Purification of Cardiomyocytes From Differentiating Pluripotent Stem Cells Using Molecular Beacons That Target Cardiomyocyte-Specific mRNA

被引:47
作者
Ban, Kiwon [1 ]
Wile, Brian [3 ,4 ]
Kim, Sangsung [1 ]
Park, Hun-Jun [1 ,5 ]
Byun, Jaemin [1 ]
Cho, Kyu-Won [1 ]
Saafir, Talib [6 ,7 ]
Song, Ming-Ke [2 ]
Yu, Shan Ping [2 ]
Wagner, Mary [6 ,7 ]
Bao, Gang [3 ,4 ]
Yoon, Young-Sup [1 ]
机构
[1] Emory Univ, Sch Med, Div Cardiol, Dept Med, Atlanta, GA 30322 USA
[2] Emory Univ, Sch Med, Dept Anesthesiol, Atlanta, GA 30322 USA
[3] Georgia Inst Technol, Dept Biomed Engn, Atlanta, GA 30332 USA
[4] Emory Univ, Atlanta, GA 30332 USA
[5] Catholic Univ Korea, Seoul St Marys Hosp, Dept Internal Med, Div Cardiol, Seoul, South Korea
[6] Emory Univ, Sch Med, Dept Pediat, Atlanta, GA USA
[7] Childrens Healthcare Atlanta, Atlanta, GA USA
基金
美国国家卫生研究院; 美国国家科学基金会;
关键词
myocytes; cardiac; oligonucleotide probes; pluripotent stem cells; regeneration; CARDIAC DIFFERENTIATION; FUNCTIONAL-PROPERTIES; HEART; TRANSPLANTATION; PROTEIN; PROGENITORS; MYOCYTES; DELIVERY; MARKERS;
D O I
10.1161/CIRCULATIONAHA.113.004228
中图分类号
R5 [内科学];
学科分类号
100201 [内科学];
摘要
Background Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. Method and Results Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and approximate to 97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. Conclusions We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.
引用
收藏
页码:1897 / 1909
页数:13
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