Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes

Hiroko Kita-Matsuo, Maria Barcova, Natalie Prigozhina, Nathan Salomonis, Karen Wei, Jeffrey G. Jacot, Brandon Nelson, Sean Spiering, René Haverslag, Changsung Kim, Maria Talantova, Ruchi Bajpai, Diego Calzolari, Alexey Tershikh, Andrew D. McCulloch, Jeffrey H. Price, Bruce R. Conklin, H. S.Vincent Chen, Mark Mercola

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164 Scopus citations

Abstract

Background: Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes. Methodology/Principal Findings: Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and α-myosin heavy chain (αMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function. Conclusion/Significance: The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.

Original languageEnglish (US)
Article numbere5046
JournalPLoS ONE
Volume4
Issue number4
DOIs
StatePublished - Apr 8 2009

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

Cite this

Kita-Matsuo, H., Barcova, M., Prigozhina, N., Salomonis, N., Wei, K., Jacot, J. G., Nelson, B., Spiering, S., Haverslag, R., Kim, C., Talantova, M., Bajpai, R., Calzolari, D., Tershikh, A., McCulloch, A. D., Price, J. H., Conklin, B. R., Chen, H. S. V., & Mercola, M. (2009). Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes. PLoS ONE, 4(4), [e5046]. https://doi.org/10.1371/journal.pone.0005046