103 96

Cited 29 times in

Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA

Authors
 Kiwon Ban  ;  Brian Wile  ;  Sangsung Kim  ;  Hun-Jun Park  ;  Jaemin Byun  ;  Kyu-Won Cho  ;  Talib Saafir  ;  Ming-Ke Song  ;  Shan Ping Yu  ;  Mary Wagner  ;  Gang Bao  ;  Young-Sup Yoon 
Citation
 CIRCULATION, Vol.128(17) : 1897-1909, 2013 
Journal Title
 CIRCULATION 
ISSN
 0009-7322 
Issue Date
2013
MeSH
Action Potentials/physiology ; Animals ; Biomarkers ; Cell Differentiation/physiology ; Cell Line ; Cell Transplantation/methods ; Cells, Cultured ; Flow Cytometry/methods ; Humans ; Mice ; Myocardial Infarction/therapy ; Myocytes, Cardiac/cytology ; Myocytes, Cardiac/physiology ; Myosin Heavy Chains/genetics ; Nanotechnology ; Nucleic Acid Conformation ; Pluripotent Stem Cells/cytology ; Pluripotent Stem Cells/physiology ; RNA probes/chemistry ; RNA Probes/isolation & purification ; RNA, Messenger/chemistry ; RNA, Messenger/isolation & purification ; Troponin I/genetics ; Troponin T/genetics
Keywords
myocytes ; cardiac ; oligonucleotide probes ; pluripotent stem cells ; regeneration
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.
Files in This Item:
T201306250.pdf Download
DOI
10.1161/CIRCULATIONAHA.113.004228
Appears in Collections:
1. College of Medicine (의과대학) > BioMedical Science Institute (의생명과학부) > 1. Journal Papers
Yonsei Authors
Yoon, Young Sup(윤영섭)
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/158499
사서에게 알리기
  feedback

qrcode

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse