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

PloS one | Apr 8, 2009

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.

Pubmed ID: 19352491 RIS Download

Mesh terms: Adult | Base Sequence | Cell Differentiation | DNA Primers | Drug Resistance | Embryonic Stem Cells | Fetal Proteins | Flow Cytometry | Gene Expression Profiling | Genetic Vectors | Green Fluorescent Proteins | Humans | Immunohistochemistry | Lentivirus | Myocardium | Promoter Regions, Genetic | Reverse Transcriptase Polymerase Chain Reaction | T-Box Domain Proteins

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Associated grants

  • Agency: NHLBI NIH HHS, Id: R33HL088266
  • Agency: NHLBI NIH HHS, Id: HL66621
  • Agency: NHLBI NIH HHS, Id: U01 HL066621
  • Agency: NHGRI NIH HHS, Id: HG003053
  • Agency: NHLBI NIH HHS, Id: 5P01HL46345-12
  • Agency: NHGRI NIH HHS, Id: R01 HG003053
  • Agency: NHLBI NIH HHS, Id: P01 HL046345
  • Agency: NHLBI NIH HHS, Id: R37 HL059502
  • Agency: NHLBI NIH HHS, Id: R33 HL088266
  • Agency: NHLBI NIH HHS, Id: R37HL059502

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