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

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.


Introduction
Many studies have emphasized the clinical importance of mesenchymal stem cells (MSCs) in degenerative and regenerative medicine. Recently, extensive clinical trials of MSCs have been conducted. However, their efficacy depreciates because of poor survival and engraftment in injured organs. In particular, the therapeutic mechanism or "mode of action" is lacking. MSC-based therapy using a gene delivery system shows enhanced efficacy due to improvement in cell survival and secretion of numerous growth factors (1). In general, viral transfection has advantages such as high efficiency in genetic transfer and stable gene expression. Previously, an optimal protocol was developed using lentiviral transfection in bone marrow-derived MSCs (BM-MSCs), resulting in higher efficiency and lower cell toxicity (2,3). However, viral vectors have several limitations, including their risk of immunogenicity and chromosomal integration in clinical applications (4). The current strategy, utilizing Nucleofection TM for the nonviral gene delivery, is not only acceptable with regard to safety issues but also is easy to handle, indicating possible applications in patient. In particular, this method has been successfully applied to difficult-to-transfect primary cells, including MSCs. Nucleofection TM platforms could potentially affect the innate abilities of MSCs (5). In our previous reports, naïve placenta-derived MSCs (PD-MSCs) were transfected using Nucleofection TM and subjected to genetic analyses (6). However, the Nucleofection TM non-viral gene delivery in MSCs is still being developed, requiring their optimization to safely establish a cell line, maintain the characteristics of naïve MSCs, and promote therapeutic efficacy. In this report, we will highlight the optimal protocol for the generation of functionally enhanced MSCs using a nonviral gene delivery system.

Primary cells
Naïve PD-MSCs were isolated as previously described using placentas for all participating women at term (≥37 gestational weeks) (7). The protocol was approved by the Institutional Review Board (IRB) of CHA General Hospital, Seoul, Republic of Korea (IRB 07-18).
►Note: Transfection using naïve PD-MSCs may be donor-dependent. Thus, it is recommended to use passages earlier than 9.
1.3 Remove the medium from the cultured naïve PD-MSCs and wash the cells with DPBS 2 times.
1.4 Add 2 ml of TrypLE TM Express (＋) Phenol Red and incubate the cells for 2 min at 37℃ in a 5% CO2 incubator.
1.5 Harvest cells with prewarmed complete medium and obtain an aliquot of cells.
1.6 Count the Trypan blue-stained cells using hemocytometer and divide the cell suspension (5×10 5 cells/tube) in each 15 ml conical tube. 1.7 Centrifuge at 200 x g for 10 min at room temperature. ►Note: To avoid the cell damage, speed should not exceed 200 x g.
Nucleofection TM : 2.1 Ensure that the entire supplement solution is added to the Nucleofection TM solution.
►Note: The ratio of the Nucleofection TM solution to the supplement solution is 4.5：1. The mixture is stable for 3 months at 4℃. For a single reaction, mix 82 μl of Nucleofection TM solution and 18 μl of supplement solution.
2.2 Operate the 4D Nucleofector TM System and set the appropriate Nucleofector TM program.
►Note: Turn on the 4D Nucleofector TM System → choose a X unit device → choose a cuvette vessel → choose the number of cuvettes → Select a cell type program "MSC, human" → pulse code was set up as FF-104 → press OK ►Note: Pulse code as FF-104 for high efficiency. 2.3 Prepare complete medium in 100 mm cell culture dish and preincubate/equilibrate each plate without cells at 37℃ in a 5% CO2 incubator.
►Note: For the stabilization of transfected PD-MSCs, its step is important to improve transfection efficiency and viability. Primer sequences Tm 2.4 After centrifugation at 200 x g for 10 min at room temperature, carefully resuspend the cell pellet in 100 μl of Nucleofection TM solution.
2.5 Add an appropriate volume of plasmid in each tube and mix well.
►Note: Make sure that matermixtures must mix well while pipetting.
►Note: Volume of mastermixtures should not exceed maximum 10 % of the total reaction volume (10 μl for 100 μl reaction). It is recommended that the concentration of plasmid is suitable for 1 μg/μl.
2.6 Transfer mastermixtures into the Nucleocuvette TM Vessels and place Nucleocuvette TM Vessels in the retainer of the 4D Nucleofector TM X unit device.
►Note: In order to prevent an electrical signal error, avoid air bubbles while transferring mastermixtures. 2.7 Start Nucleofection process according to the presetting value (refer to 2.2).
2.8 After running, remove Nucleocuvette TM Vessels in the retainer of the device and incubate Nucleocuvette TM for 10 min at room temperature.
2.9 Add 500 μl of prewarmed PD-MSCs medium and gently resuspend the transfected cells.
2.10 Plate the transfected cells in preincubated cell culture plates.
►Note: Gene expression is often detectable after 4∼8 h. However, gene or GFP expression in PD-MSCs recommended 24 h post Nucleofection TM . 3.2 After 24 h, remove medum and change selection medium including 1.5 mg/ml neomycin for 7 days.
3.3 Confirm the gene or GFP expression.

Characterizations
Cell viability and transfection efficiency using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting: PRL-1-overexpressing PD-MSCs (PD-MSCs PRL-1 , PRL-1＋) were counted 24 h post transfection to examine the transfected cell viability. For analysis of the efficiency of PD-MSCs PRL-1 post-transfection, total RNA and protein lysate were extracted from the cell pellet. qRT-PCR analysis was performed with primers (Table 1) using the CFX Connect TM Real-Time System (Bio-Rad, USA). Western blotting was performed with primary monoclonal anti-turboGFP (1：500, Origene, Inc.) and horseradish peroxidase (HRP)-conjugated anti-goat IgG (Santa Cruz Biotechnology, USA).
Expression of stemness markers by reverse transcription (RT)-PCR: For analysis of stemness markers in PD-MSCs PRL-1 , PCR amplification was conducted with Taq DNA polymerase (Solgent, Korea) according to the manufacturer's instructions. Each primer is provided in Table 1.
Karyotyping: For analysis of karyotypes of naïve and PD-MSCs PRL-1 , G-banding techniques were performed, and the karyotype was elucidated by the International System for Human Cytogenomic Nomenclature (ISCN 2016) using an Axipskop 2 plus light microscope (Zeiss, Germany).

Safety evaluation of PD-MSCs PRL-1 using the nonviral electroporation technique
The expression of PRL-1 in PD-MSCs PRL-1 was significantly increased until passage 8 compared to that of the naïve PD-MSCs (Fig. 3A, *p＜0.05). The karyotypes of the naïve and PD-MSCs PRL-1 were diploid and stable (Fig. 3B). In NOD/SCID mouse testes, there was no teratoma formation (Fig. 3C), and human Alu expression was significantly increased after PD-MSC PRL-1 transplantation (Fig. 3D, *p＜0.05). Therefore, these data suggest that PD-MSCs PRL-1 generated by a nonviral electroporation system showed efficacy as well as safety.

Conclusion
An optimized protocol for PD-MSCs PRL-1 using nonviral AMAXA electroporation was established, and its efficiency was confirmed. In addition, the characteristics of PD-MSCs PRL-1 were similar to those of the naïve MSCs, and a safety evaluation was accomplished. Therefore, this nonviral electroporation technique could be a new strategy for the clinical application of stem cell therapy.