A highly sensitive and accurate method to quantify absolute numbers of c-kit+ cardiac stem cells following transplantation in mice.
A highly sensitive and accurate method to quantify absolute numbers of c-kit+ cardiac stem cells following transplantation in mice.
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DOI：
10.1007/s00395-013-0346-0
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发表时间：
2013-05
影响因子：
9.5
通讯作者：
Bolli R
中科院分区：
文献类型：
--
作者：
Hong KU;Li QH;Guo Y;Patton NS;Moktar A;Bhatnagar A;Bolli R
尽管c-kit+心脏干细胞（CSCs）的移植可缓解心肌梗死后左心室功能障碍，但没有可靠的方法能够测量在受体心脏中持续存在的CSCs的绝对数量。为了克服这一局限性，我们开发了一种高度灵敏和准确的方法来量化移植后小鼠CSC的绝对数量。该方法具有两个独特的特征：i）基于实时PCR检测新的雄性特异性多拷贝基因Rbmy，其显著增加了在雌性受体中检测雄性供体细胞的灵敏度，和ii）内标，其允许定量受体器官中CSC的绝对数量以及细胞的总数。雌性C57 BL/6小鼠接受冠状动脉闭塞和再灌注; 2天后，心肌内注射105只雄性小鼠CSC。在连续时间点通过实时PCR分析组织。在风险区域中，在5分钟时存在的>75%的CSC在随后的24小时内丢失;在移植后7天，仅7.6±2.1%的5分钟时存在的CSC仍然可以被发现，并且仅2.8±0.5%（即，1，224 ±230个细胞/心脏）。因此，即使在直接心肌内注射后，保留在鼠心脏中的CSC的总数也是最小的（在24小时，约10%的注射细胞;在35天，约1%）。这种新的干细胞定量检测方法能够测量绝对细胞数量，应该有助于优化基于细胞的疗法，不仅适用于CSC，而且适用于其他干细胞和其他器官。
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: i) 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 ii) 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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影响因子：
9.5
作者：
Li Q;Guo Y;Ou Q;Chen N;Wu WJ;Yuan F;O'Brien E;Wang T;Luo L;Hunt GN;Zhu X;Bolli R
通讯作者：
Bolli R
影响因子：
6
作者：
Laflamme, MA;Gold, J;Murry, CE
通讯作者：
Murry, CE
影响因子：
4.4
作者：
Chai, NN;Zhou, HY;Yen, PH
通讯作者：
Yen, PH
影响因子：
37.8
作者：
Li Q;Guo Y;Ou Q;Cui C;Wu WJ;Tan W;Zhu X;Lanceta LB;Sanganalmath SK;Dawn B;Shinmura K;Rokosh GD;Wang S;Bolli R
通讯作者：
Bolli R
影响因子：
37.8
作者：
Reinecke, H;Zhang, M;Murry, CE
通讯作者：
Murry, CE