Stable transfection of MSCs by electroporation

Peister, Alexandra; Mellad, J A; Wang, M; Tucker, H. Alan; Prockop, D.J.

doi:10.1038/sj.gt.3302163

Cited by 54 publications

(45 citation statements)

References 12 publications

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“…More recently, human MSC electrotransfection was maximal after application of a single electric pulse 120 ms in length at an amplitude of 3000 V cm À1 . 34 Taken together, these data suggest that increasing the number of pulses reduces the need for increasing the pulse amplitude for maximal MSC electrotransfection. Although lacZ electrotransfer into MSCs at 1700 V cm À1 resulted in maximal b-galactosidase activity, it also resulted in important cell death and lack of reproducibility.…”

Section: Discussionmentioning

confidence: 81%

“…Previously, the transfection rate of human MSCs by electroporation (cell recovery was not documented) using a commercially available device reached 12% when the quantity of DNA the intensity, and length of the electric pulses were optimized. 34 Recently, an electroporation-based technology called nucleofector, which elicits the destabilization of both cytoplasmic and nuclear membranes, has been proposed as an alternative tool for gene transfer. 47 Although MSC nucleofection resulted in high transfection efficiency rates (450%), [48][49][50] it unfortunately led to limited cell recovery, 51-53 reflecting a major drawback of most electroporation techniques employed to facilitate the transplantation of in vitro genetically modified cells.…”

Section: Discussionmentioning

confidence: 99%

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Optimization of a gene electrotransfer method for mesenchymal stem cell transfection

et al. 2008

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Gene electrotransfer is an efficient and reproducible nonviral gene transfer technique useful for the nonpermanent expression of therapeutic transgenes. The present study established optimal conditions for the electrotransfer of reporter genes into mesenchymal stem cells (MSCs) isolated from rat bone marrow by their selective adherence to tissue-culture plasticware. The electrotransfer of the lacZ reporter gene was optimized by adjusting the pulse electric field intensity, electric pulse type, electropulsation buffer conductivity and electroporation temperature. LacZ electrotransfection into MSCs was optimal at 1500 V cm À1 with pre-incubation in Spinner's minimum essential medium buffer at 22 1C. Under these conditions b-galactosidase expression was achieved in 29 ± 3% of adherent cells 48 h post transfection. The kinetics of b-galactosidase activity revealed maintenance of b-galactosidase production for at least 10 days. Moreover, electroporation did not affect the MSC potential for multidifferentiation; electroporated MSCs differentiated into osteoblastic, adipogenic and chondrogenic lineages to the same extent as cells that were not exposed to electric pulses. Thus, this study demonstrates the feasibility of efficient transgene electrotransfer into MSCs while preserving cell viability and multipotency.

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Section: Discussionmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Optimization of a gene electrotransfer method for mesenchymal stem cell transfection

et al. 2008

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“…Differentiated mesenchymal stem cells from human bone marrow were prepared as previously described [6,7]. Briefly, bone marrow (10-20 ml) was aspirated from the posterior iliac crest of healthy, adult volunteers.…”

Section: Cell Culturesmentioning

confidence: 99%

“…Incubation of mononuclear cells isolated from adult, human bone marrow with mesenchymal stem cell-stimulating medium followed by osteogenic differentiation medium [6,7] gave rise to cells with osteoblastic characteristics, for example mineral deposition (Fig. 3d, e) and alkaline phosphatasesecreting activity (not shown), to be used in coculture with LNCaP cells (Fig.…”

Section: Egfr/erbb2 Phosphorylation In Supplementary Biological Modelsmentioning

confidence: 99%

Osteoblast-induced EGFR/ERBB2 signaling in androgen-sensitive prostate carcinoma cells characterized by multiplex kinase activity profiling

Bratland

Boender

Høifødt

et al. 2009

Clin Exp Metastasis

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Bone metastases in prostate cancer are predominantly osteoblastic. To study regulatory mechanisms underlying the establishment of prostate cancer within an osteoblastic microenvironment, human androgen-sensitive prostate carcinoma cells (LNCaP) were treated with culture medium conditioned by human osteoblast-derived sarcoma cells (OHS), and activated signalling pathways in the carcinoma cells were analyzed using microarrays with tyrosine kinase substrates. Network interaction analysis of substrates with significantly increased phosphorylation levels revealed that signalling pathways mediated by EGFR and ERBB2 were activated in LNCaP cells under OHS influence but also by androgen treatment. Activation of EGFR/ERBB2 signalling was also found in LNCaP cells in cocultures with OHS cells or osteoblastic cells that had been differentiated from human mesenchymal stem cells. Our experimental data suggests osteoblast-directed induction of signalling activity via EGFR and ERBB2 in prostate carcinoma cells and may provide a rationale for the use of EGFR or ERBB2 inhibition in systemic prevention or treatment of metastatic prostate cancer in the androgensensitive stage of the disease.

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“…Peister et al [37] developed improved conditions for stable transfection of human MSCs by electroporation. Following selection using G418, the transfected MSCs could be expanded 300-fold in 14 days and 98% of the progeny cells expressed the transgene.…”

Section: Transgene Delivery Into Mesenchymal Stem Cells Using Non-virmentioning

confidence: 99%

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases

Reiser

Zhang

Hemenway

et al. 2005

Expert Opinion on Biological Therapy

166

114

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The intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells, including the pluripotent haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and possibly the multipotent adherent progenitor cells (MAPCs), are being considered as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineagesn and, at least in vitro, have significant expansion capability. The apparently high self-renewal potential makes them strong candidates for delivering genes and restoring organ systems function. However, the high proliferative potential of MSCs, now presumed to be self-renewal, may be more apparent than real. Although expanded MSCs have great proliferation and differentiation potential in vitro, there are limitations with the biology of these cells in vivo. So far, expanded MSCs have failed to induce durable therapeutic effects expected from a true self-renewing stem cell population. The loss of in vivo self-renewal may be due to the extensive expansion of MSCs in existing in vitro expansion systems, suggesting that the original stem cell population and/or properties may no longer exist. Rather, the expanded population may indeed be heterogeneous and represents several generations of different types of mesenchymal cell progeny that have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and non-mesenchymal lineages. Novel technology that allows MSCs to maintain their stem cell function in vivo is critical for distinguishing the elusive stem cell from its progenitor cell populations. The ultimate dream is to use MSCs in various forms of cellular therapies, as well as genetic tools that can be used to better understand the mechanisms leading to repair and regeneration of damaged or diseased tissues and organs.

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Stable transfection of MSCs by electroporation

Cited by 54 publications

References 12 publications

Optimization of a gene electrotransfer method for mesenchymal stem cell transfection

Optimization of a gene electrotransfer method for mesenchymal stem cell transfection

Osteoblast-induced EGFR/ERBB2 signaling in androgen-sensitive prostate carcinoma cells characterized by multiplex kinase activity profiling

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases

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