한빛사 논문
Abstract
Kiwon Ban1; Brian Wile2; Sangsung Kim1; Hun-Jun Park3; Jaemin Byun1; Kyu-Won Cho1; Talib Saafir4; Ming-Ke Song1; Shan Ping Yu1; Mary Wagner4; Gang Bao2; Young-Sup Yoon1*
1Emory University School of Medicine, Atlanta, GA
2Georgia Institute of Technology and Emory University, Atlanta, GA
3Emory University School of Medicine, Atlanta, GA & Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea
4Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA
*Corresponding author
Abstract
Background - While methods for generating cardiomyocytes (CMs) from pluripotent stem cells (PSCs) have been reported, current methods produce heterogeneous mixtures of CMs and non-CM cells. Here, we report an entirely novel system in which PSC-derived CMs are purified by CM-specific molecular beacons (MBs). MBs are nano-scale 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 five MBs, a MB targeting myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 CMs, a mouse CM cell line, but < 3% of four non-CM cell types in flow cytometry analysis, indicating that MHC1-MB is specific for identifying CMs. We delivered MHC1-MB into cardiomyogenically differentiated PSCs through nucleofection. The detection rate of CMs was similar to the percentages of cardiac troponin T (TNNT2) or cardiac troponin I (TNNI3)-positive CMs, supporting the specificity of MBs. Finally, MHC1-MB-positive cells were FACS-sorted from mouse and human PSC differentiating cultures and ~97% cells expressed TNNT2- or TNNI3 determined by flow cytometry. These MB-based sorted cells maintained their CM characteristics verified by spontaneous beating, electrophysiologic studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified CMs improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors.
Conclusions - We developed a novel CM selection system that allows production of highly purified CMs. These purified CMs and this system can be valuable for cell therapy and drug discovery.
Key Words: molecular beacon, cell selection, cardiac regeneration, embryonic stem cell, cardiomyocyte, cardiac differentiation, cell transplantation
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