Validation of the cardiosphere method to culture cardiac progenitor cells from myocardial tissue.
BACKGROUND
At least four laboratories have shown that endogenous cardiac progenitor cells (CPCs) can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs). Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing) to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential) of CDCs.


RESULTS
CPCs were expanded from human endomyocardial biopsies (n = 160), adult bi-transgenic MerCreMer-Z/EG mice (n = 6), adult C57BL/6 mice (n = 18), adult GFP(+) C57BL/6 transgenic mice (n = 3), Yucatan mini pigs (n = 67), adult SCID beige mice (n = 8), and adult Wistar-Kyoto rats (n = 80). Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit(+)), endothelial cells (CD31(+), CD34(+)), and mesenchymal cells (CD90(+)). Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential.


CONCLUSIONS
This study demonstrates that direct culture and expansion of CPCs from myocardial tissue is simple, straightforward, and reproducible when appropriate techniques are used.
Davis
Darryl R
DR
Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.
Zhang
Yiqiang
Y
Smith
Rachel R
RR
Cheng
Ke
K
Terrovitis
John
J
Malliaras
Konstantinos
K
Li
Tao-Sheng
TS
White
Anthony
A
Makkar
Raj
R
Marbán
Eduardo
E
D000328:Adult; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D019070:Cell Lineage; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D008822:Mice, Transgenic; D008875:Middle Aged; D009206:Myocardium / Q000378:metabolism / Q000473:pathology*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D011921:Rats, Inbred WKY; D013234:Stem Cells / Q000166:cytology*