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A highly sensitive and accurate method to quantify absolute numbers of c-kit+ cardiac stem cells following transplantation in mice

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Abstract

Although transplantation of c-kit+ cardiac stem cells (CSCs) alleviates post-myocardial infarction left ventricular dysfunction, there are no reliable methods that enable measurement of the absolute number of CSCs that persist in the recipient heart. To overcome this limitation, we developed a highly sensitive and accurate method to quantify the absolute number of murine CSCs after transplantation. This method has two unique features: (1) real-time PCR-based detection of a novel male-specific, multiple-copy gene, Rbmy, which significantly increases the sensitivity of detection of male donor cells in a female recipient, and (2) an internal standard, which permits quantification of the absolute number of CSCs as well as the total number of cells in the recipient organ. Female C57BL/6 mice underwent coronary occlusion and reperfusion; 2 days later, 105 male mouse CSCs were injected intramyocardially. Tissues were analyzed by real-time PCR at serial time points. In the risk region, >75 % of CSCs present at 5 min were lost in the ensuing 24 h; only 7.6 ± 2.1 % of the CSCs present at 5 min could still be found at 7 days after transplantation and only 2.8 ± 0.5 % (i.e., 1,224 ± 230 cells/heart) at 35 days. Thus, even after direct intramyocardial injection, the total number of CSCs that remain in the murine heart is minimal (at 24 h, ~10 % of the cells injected; at 35 days, ~1 %). This new quantitative method of stem cell detection, which enables measurement of absolute cell number, should be useful to optimize cell-based therapies, not only for CSCs but also for other stem cells and other organs.

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Acknowledgments

This study was supported in part by NIH grants R01 HL55757, HL-70897, HL-76794, and P01HL78825.

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

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Authors and Affiliations

  1. Department of Medicine, Institute of Molecular Cardiology, University of Louisville, Louisville, KY, 40202, USA

    Kyung U. Hong, Qian-Hong Li, Yiru Guo, Nikita S. Patton, Afsoon Moktar, Aruni Bhatnagar & Roberto Bolli

  2. Division of Cardiovascular Medicine, University of Louisville School of Medicine, Ambulatory Care Building, 3rd Floor, 550 S. Jackson St., Louisville, KY, 40202, USA

    Roberto Bolli

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  1. Kyung U. Hong
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  2. Qian-Hong Li
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Correspondence to Roberto Bolli.

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Hong, K.U., Li, QH., Guo, Y. et al. A highly sensitive and accurate method to quantify absolute numbers of c-kit+ cardiac stem cells following transplantation in mice. Basic Res Cardiol 108, 346 (2013). https://doi.org/10.1007/s00395-013-0346-0

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