Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes

Abstract

BackgroundDevelopmental, 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 FindingsHere 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/SignificanceThe protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.

Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors

“…None of the cells displayed only PECAM1 marker, making it unlikely that NKX2.5 was expressed in endothelial cells. To determine whether the ETS2/ MESP1 cell treatment could generate functional cardiac myocytes, NHDFs were first preinfected with α-MHC-Puro selection lentivector (30) followed by the optimized conditions for TAT-protein transduction. After 8 d, cells were treated with 10 μg/mL puromycin to select converted cells.…”

Stem cell therapy for cardiac disease

“…Injection of neomycin resistance-selected hEBs into the hind limb muscles of SCID mice resulted in no teratoma formation after 23 wk (58). Contractile forces in puromycin resistance-selected CMs were similar to those generated by rat neonatal ventricular CMs (59). Whereas isolation of hESC-derived CMs from these lines was based on positive selection, Anderson et al implemented a negative selection strategy to deplete undifferentiated, proliferating hESCs from cultures of hESC-derived CMs (61).…”

“…Xu et al generated stable hESC lines using a reporter consisting of the cardiac-specific mouse α-myosin heavy chain promoter driving expression of the neomycin resistance gene (58). Kita-Matsuo et al designed a set of lentiviral vectors to generate multiple stable hESC lines with eGFP and mCherry reporters or with puromycin resistance downstream of the mouse α-myosin heavy chain promoter (59). Recently, Ritner et al generated a cardiac-specific hESC reporter line using a lentiviral construct consisting of a fragment of the mouse α-myosin heavy chain promoter upstream of eGFP.…”

Lentiviral vectors and cardiovascular diseases: a genetic tool for manipulating cardiomyocyte differentiation and function

“…25,26 Our group has also given a contribution to this field: we have constructed a cardiacspecific LVV in which expression of the transgene is driven by a short fragment of the cardiac troponin I promoter (TNNI3) with a human cardiac a-actin enhancer (hEnAct). 27 Using an enhanced green fluorescence protein reporter, the TNNI3-LVV has been demonstrated to be effective in tracking cardiac lineage induction during differentiation in both mouse and human embryonic stem cells; moreover, the addition of hEnAct conferred a further increase in expression specificity to the TNNI3-LVV in human embryonic stem cells.…”

“…19,22 Kita-Matsuo et al 26 recently proposed vectors carrying T/Brachyury and alpha-myosin heavy chain (aMHC) promoters driving drug-resistance expression in early mesodermal cells and CMCs, respectively: this drug-selection protocol yielded 96% pure CMCs, which had a molecular profile and electrophysiological properties similar to those of human CMCs.…”