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

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, Youngsup Yoon

Research output: Contribution to journalArticle

29 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1897-1909
Number of pages13
JournalCirculation
Volume128
Issue number17
DOIs
Publication statusPublished - 2013 Oct 22

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Pluripotent Stem Cells
Cardiac Myocytes
Messenger RNA
Troponin T
Troponin I
Myosin Heavy Chains
Flow Cytometry
Fluorescence
Molecular Models
Drug Discovery
Cell- and Tissue-Based Therapy

All Science Journal Classification (ASJC) codes

  • Physiology (medical)
  • Cardiology and Cardiovascular Medicine

Cite this

Ban, Kiwon ; Wile, Brian ; Kim, Sangsung ; Park, Hun Jun ; Byun, Jaemin ; Cho, Kyu Won ; Saafir, Talib ; Song, Ming Ke ; Yu, Shan Ping ; Wagner, Mary ; Bao, Gang ; Yoon, Youngsup. / Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA. In: Circulation. 2013 ; Vol. 128, No. 17. pp. 1897-1909.
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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.",
author = "Kiwon Ban and Brian Wile and Sangsung Kim and Park, {Hun Jun} and Jaemin Byun and Cho, {Kyu Won} and Talib Saafir and Song, {Ming Ke} and Yu, {Shan Ping} and Mary Wagner and Gang Bao and Youngsup Yoon",
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Ban, K, Wile, B, Kim, S, Park, HJ, Byun, J, Cho, KW, Saafir, T, Song, MK, Yu, SP, Wagner, M, Bao, G & Yoon, Y 2013, 'Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA', Circulation, vol. 128, no. 17, pp. 1897-1909. https://doi.org/10.1161/CIRCULATIONAHA.113.004228

Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA. / Ban, Kiwon; Wile, Brian; Kim, Sangsung; Park, Hun Jun; Byun, Jaemin; Cho, Kyu Won; Saafir, Talib; Song, Ming Ke; Yu, Shan Ping; Wagner, Mary; Bao, Gang; Yoon, Youngsup.

In: Circulation, Vol. 128, No. 17, 22.10.2013, p. 1897-1909.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA

AU - Ban, Kiwon

AU - Wile, Brian

AU - Kim, Sangsung

AU - Park, Hun Jun

AU - Byun, Jaemin

AU - Cho, Kyu Won

AU - Saafir, Talib

AU - Song, Ming Ke

AU - Yu, Shan Ping

AU - Wagner, Mary

AU - Bao, Gang

AU - Yoon, Youngsup

PY - 2013/10/22

Y1 - 2013/10/22

N2 - 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.

AB - 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.

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U2 - 10.1161/CIRCULATIONAHA.113.004228

DO - 10.1161/CIRCULATIONAHA.113.004228

M3 - Article

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EP - 1909

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SN - 0009-7322

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