Staff View: Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells

Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells

Human mesenchymal stromal cell (hMSC) is a potential target for cell and gene therapy-based approaches against a variety of different diseases. Whilst cationic lipofection has been widely experimented, the Nucleofector technology is a relatively new non-viral transfection method designed for primary...

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Main Authors:	P. L., Mok,, C. F., Leong,, O., Ainoon,, S. K., Cheong,, K. H., Chua,
Format:	Article
Language:	English
Published:	Springer 2015
Subjects:	Bone Marrow Mesenchymal Stromal Cells Nucleofection Cationic Lipofection MIDGE Erythropoietin
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spelling	my.usim-81352015-12-29T08:17:20Z Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells P. L., Mok, C. F., Leong, O., Ainoon, S. K., Cheong, K. H., Chua, Bone Marrow Mesenchymal Stromal Cells Nucleofection Cationic Lipofection MIDGE Erythropoietin Human mesenchymal stromal cell (hMSC) is a potential target for cell and gene therapy-based approaches against a variety of different diseases. Whilst cationic lipofection has been widely experimented, the Nucleofector technology is a relatively new non-viral transfection method designed for primary cells and hard-to-transfect cell lines. Herein, we compared the efficiency and viability of nucleofection with cationic lipofection, and used the more efficient transfection method, nucleofection, to deliver a construct of minimalistic, immunologically defined gene expression encoding the erythropoietin (MIDGE-EPO) into hMSC. MIDGE construct is relatively safer than the viral and plasmid expression systems as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. Using a plasmid encoding the luciferase gene, we demonstrated a high transfection efficiency using the U-23 (21.79 +/- 1.09%) and C-17 (5.62 +/- 1.09%) pulsing program in nucleofection. The cell viabilities were (44.93 +/- 10.10)% and (21.93 +/- 5.72)%, respectively 24 h post-nucleofection. On the other hand, lipofection treatment only yielded less than 0.6% efficiencies despite showing higher viabilities. Nucleofection did not affect hMSC renewability, immunophenotype and differentiation potentials. Subsequently, we nucleofected MIDGE-EPO using the U-23 pulsing program into hMSC. The results showed that, despite a low nucleofection efficiency with this construct, the EPO protein was stably expressed in the nucleofected cells up to 55 days when determined by ELISA or immunocytochemical staining. In conclusion, nucleofection is an efficient non-viral transfection approach for hMSC, which when used in conjunction with a MIDGE construct, could result in extended and stable transgene expression in hMSC. 2015-05-18T06:34:01Z 2015-05-18T06:34:01Z 2012 Article 0920-9069 en Springer
institution	Universiti Sains Islam Malaysia
building	USIM Library
collection	Institutional Repository
continent	Asia
country	Malaysia
content_provider	Universit Sains Islam i Malaysia
content_source	USIM Institutional Repository
url_provider	http://ddms.usim.edu.my/
language	English
topic	Bone Marrow Mesenchymal Stromal Cells Nucleofection Cationic Lipofection MIDGE Erythropoietin
spellingShingle	Bone Marrow Mesenchymal Stromal Cells Nucleofection Cationic Lipofection MIDGE Erythropoietin P. L., Mok, C. F., Leong, O., Ainoon, S. K., Cheong, K. H., Chua, Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells
description	Human mesenchymal stromal cell (hMSC) is a potential target for cell and gene therapy-based approaches against a variety of different diseases. Whilst cationic lipofection has been widely experimented, the Nucleofector technology is a relatively new non-viral transfection method designed for primary cells and hard-to-transfect cell lines. Herein, we compared the efficiency and viability of nucleofection with cationic lipofection, and used the more efficient transfection method, nucleofection, to deliver a construct of minimalistic, immunologically defined gene expression encoding the erythropoietin (MIDGE-EPO) into hMSC. MIDGE construct is relatively safer than the viral and plasmid expression systems as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. Using a plasmid encoding the luciferase gene, we demonstrated a high transfection efficiency using the U-23 (21.79 +/- 1.09%) and C-17 (5.62 +/- 1.09%) pulsing program in nucleofection. The cell viabilities were (44.93 +/- 10.10)% and (21.93 +/- 5.72)%, respectively 24 h post-nucleofection. On the other hand, lipofection treatment only yielded less than 0.6% efficiencies despite showing higher viabilities. Nucleofection did not affect hMSC renewability, immunophenotype and differentiation potentials. Subsequently, we nucleofected MIDGE-EPO using the U-23 pulsing program into hMSC. The results showed that, despite a low nucleofection efficiency with this construct, the EPO protein was stably expressed in the nucleofected cells up to 55 days when determined by ELISA or immunocytochemical staining. In conclusion, nucleofection is an efficient non-viral transfection approach for hMSC, which when used in conjunction with a MIDGE construct, could result in extended and stable transgene expression in hMSC.
format	Article
author	P. L., Mok, C. F., Leong, O., Ainoon, S. K., Cheong, K. H., Chua,
author_facet	P. L., Mok, C. F., Leong, O., Ainoon, S. K., Cheong, K. H., Chua,
author_sort	P. L., Mok,
title	Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells
title_short	Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells
title_full	Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells
title_fullStr	Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells
title_full_unstemmed	Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells
title_sort	extended and stable gene expression via nucleofection of midge construct into adult human marrow mesenchymal stromal cells
publisher	Springer
publishDate	2015
_version_	1645152346121961472
score	13.222552

Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells

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