Suite of lentiviral vectors for fluorescent tracking and drug-based purification of hESC-derived cardiomyocytes. Core architecture uses the PGK promoter (−528 to −13 bp) for ubiquitous nuclear H2B-fluorescent reporters, αMHC for cardiomyocyte-selective expression, and Rex-1 for hESC-stage selection. Dual selection (G418/Blasticidin + Puromycin at day 12) yielded >96% pure cardiomyocyte populations. Key finding: unsorted lentiviral populations progressively silence transgene expression from >80% to <5% positive cells by passage 6, necessitating FACS enrichment or clonal selection for stable long-term reporter lines.
Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes
Cardiomyocyte yield from differentiating stem cells was low, and uniform reporter lines were hard to make. This paper built lentiviral vectors with lineage promoters driving puromycin resistance to purify cardiomyocytes. Spheroids were 96% pure by Troponin-I staining.
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Key findings
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.