Minicircle microporation-based non-viral gene delivery improved the targeting of mesenchymal stem cells to an injury site.
Genetic engineering approaches to improve the therapeutic potential of mesenchymal stem cells (MSCs) have been made by viral and non-viral gene delivery methods. Viral methods have severe limitations in clinical application because of potential oncogenic, pathogenic, and immunogenic risks, while non-viral methods have suffered from low transfection efficiency and transient weak expression as MSCs are hard-to-transfect cells. In this study, minicircle, which is a minimal expression vector free of bacterial sequences, was employed for MSC transfection as a non-viral gene delivery method. The conventional cationic liposome method was not effective for MSC transfection as it resulted in very low transfection efficiency (less than 5%). Microporation, a new electroporation method, greatly improved the transfection efficiency of minicircles by up to 66% in MSCs without any significant loss of cell viability. Furthermore, minicircle microporation generated much stronger and prolonged transgene expression compared with plasmid microporation. When MSCs microporated with minicircle harboring firefly luciferase gene were subcutaneously injected to mice, the bioluminescence continued for more than a week, whereas the bioluminescence of the MSCs induced by plasmid microporation rapidly decreased and disappeared in mice within three days. By minicircle microporation as a non-viral gene delivery, MSCs engineered to overexpress CXCR4 showed greatly increased homing ability toward an injury site as confirmed through in vivo bioluminescence imaging in mice. In summary, the engineering of MSCs through minicircle microporation is expected to enhance the therapeutic potential of MSCs in clinical applications.
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, Republic of Korea.
Ji-Young
JY
Mun
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, Republic of Korea.
Keun Koo
KK
Shin
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, Republic of Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon, Republic of Korea.
Ohsuk
O
Kwon
SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Republic of Korea. Electronic address: yongtaik@skku.edu.
Yong Taik
YT
Lim
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, Republic of Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon, Republic of Korea. Electronic address: dboh@kribb.re.kr.
Doo-Byoung
DB
Oh
D000818:Animals; D002460:Cell Line; D002465:Cell Movement; D006801:Humans; D008163:Luminescent Measurements; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D008807:Mice, Inbred BALB C; D061848:Optical Imaging; D010957:Plasmids / Q000235:genetics*; D019718:Receptors, CXCR4 / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D019076:Transgenes; D014945:Wound Healing
D019718:Receptors, CXCR4
Bioluminescence imaging; CXCR4; Mesenchymal stem cells; Microporation; Minicircle; Non-viral gene delivery