Riley, P.R. 2008. The stem cell movement. Circ. Res. 102:1155‐1168.
   Smart, N., Risebro, C.A., Melville, A.A., Moses, K., Schwartz, R.J., Chien, K.R., and Riley, P.R. 2007. Thymosin beta‐4 is essential for coronary vessel development and promotes neovascularization via adult epicardium. Ann. N.Y. Acad. Sci. 1112:171‐188.
   Srivastava, D. and Ivey, K.N. 2006. Potential of stem‐cell‐based therapies for heart disease. Nature 441:1097‐1099.
   van Tuyn, J., Atsma, D.E., Winter, E.M., van der Velde‐van Dijke, I., Pijnappels, D.A., Bax, N.A.M., Knaan‐Shanzer, S., Gittenberger–de Groot, A.C., Poelmann, R.E., van der Laarse, A., van der Wall, E.E., Schalij, M.J., and de Vries, A.A. 2006. Epicardial cells of human adults can undergo an epithelial‐to‐mesenchymal transition and obtain characteristics of smooth muscle cells in vitro. Stem Cells 2006‐0366.
   Zhou, B., Ma, Q., Rajagopal, S., Wu, S.M., Domian, I., Rivera‐Feliciano, J., Jiang, D., von, G.A., Ikeda, S., Chien, K.R., and Pu, W.T. 2008. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature 454:109‐113.
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