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

• Type of Material: Article
• Authors: Kiwon Ban, Emory University; Brian Wile, Georgia Institute of Technology; Sangsung Kim, Emory University; Hun-Jun Park, Emory University; Jaemin Byun, Emory University; Kyu-Won Cho, Emory University; Talib Saafir, Emory University; Ming-Ke Song, Emory University; Shan Ping Yu, Emory University; Mary Wagner, Emory University; Gang Bao, Emory University; Young-sup Yoon, Emory University
• Language: English
• Date: 2013-10-22
• Publisher: American Heart Association
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2013 American Heart Association, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1161/CIRCULATIONAHA.113.004228
• Title of Journal or Parent Work: Circulation
• ISSN: 0009-7322
• Volume: 128
• Issue: 17
• Start Page: 1897
• End Page: 1909
• Grant/Funding Information: This work was supported in part by the NHLBI of the NIH as a Program of Excellence in Nanotechnology award (HHSN268201000043C to YSY and GB), by grants from NIDDK (DP3DK094346 to YSY), NHLBI (R01HL088488 to MBW), ACTSI (PHS grant UL1 RR025008 from the CTSA program, National Center for Research Resources, NIH to YSY) and the National Science Foundation STC award (CBET-0939511).
• Keywords: myocytes; CARDIAC DIFFERENTIATION; MYOCYTES; oligonucleotide probes; FUNCTIONAL-PROPERTIES; Cardiovascular System & Cardiology; Science & Technology; cardiac; MARKERS; pluripotent stem cells; HEART; TRANSPLANTATION; PROTEIN; Life Sciences & Biomedicine; regeneration; PROGENITORS; Cardiac & Cardiovascular Systems; Peripheral Vascular Disease; DELIVERY
• Research Categories: Engineering, Biomedical; Biology, Molecular; Health Sciences, Medicine and Surgery

Abstract

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 ≈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.