Isolating Embryonic Cardiac Progenitors and Cardiac Myocytes by Fluorescence-Activated Cell Sorting

  • Abdalla Ahmed
  • Paul Delgado-Olguin
Part of the Methods in Molecular Biology book series (MIMB, volume 1752)


Isolation of highly purified populations of embryonic cardiomyocytes enables the study of congenital cardiac phenotypes at the cellular level. Fluorescent-activated cell sorting (FACS) is normally used to isolate fluorescently tagged cells. Here we describe the isolation of differentiating mouse embryonic cardiac progenitors and cardiomyocytes at embryonic day (E) 9.5 and E13.5, respectively by FACS. Over 50,000 differentiating cardiac progenitors and 200,000 cardiomyocytes can be obtained in a single prep using the methods described.

Key words

Fluorescent-activated cell sorting Cardiac progenitor isolation Cardiomyocyte isolation Mouse embryo 



We thank The SickKids-UHN Flow Cytometry Facility for help with FACS, and The Centre for Phenogenomics (TCP) for mouse husbandry. This work was supported by the Heart and Stroke Foundation of Canada (G-17-0018613), the Natural Sciences and Engineering Research Council of Canada (NSERC) (500865), the Canadian Institutes of Health Research (CIHR) (PJT-149046), and Operational Funds from the Hospital for Sick Children to P.D.-O.


  1. 1.
    Loken MR, Herzenber LA (1975) Analysis of cell populations with a fluorescence-activated cell sorter. Ann N Y Acad Sci 254:163–171CrossRefPubMedGoogle Scholar
  2. 2.
    Han Y, Gu Y, Zhang AC, Lo YH (2016) Review: imaging technologies for flow cytometry. Lab Chip 16(24):4639–4647CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Herzenberg LA, Parks D, Sahaf B, Perez O, Roederer M, Herzenberg LA (2002) The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem 48(10):1819–1827PubMedGoogle Scholar
  4. 4.
    Pontén A, Walsh S, Malan D, Xian X, Schéele S, Tarnawski L, Fleischmann BK, Jovinge S (2013) FACS-based isolation, propagation and characterization of mouse embryonic cardiomyocytes based on VCAM-1 surface marker expression. PLoS One 8(12):e82403CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Dubois NC, Craft AM, Sharma P, Elliott DA, Stanley EG, Elefanty AG, Gramolini A, Keller G (2011) SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat Biotechnol 29(11):1011–1018CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Gantz JA, Palpant NJ, Welikson RE, Hauschka SD, Murry CE, Laflamme MA (2012) Targeted genomic integration of a selectable floxed dual fluorescence reporter in human embryonic stem cells. PLoS One 7(10):e46971CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lee MY, Sun B, Schliffke S, Yue Z, Ye M, Paavola J, Bozkulak EC, Amos PJ, Ren Y, Ju R, Jung YW, Ge X, Yue L, Ehrlich BE, Qyang Y (2012) Derivation of functional ventricular cardiomyocytes using endogenous promoter sequence from murine embryonic stem cells. Stem Cell Res 1:49–57CrossRefGoogle Scholar
  8. 8.
    Bergmann O, Zdunek S, Alkass K, Druid H, Bernard S, Frisén J (2011) Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover. Exp Cell Res 317(2):188–194CrossRefPubMedGoogle Scholar
  9. 9.
    Gilsbach R, Preissl S, Grüning BA, Schnick T, Burger L, Benes V, Würch A, Bönisch U, Günther S, Backofen R, Fleischmann BK, Schübeler D, Hein L (2014) Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease. Nat Commun 5:5288CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Delgado-Olguín P, Huang Y, Li X, Christodoulou D, Seidman CE, Seidman JG, Tarakhovsky A, Bruneau BG (2012) Epigenetic repression of cardiac progenitor gene expression by Ezh2 is required for postnatal cardiac homeostasis. Nat Genet 44(3):343–347CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Sah R, Mesirca P, Mason X, Gibson W, Bates-Withers C, Van den Boogert M, Chaudhuri D, Pu WT, Mangoni ME, Clapham DE (2013) Timing of myocardial trpm7 deletion during cardiogenesis variably disrupts adult ventricular function, conduction, and repolarization. Circulation 128(2):101–114CrossRefPubMedGoogle Scholar
  12. 12.
    Hamdani N, Kooij V, van Dijk S, Merkus D, Paulus WJ, Remedios CD, Duncker DJ, Stienen GJ, van der Velden J (2008) Sarcomeric dysfunction in heart failure. Cardiovasc Res 77(4):649–658CrossRefPubMedGoogle Scholar
  13. 13.
    Song L, Zhao M, Wu B, Zhou B, Wang Q, Jiao K (2011) Cell autonomous requirement of endocardial Smad4 during atrioventricular cushion development in mouse embryos. Dev Dyn 240(1):211–220CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Fan D, Takawale A, Lee J, Kassiri Z (2012) Cardiac fibroblasts, fibrosis and extracellular matrix remodeling in heart disease. Fibrogenesis Tissue Repair 5(1):15CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Lints TJ, Parsons LM, Hartley L, Lyons I, Harvey RP (1993) Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119(2):419–431PubMedGoogle Scholar
  16. 16.
    Hsiao EC, Yoshinaga Y, Nguyen TD, Musone SL, Kim JE, Swinton P, Espineda I, Manalac C, deJong PJ, Conklin BR (2008) Marking embryonic stem cells with a 2A self-cleaving peptide: a NKX2-5 emerald GFP BAC reporter. PLoS One 3(7):e2532CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Translational MedicineThe Hospital for Sick Children, Peter Gilgan Centre for Research and LearningTorontoCanada
  2. 2.Department of Molecular GeneticsUniversity of TorontoTorontoCanada
  3. 3.Translational MedicineThe Hospital for Sick ChildrenTorontoCanada
  4. 4.Department of Molecular GeneticsUniversity of TorontoTorontoCanada
  5. 5.Heart & Stroke/Richard Lewar Centres of Excellence in Cardiovascular ResearchTorontoCanada

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