Nucleofection-Based<i>Ex Vivo</i>Nonviral Gene Delivery to Human Stem Cells as a Platform for Tissue Regeneration

Aslan, Hadi; Zilberman, Yoram; Arbeli, Vered; Sheyn, Dima; Matan, Y.; Liebergall, Meir; Li, Jin Zhong; Helm, Gregory A.; Gazit, Dan; Gazit, Zulma

doi:10.1089/ten.2006.12.877

Cited by 136 publications

(132 citation statements)

References 66 publications

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“…26,27 It was recently shown that the BMP-2, -6, -7 and -9 genes are the most potent inducers of osteogenic differentiation among the BMP family. [28][29][30] In this study we used the BMP-9 gene, since our previous observations indicated it could lead to more significant osteogenesis than the human BMP-2 plasmid. 28 We hypothesized that, in optimized acoustic conditions, the direct in vivo sonoporation of naked DNA encoding an osteogenic gene recombinant human BMP-9 (rhBMP-9) would induce bone formation.…”

Section: Introductionmentioning

confidence: 99%

Ultrasound-based nonviral gene delivery induces bone formation in vivo

et al. 2007

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Nonviral gene delivery is a promising, safe, therapeutic tool in regenerative medicine. This study is the first to achieve nonviral, ultrasound-based, osteogenic gene delivery that leads to bone tissue formation, in vivo. We hypothesized that direct in vivo sonoporation of naked DNA encoding for the osteogenic gene, recombinant human bone morphogenetic protein-9 (rhBMP-9) would induce bone formation. A luciferase plasmid (Luc), encoding rhBMP-9 or empty pcDNA3 vector mixed with microbubbles, was injected into the thigh muscles of mice. After injection, noninvasive sonoporation was applied. Luc activity was monitored noninvasively, and quantitatively using bioluminescence imaging in vivo, and found for 14 days with a peak expression on day 7. To examine osteogenesis in vivo, rhBMP-9 plasmid was sonoporated into the thigh muscles of transgenic mice that express the Luc gene under the control of a human osteocalcin promoter. Following rhBMP-9 sonoporation, osteocalcin-dependent Luc expression lasted for 24 days and peaked on day 10. Bone tissue was formed in the site of rhBMP-9 delivery, as was shown by microcomputerized tomography and histology. The sonoporation method was also compared with previously developed electrotransfer-based gene delivery and was found significantly inferior in its efficiency of gene delivery. We conclude that ultrasound-mediated osteogenic gene delivery could serve as a therapeutic solution in conditions requiring bone tissue regeneration after further development that will increase the transfection efficiency.

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Section: Introductionmentioning

confidence: 99%

Ultrasound-based nonviral gene delivery induces bone formation in vivo

et al. 2007

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“…Nucleofection has been used successfully for transient transfection of human, bovine, rat and porcine MSCs, but has never been tested in BM-derived mMSCs. [34][35][36][37] Magnetically enhanced nucleic acid delivery (magnetofection) has not explored for any type of MSCs to date. We found that nucleofection is a very efficient transfection technique for mMSCs with transfection rates of B60%.…”

Section: Nonviral Gene Transfer Into Murine Mscsmentioning

confidence: 99%

“…This is in line with nucleofection data obtained for human MSCs, where transfection rates ranged from 42 to 88%. 36,38,39 Magnetofection yielded transient transfection rates of 30% in mMSCs, whereas lipofection only reached B20%. Earlier reports on lipofection of human and rat MSCs with Lipofectamine described transfection rates of 2% and 20%, respectively.…”

Section: Nonviral Gene Transfer Into Murine Mscsmentioning

confidence: 99%

Nonviral gene delivery of erythropoietin by mesenchymal stromal cells

et al. 2011

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Erythropoietin (EPO) acts on erythroblasts in the bone marrow (BM) to stimulate the formation of red blood cells. In this study, we wanted to determine whether BM-derived mesenchymal stromal cells (MSCs) can be used as cellular vehicles to deliver EPO in mice without the use of viral vectors. After isolation and characterization of murine MSCs (mMSCs), different transient transfection procedures were compared for their efficacy of gene transfer of the pEGFP-N2 plasmid. Nucleofection outperformed magnetofection and lipofection. Stably transfected mMSCs were generated by selection with G418-disulfate and single-cellcolony-forming unit (sc-CFU) assays without changes in their proliferation capacity and osteogenic/adipogenic differentiation potential. Next, murine EPO was stably introduced into mMSCs by nucleofection of a plasmid encoding the epo and egfp genes. Intraperitoneal transplantation of EPO-expressing mMSCs increased serum EPO levels, hematocrit and hemoglobin of C57BL/6 mice within 1 week. The hematocrit remained elevated for 5 weeks, but production of antibodies against both transgenes was detected in the hosts and serum EPO levels normalized. Our results suggest that nonviral gene delivery into MSCs can be used to enhance the known beneficial effects MSCs by additional production of therapeutic factors like EPO in vivo.

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“…Nucleofection is a technique whereby the plasmid DNA is transfected directly into the cell nucleus through electrical pulse. Aslan et al 17 showed that human bone morphogenetic protein-2 or 9-nucleofected BM-hMSCs were able to induce bone formation 4 weeks after in vivo engraftment. However, the low level of gene expression signifies the need for long-term selection of stable clones.…”

Section: Introductionmentioning

confidence: 99%

HSV-1 amplicon viral vector-mediated gene transfer to human bone marrow-derived mesenchymal stem cells

Chan

Miao

et al. 2008

Cancer Gene Ther

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Human bone marrow-derived mesenchymal stem cells (BM-hMSCs) are nonhematopoietic stem cells that have the potential to differentiate into adipocytes, osteocytes and chondrocytes. Because of its propensity to migrate to the sites of injury and the ability to expand them rapidly, BM-hMSCs have been exploited as potential gene transfer vehicles to deliver therapeutic genes. Herein, we evaluated the feasibility of employing herpes simplex virus type I (HSV-1) amplicon viral vector as a gene delivery vector to BM-hMSCs. High transduction efficiencies were consistently observed in different isolates of BM-hMSCs following infection with HSV-1 amplicon viral vectors. Furthermore, we demonstrated that transduction with HSV-1 amplicon viral vector did not alter the intrinsic properties of the BM-hMSCs. The morphology and cellular proliferation of the transduced BM-hMSCs were not altered. Chromosomal stability, as confirmed by karyotyping and soft agar colony assays, of the transduced BM-hMSCs was not affected. Similarly, transduction with HSV-1 amplicon viral vectors has no effect on the pluripotent differentiation potential and the tumor tropism of BM-hMSCs. Taken together, these results demonstrated that BM-hMSCs could be transduced efficiently by HSV-1 amplicon viral vector in an 'inert' manner and thus enable strategies to express potential therapeutic genes in BM-hMSCs.

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Nucleofection-BasedEx VivoNonviral Gene Delivery to Human Stem Cells as a Platform for Tissue Regeneration

Cited by 136 publications

References 66 publications

Ultrasound-based nonviral gene delivery induces bone formation in vivo

Ultrasound-based nonviral gene delivery induces bone formation in vivo

Nonviral gene delivery of erythropoietin by mesenchymal stromal cells

HSV-1 amplicon viral vector-mediated gene transfer to human bone marrow-derived mesenchymal stem cells

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