Immunohistochemical and flow cytometric analysis of long-term label-retaining cells in the adult heart

Stem Cells Dev. 2011 Feb;20(2):211-22. doi: 10.1089/scd.2009.0203. Epub 2010 Sep 14.


Cardiac-resident stem/progenitor cells have been identified based on expression of stem cell-associated antigens. However, no single surface marker allows to identify a definite cardiac stem/progenitor cell entity. Hence, functional stem cell markers have been extensively searched for. In homeostatic systems, stem cells divide infrequently and therefore retain DNA labels such as 5-bromo-2'-deoxyuridine, which are diluted with division. We used this method to analyze long-term label-retaining cells in the mouse heart after 14 days of 5-bromo-2'-deoxyuridine administration. Labeled cells were detected using immunohistochemical and flow-cytometric methods after varying chasing periods up to 12 months. Using mathematical models, the observed label dilution could consistently be described in the context of a 2-population model, whereby a population of rapidly dividing cells accounted for an accelerated early decline, and a population of slowly dividing cells accounted for decelerated dilution on longer time scales. Label-retaining cells were preferentially localized in the atria and apical region and stained negative for markers of the major cell lineages present in the heart. Most cells with long-term label-retention expressed stem cell antigen-1 (Sca-1). Sca-1(+)CD31(-) cells formed cell aggregates in culture, out of which lineage-negative (Lin(-))Sca-1(+)CD31(-) cells emerged, which could be cultured for many passages. These cells formed cardiospheres and showed differentiation potential into mesenchymal cell lineages. When cultured in cardiomyogenic differentiation medium, they expressed cardiac-specific genes. In conclusion, recognition of slow-cycling cells provides functional evidence of stem/progenitor cells in the heart. Lin(-)Sca-1(+)CD31(-) cardiac-derived progenitors have a potential for differentiation into cardiomyogenic and mesenchymal cell lineages.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Differentiation / metabolism
  • Antigens, Ly / metabolism
  • Bromodeoxyuridine*
  • Cell Differentiation
  • Cell Lineage
  • Cells, Cultured
  • Flow Cytometry
  • Immunohistochemistry
  • Male
  • Membrane Proteins / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Myocardium / cytology
  • Myocardium / metabolism*
  • Staining and Labeling
  • Stem Cells / metabolism


  • Antigens, Differentiation
  • Antigens, Ly
  • Ly6a protein, mouse
  • Membrane Proteins
  • Bromodeoxyuridine