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

PLoS One. 2009;4(4):e5046. doi: 10.1371/journal.pone.0005046. Epub 2009 Apr 8.

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 alpha-myosin heavy chain (alphaMHC) 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Base Sequence
  • Cell Differentiation
  • DNA Primers
  • Drug Resistance
  • Embryonic Stem Cells / cytology*
  • Embryonic Stem Cells / metabolism
  • Fetal Proteins / genetics
  • Flow Cytometry
  • Gene Expression Profiling
  • Genetic Vectors*
  • Green Fluorescent Proteins / genetics
  • Humans
  • Immunohistochemistry
  • Lentivirus / genetics*
  • Myocardium / cytology*
  • Myocardium / metabolism
  • Promoter Regions, Genetic
  • Reverse Transcriptase Polymerase Chain Reaction
  • T-Box Domain Proteins / genetics

Substances

  • DNA Primers
  • Fetal Proteins
  • T-Box Domain Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Brachyury protein