The Derivation of Primary Human Epicardium‐Derived Cells

Caitlin Clunie‐O'Connor1, Anke M. Smits1, Charalambos Antoniades2, Angela J. Russell3, Derek M. Yellon4, Marie‐José Goumans5, Paul R. Riley6

1 These authors contributed equally to this study, 2 Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, 3 Department of Pharmacology, University of Oxford, Oxford, 4 The Hatter Cardiovascular Institute, University College, London, 5 Department of Molecular Cell Biology, Leiden University Medical Centre, Leiden, 6 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2C.5
DOI:  10.1002/9780470151808.sc02c05s35
Online Posting Date:  November, 2015
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Abstract

To develop therapeutic strategies for the regeneration of lost heart muscle after myocardial infarction (MI), a source of functional new muscle cells and associated coronary vessels must be identified. The epicardium is a source of several cardiovascular cell types during heart development and is widely regarded as a resident progenitor population, which becomes dormant during adulthood. In adult mice, MI induces epicardial reactivation characterized by an upregulation of fetal genes and subsequent epicardium derived cell (EPDC) proliferation, migration, and differentiation. Determining whether the epicardium can be therapeutically targeted following cardiovascular disease requires an in vitro system for the study of adult human EPDCs (hEPDCs). This protocol describes techniques to establish and maintain human epicardium explant cultures from patient‐derived right atrial appendage biopsies and documents methods to probe the resultant outgrowth of hEPDCs. The model facilitates a high‐throughput approach to either genetic or chemical phenotypic screening for drug‐like modifiers of hEPDC activation and potential cell fate. © 2015 by John Wiley & Sons, Inc.

Keywords: human; heart; epicardium; hEPDCs; regeneration

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Establishing hEPDC Cultures from Human Right Atrial Appendage Samples
  • Basic Protocol 2: hEPDC Culture and Cell Passaging
  • Support Protocol 1: Induction of hEPDC EMT with TGFβ and Characterization of Phenotype
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Establishing hEPDC Cultures from Human Right Atrial Appendage Samples

  Materials
  • 0.1% gelatin solution (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS) supplemented with penicillin/streptomycin (pen/strep) (see recipe)
  • Samples: this protocol utilizes human right atrial appendage samples excised from patients undergoing cardiac surgery collected in hEPDC culture medium supplemented with serum and antibiotics
  • hEPDC culture medium (see recipe), warmed (37°C)
  • 6‐well Corning Costar cell culture plates (34.8‐mm diameter; Corning, cat. no. 3516) or Primaria tissue culture plates (35‐mm diameter; Corning, cat. no. 353846)
  • 37°C incubator
  • 100‐mm × 25‐mm petri dishes for dissection, sterile
  • Stereomicroscope
  • Two sets of forceps (approximately 0.5‐mm tip size)
  • Swann Morton no. 21 disposable scalpels, sterile
  • Round VWR glass coverslips (30‐mm diameter; VWR, cat. no. 631‐1585)

Basic Protocol 2: hEPDC Culture and Cell Passaging

  Materials
  • 0.1% gelatin solution (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS; ThermoFisher Scientific, cat. no. 14190‐094)
  • hEPDC cultures (see protocol 1)
  • hEPDC culture medium (see recipe), warmed (37°C)
  • 0.05% Trypsin‐EDTA, phenol red, warmed (37°C)
  • SB‐435142 (Alk‐5 kinase inhibitor; see recipe)
  • Appropriate‐sized tissue culture plates or flasks
  • 37°C incubator
NOTE: When splitting hEPDC cultures, the passage ratio should be no higher than 1:2. Passaging hEPDCs at lower concentrations induces spontaneous EMT and loss of epithelial morphologyNOTE: Cultures should be 95% to 100% confluent (i.e., an epithelial sheet) before passaging.NOTE: Sometimes patches of the explant cultures may begin to spontaneously develop a mesenchymal phenotype. Scratching away the differentiated cells with a 200‐μl pipet tip and replacing with fresh hEPDC culture medium can help prevent further EMT.NOTE: Adding the Alk‐5 kinase inhibitor SB‐435142 to the medium at a concentration of 10 μM can help prevent EMT.

Support Protocol 1: Induction of hEPDC EMT with TGFβ and Characterization of Phenotype

  Materials
  • 0.1% gelatin solution (see recipe)
  • 0.05% Trypsin‐EDTA, phenol red, warmed (37°C)
  • hEPDC culture medium (see recipe), warmed (37°C)
  • Alk‐5 kinase inhibitor SB‐435142 (see recipe)
  • Recombinant Human TGFβ‐1 (R&D Systems, cat. no. 240‐B); working concentration 2 ng/ml (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 4% paraformaldehyde in PBS
  • 0.1% Triton‐X 100 in PBS (PBT; see recipe)
  • Block (see recipe)
  • Primary antibodies of choice (Table 2.5.1)
  • Appropriate secondary antibodies of choice
  • DAPI (5 μg/ml in PBS)
  • 50% glycerol in PBS (see recipe)
  • Clear nail polish
  • 24‐well Corning Costar cell culture plates (15.9‐mm diameter; Corning, cat. no. 3527)
  • 37°C incubator
  • Round 13‐mm sterile glass coverslips (Fisher Scientific, cat. no. 12392128)
  • Light‐proof microscope slide box
  • Parafilm (Sigma Aldrich, cat. no. P7793)
  • Superfrost Plus microscope slides (Thermo Scientific, cat. no. 4951PLUS4)
  • Fluorescence microscope
Table 2.0.1   MaterialsAntibodies and Stains Used for hEPDC Immunohistochemical Staining (see Fig. )

Antibody Supplier Clonality Source Dilution
B‐catenin Abcam Monoclonal Rabbit 1: 250
WT1 Abcam Monoclonal Rabbit 1:50
Alexa Fluor(R) 594 phalloidin Invitrogen Not applicable Not applicable 1:150

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Figures

Videos

Literature Cited

Literature Cited
  Bax, N.A.M., van Oorschot, A.A.M., Maas, S., Braun, J., van Tuyn, J., de Vries, A.A.F., Groot, A.C.G., and Goumans, M.‐J. 2011. In vitro epithelial‐to‐mesenchymal transformation in human adult epicardial cells is regulated by TGFβ‐signaling and WT1. Basic Res. Cardiol. 106:829‐847. doi: 10.1007/s00395‐011‐0181‐0.
  Braitsch, C.M., Kanisicak, O., van Berlo, J.H., Molkentin, J.D., and Yutzey, K.E. 2013. Differential expression of embryonic epicardial progenitor markers and localization of cardiac fibrosis in adult ischemic injury and hypertensive heart disease. J. Mol. Cell. Cardiol. 65C:108‐119. doi: 10.1016/j.yjmcc.2013.10.005.
  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. doi: 10.1038/nature06969.
  Chen, T.H.‐P., 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. Dev. Biol. 250:198‐207. doi: 10.1006/dbio.2002.0796.
  Limana, F., Zacheo, A., Mocini, D., Mangoni, A., Borsellino, G., Diamantini, A., De Mori, R., Battistini, L., Vigna, E., Santini, M., Loiaconi, V., Pompilio, G., Germani, A., and Capogrossi, M.C. 2007. Identification of myocardial and vascular precursor cells in human and mouse epicardium. Circ. Res. 101:1255‐1265. doi: 10.1161/CIRCRESAHA.107.150755.
  Moon, S.‐H., Kang, S.‐W., Park, S.‐J., Bae, D., Kim, S.J., Lee, H.‐A., Kim, K.S., Hong, K.‐S., Kim, J.S., Do, J.T., Byun, K.H., and Chung, H.M. 2013. The use of aggregates of purified cardiomyocytes derived from human ESCs for functional engraftment after myocardial infarction. Biomaterials 34:4013‐4026. doi: 10.1016/j.biomaterials.2013.02.022.
  Smart, N. and Riley, P.R. 2009. Derivation of epicardium‐derived progenitor cells (EPDCs) from adult epicardium. Curr. Protoc. Stem Cell Biol. Chapter 2:Unit2C.2.
  Smart, N., Bollini, S., Dubé, K.N., Vieira, J.M., Zhou, B., Davidson, S., Yellon, D., Riegler, J., Price, A.N., Lythgoe, M.F., Pu, W.T., and Riley, P.R. 2011. De novo cardiomyocytes from within the activated adult heart after injury. Nature 474:640‐644. doi: 10.1038/nature10188.
  Van Tuyn, J., Atsma, D.E., Winter, E.M., van der Velde‐van Dijke, I., Pijnappels, D.A., Bax, N.A.M., Knaän‐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. 2007. Epicardial cells of human adults can undergo an epithelial‐to‐mesenchymal transition and obtain characteristics of smooth muscle cells in vitro. Stem Cells 25:271‐278. doi: 10.1634/stemcells.2006‐0366.
  Van Wijk, B., Gunst, Q.D., Moorman, A.F.M., and van den Hoff, M.J.B. 2012. Cardiac Regeneration from Activated Epicardium. PLoS ONE 7:e44692. doi: 10.1371/journal.pone.0044692.
  Wessels, A. and Pérez‐Pomares, J.M. 2004. The epicardium and epicardially derived cells (EPDCs) as cardiac stem cells. Anat. Rec. A Discov. Mol. Cell Evol. Biol. 276:43‐57. doi: 10.1002/ar.a.10129.
  Winter, E.M. and Groot, A.C. G. 2007. Cardiovascular development: Towards biomedical applicability. Cell. Mol. Life Sci. 64:692‐703. doi: 10.1007/s00018‐007‐6522‐3.
  Yamaguchi, Y., Cavallero, S., Patterson, M., Shen, H., Xu, J., Kumar, S.R., and Sucov, H.M. 2015. Adipogenesis and epicardial adipose tissue: A novel fate of the epicardium induced by mesenchymal transformation and PPARγ activation. Proc. Natl. Acad. Sci. 112:2070‐2075. doi: 10.1073/pnas.1417232112.
  Zhou, B., Ma, Q., Rajagopal, S., Wu, S.M., Domian, I., Rivera‐Feliciano, J., Jiang, D., Gise, A. von, 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. doi: 10.1038/nature07060.
  Zhou, B., Honor, L.B., He, H., Ma, Q., Oh, J.‐H., Butterfield, C., Lin, R.‐Z., Melero‐Martin, J.M., Dolmatova, E., Duffy, H.S., Gise, A. von, Zhou, P., Hu, Y.W., Wang, G., Zhang, B., Wang, L., Hall, J.L., Moses, M.A., McGowan, F.X., and Pu, W.T. 2011. Adult mouse epicardium modulates myocardial injury by secreting paracrine factors. J. Clin. Invest. 121:1894‐1904. doi: 10.1172/JCI45529.
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