Int J Stem Cells.  2021 Feb;14(1):112-118. 10.15283/ijsc20117.

Efficacy of Gene Modification in Placenta-Derived Mesenchymal Stem Cells Based on Nonviral Electroporation

• Kim JY
• Choi JH
• Kim SH
• Park H
• Lee D
• Kim GJ

• KMID: 2513088
• DOI: http://doi.org/10.15283/ijsc20117

Abstract

Mesenchymal stem cell (MSC)-based therapy using gene delivery systems has been suggested for degenerative diseases. Although MSC-based clinical applications are effective and safe, the mode of action remains unclear. Researchers have commonly applied viral-based gene modification because this system has efficient vehicles. While viral transfection carries many risks, such as oncogenes and chromosomal integration, nonviral gene delivery techniques are less expensive, easier to handle, and safe, although they are less efficient. The electroporation method, which uses Nucleofection technology, provides critical opportunities for hard-to-transfect primary cell lines, including MSCs. Therefore, to improve the therapeutic efficacy using genetically modified MSCs, researchers must determine the optimal conditions for the introduction of the Nucleofection technique in MSCs. Here, we suggest optimal methods for gene modification in PD-MSCs using an electroporation gene delivery system for clinical application.

Keyword

Electroporation; Gene modification; Placenta-derived mesenchymal stem cells; Nonviral gene delivery system

Figure

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  Fig. 1 Optimization of transfection in PD-MSCs. (A) Maps of GFP, lentiviral, and nonviral plasmid vectors. (B) GFP expression using 2 and 5 μg of PRL-1 plasmid using nonviral electroporation as well as the lentiviral system. Scale bar=100 μm. (C) Cell viability at 24 h after transfection. (D) mRNA expression of PRL-1 in PD-MSCsPRL-1. *p<0.05 versus GFP; **p<0.05 versus 2 μg; #p<0.05 versus lentiviral system. (E) Stemness markers (e.g., Oct4, Nanog, Sox2), TERT, and HLA-G expression in PD-MSCsPRL-1 were determined by RT-PCR. Data from each group are expressed as the mean±SD.

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  Fig. 2 Characterization of PD-MSCsPRL-1 using a nonviral electroporation system. (A) Hydrophobic PRL-1 levels in the cell culture supernatant. *p<0.05 versus naïve. (B) GFP fusion protein expression after transfection shown by western blotting. (C) FACS analysis of surface markers (positive: CD13, CD90, CD105, HLA-ABC, and HLA-G; negative: HLA-DR) in PD-MSCsPRL-1. (D) Osteogenic differentiation (Diff) by von Kossa staining and mRNA of BGLAP and COL1A1. (E) Adipogenic differentiation by oil red O staining and mRNA of adipsin and PPARG. (F) Hepatogenic differentiation by ICG uptake and mRNA of albumin and TAT by qRT-PCR. Scale bar=50 μm. Data from each group are expressed as the mean±SD. *p<0.05 versus undifferentiated (Undiff).

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  Fig. 3 Safety evaluation of PD- MSCsPRL-1 using the nonviral electroporation technique. (A) mRNA expression of PRL-1 in PD-MSCsPRL-1 from 10 passages by qRT-PCR. *p<0.05 versus naïve. (B) Karyotyping of naïve and PD-MSCsPRL-1. (B) H&E staining in normal (Con) and PD- MSCPRL-1 transplanted testes (Tx) in NOD/SCID mice. (C) Human Alu expression in gDNA from each testis. Data from each group are expressed as the mean±SD. *p<0.05 versus Con.

Reference

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