Comparison of Gene-Transfer Efficiency in Human Embryonic Stem Cells

Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Edwards Building R354, Stanford, CA 94305-5344, USA.
Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging (Impact Factor: 2.77). 06/2009; 12(1):15-24. DOI: 10.1007/s11307-009-0236-x
Source: PubMed


Technologies designed to allow manipulation and modification of human embryonic stem (hES) cells are numerous and vary in the complexity of their methods, efficiency, reliability, and safety. The most commonly studied and practiced of these methods include electroporation, lipofection, nucleofection, and lentiviral transduction. However, at present, it is unclear which protocol offers the most efficient and reliable method of gene transfer to hES cells. In this study, a bi-fusion construct with ubiquitin promoter driving enhanced green fluorescent protein reporter and the firefly luciferase (pUb-eGFP-Fluc) along with neomycin selection marker was used for in vitro and in vivo studies. In vitro studies examined the transfection efficiency and viability of each technique using two hES cell lines (male H1 and female H9 cells). Lentiviral transduction demonstrated the highest efficiency (H1: 25.3 +/- 4.8%; H9: 22.4 +/- 6.5%) with >95% cell viability. Nucleofection demonstrated transfection efficiency of 16.1 +/- 3.6% (H1) and 5.8 +/- 3.2% (H9). However, minimal transfection efficiency was observed with electroporation (2.1 +/- 0.4% (H1) and 1.9 +/- 0.3% (H9)) and lipofection (1.5 +/- 0.5% (H1) and 1.3 +/- 0.2% (H9); P < 0.05 vs. lentiviral transduction). Electroporation also demonstrated the highest cell death (62 +/- 11% (H1) and 42 +/- 10% (H9)) followed by nucleofection (25 +/- 9% (H1) and 30 +/- 15 (H9)). Importantly, lentiviral transduction generated a greater number of hES cell lines stably expressing the double-fusion reporter gene (hES-DF) compared to other transfection techniques. Finally, following subcutaneous transplantation into immunodeficient nude mice, the hES-eGFP-Fluc cells showed robust proliferation as determined by longitudinal bioluminescence imaging. In summary, this study demonstrates that lentiviral transduction and nucleofection are efficient, simple, and safe techniques for reliable gene transfer in hES cells. The double-fusion construct provides an attractive approach for generating stable hES cell lines and monitoring engraftment and proliferation in vitro and in vivo.

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    • "Careful and accurate genome editing is particularly attractive for autologous stem cells, which after ex vivo gene correction can be transplanted back into the patient. However, current gene transfer methods, which enable the transient expression of designer nucleases in human stem cells, can be associated with high toxicities and/or low delivery efficiencies, thus presenting a major hurdle in the preparation of autologous gene corrected cells21. To overcome this obstacle, viral vector systems, like integrase-deficient lentiviral vectors (IDLVs), adenoviral vectors (AdV), and vectors based on adeno-associated viruses (AAVs) have been successfully employed1422232425. "
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    ABSTRACT: Designer nucleases, like zinc-finger nucleases (ZFNs), represent valuable tools for targeted genome editing. Here, we took advantage of the gamma-retroviral life cycle and produced vectors to transfer ZFNs in the form of protein, mRNA and episomal DNA. Transfer efficacy and ZFN activity were assessed in quantitative proof-of-concept experiments in a human cell line and in mouse embryonic stem cells. We demonstrate that retrovirus-mediated protein transfer (RPT), retrovirus-mediated mRNA transfer (RMT), and retrovirus-mediated episome transfer (RET) represent powerful methodologies for transient protein delivery or protein expression. Furthermore, we describe complementary strategies to augment ZFN activity after gamma-retroviral transduction, including serial transduction, proteasome inhibition, and hypothermia. Depending on vector dose and target cell type, gene disruption frequencies of up to 15% were achieved with RPT and RMT, and >50% gene knockout after RET. In summary, non-integrating gamma-retroviral vectors represent a versatile tool to transiently deliver ZFNs to human and mouse cells.
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    • "Apart from the effects of vector structure (i.e., type and arrangement of promoter and reporter transgenes), a transient transfection efficiency of up to *45% for eGFP and *65% for nRed- Star was observed for hiPSC lines whereas *40% eGFP positivity was observed for hES3. This is significantly higher compared with previous reports for electroporation (2 – 0.4%), nucleofection (16 – 3.6%), and lentiviral transduction (25 – 4.8%) (Cao et al., 2010) and equivalent to results observed for the Neon transfection system by others investigators (Moore et al., 2010). "
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    ABSTRACT: Human pluripotent stem cells (hPSCs) are a prime cell source for regenerative therapies due to their extensive expansion potential and the ability to differentiate into essentially all somatic lineages in vitro. The introduction of transgenes into hPSCs will facilitate their pre-clinical testing and other applications such as the purification of desired cell lineages during differentiation and in vivo monitoring of transplanted progenies in relevant animal models as well. To date, several limits regarding the efficient generation of transgenic cell lines exist. This includes low transfection rates via non-viral methods, inefficient recovery of engineered clones, and silencing of transgene expression. Here we describe a fast and highly efficient method for the generation of multi-transgenic hPSC lines by overcoming the need for any pre-adaption to feeder-free culture conditions before genetic manipulation. Selection for a single antibiotic resistance gene encoded on one plasmid allowed for the stable genomic integration of several independent plasmid constructs thereby generating valuable multi-transgenic cell lines.
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    • "Moreover, viral systems are suspected to have lifethreatening effects of immunogenicity and carcinogenicity. In addition, the efficiency of gene transfer in hES cells is still poor compared with other cell lines (Cao et al., 2010; Wasungu and Hoekstra, 2006). For these reasons, many scientists hesitate to use these strategies in medicine and have searched for new ways to control ES cell differentiation. "

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