Plasmid DNA Size Does Affect Nonviral Gene Delivery Efficiency in Stem Cells

Abstract Background Placenta-derived mesenchymal stem cells (PD-MSCs) have been highlighted as an alternative cell therapy agent that has become a next-generation stem cell treatment. Phosphatase of regenerating liver-1 (PRL-1), an immediate early gene, plays a critical role during liver regeneration. Here, we generated enhanced PRL-1 in PD-MSCs (PD-MSCsPRL-1, PRL-1+) using lentiviral and nonviral gene delivery systems and investigated mitochondrial functions by PD-MSCPRL-1 transplantation for hepatic functions in a rat bile duct ligation (BDL) model. Methods PD-MSCsPRL-1 were generated by lentiviral and nonviral AMAXA gene delivery systems and analyzed for their characteristics and mitochondrial metabolic functions. Liver cirrhosis was induced in Sprague-Dawley (SD) rats using common BDL for 10 days. PKH67+ naïve and PD-MSCsPRL-1 using a nonviral sysyem (2 × 106 cells/animal) were intravenously administered into cirrhotic rats. The animals were sacrificed at 1, 2, 3, and 5 weeks after transplantation and engraftment of stem cells, and histopathological analysis and hepatic mitochondrial functions were performed. Results PD-MSCsPRL-1 were successfully generated using lentiviral and nonviral AMAXA systems and maintained characteristics similar to those of naïve cells. Compared with naïve cells, PD-MSCsPRL-1 improved respirational metabolic states of mitochondria. In particular, mitochondria in PD-MSCsPRL-1 generated by the nonviral AMAXA system showed a significant increase in the respirational metabolic state, including ATP production and mitochondrial biogenesis (*p < 0.05). Furthermore, transplantation of PD-MSCsPRL-1 using a nonviral AMAXA system promoted engraftment into injured target liver tissues of a rat BDL cirrhotic model and enhanced the metabolism of mitochondria via increased mtDNA and ATP production, thereby improving therapeutic efficacy. Conclusions Our findings will further our understanding of the therapeutic mechanism of enhanced MSCs and provide useful data for the development of next-generation MSC-based cell therapy and therapeutic strategies for regenerative medicine in liver disease.

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INTRAMYOCARDIAL NONVIRAL GENE DELIVERY: THE IN VIVO EVALUATION OF TAT BASED PEPTIDES AS VECTORS FOR PLASMID DNA.

Cardiovascular Pathology ◽

10.1016/j.carpath.2004.03.543 ◽

2004 ◽

Vol 13 (3) ◽

pp. 180

Author(s):

Irina Hellgren ◽

Anwar J Siddiqui ◽

Beston Nore ◽

Karl Henrik Grinnemo ◽

Edvard Smith ◽

...

Keyword(s):

Gene Delivery ◽

Plasmid Dna ◽

Nonviral Gene Delivery ◽

In Vivo Evaluation

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Effects of calcium concentration on nonviral gene delivery to bone marrow‐derived stem cells

Journal of Tissue Engineering and Regenerative Medicine ◽

10.1002/term.2971 ◽

2019 ◽

Vol 13 (12) ◽

pp. 2256-2265 ◽

Cited By ~ 3

Author(s):

Timothy M. Acri ◽

Noah Z. Laird ◽

Sean M. Geary ◽

Aliasger K. Salem ◽

Kyungsup Shin

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Gene Delivery ◽

Calcium Concentration ◽

Nonviral Gene Delivery

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Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

Stem Cells International ◽

10.1155/2013/326828 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 27

Author(s):

Christian Bucher ◽

Amiq Gazdhar ◽

Lorin M. Benneker ◽

Thomas Geiser ◽

Benjamin Gantenbein-Ritter

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Gene Transfer ◽

Gene Delivery ◽

Intervertebral Disc ◽

Organ Culture ◽

Nonviral Gene Delivery ◽

Human Mscs ◽

Differentiation Factor ◽

Gdf5 Gene

Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved byin vitroelectroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

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Nonviral Gene Delivery in Neural Progenitors Derived from Human Pluripotent Stem Cells

Methods in Molecular Biology - Human Pluripotent Stem Cells ◽

10.1007/978-1-61779-201-4_25 ◽

2011 ◽

pp. 343-354 ◽

Cited By ~ 7

Author(s):

Sujoy K. Dhara ◽

Anirban Majumder ◽

Mahesh C. Dodla ◽

Steven L. Stice

Keyword(s):

Stem Cells ◽

Gene Delivery ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells ◽

Neural Progenitors ◽

Nonviral Gene Delivery

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Nonviral Gene Delivery to Mesenchymal Stem Cells Using Cationic Liposomes for Gene and Cell Therapy

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/735349 ◽

2010 ◽

Vol 2010 ◽

pp. 1-12 ◽

Cited By ~ 55

Author(s):

C. Madeira ◽

R. D. Mendes ◽

S. C. Ribeiro ◽

J. S. Boura ◽

M. R. Aires-Barros ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Gene Delivery ◽

Cationic Liposome ◽

Nonviral Gene Delivery ◽

Great Promise ◽

Full Potential ◽

Genetically Engineer ◽

Or Gene ◽

First Time

Mesenchymal stem cells (MSCs) hold a great promise for application in several therapies due to their unique biological characteristics. In order to harness their full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing an efficient and safe methodology to genetically engineer human bone marrow MSC (BM MSC), enhancing their therapeutic efficacy in Regenerative Medicine. The plasmid DNA delivery was optimized using a cationic liposome-based reagent. Transfection efficiencies ranged from ~2% to ~35%, resulting from using a Lipid/DNA ratio of 1.25 with a transgene expression of 7 days. Importantly, the number of plasmid copies in different cell passages was quantified for the first time and ~20,000 plasmid copies/cell were obtained independently of cell passage. As transfected MSC have shown high viabilities (>90%) and recoveries (>52%) while maintaining their multipotency, this might be an advantageous transfection strategy when the goal is to express a therapeutic gene in a safe and transient way.

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