Immunohistochemical and flow cytometric analysis of long-term label-retaining cells in the adult heart.
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.
Meinhardt
Andrea
A
Department of Cardiology, CHUV University Hospital, Lausanne, Switzerland.
Spicher
Albert
A
Roehrich
Marc-Estienne
ME
Glauche
Ingmar
I
Vogt
Pierre
P
Vassalli
Giuseppe
G
D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism; D000950:Antigens, Ly / Q000378:metabolism; D001973:Bromodeoxyuridine; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D005434:Flow Cytometry; D007150:Immunohistochemistry; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D013194:Staining and Labeling; D013234:Stem Cells / Q000378:metabolism
D000943:Antigens, Differentiation; D000950:Antigens, Ly; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D001973:Bromodeoxyuridine