Biopolym. Cell. 2020; 36(6):433-445.
http://dx.doi.org/10.7124/bc.000A41
Methods
Transfection of mesenchymal stem cells at physiological oxygen concentrations
1Shuvalova N. S., 1, 2Toporova O. K., 1, 2Kordium V. A.
  1. Institute of Genetic and Regenerative Medicine, NAMS of Ukraine
    67, Vyshgorodska Str., Kyiv, Ukraine, 04114
  2. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143

Abstract

Aim. Numerous works showed the beneficial effect of oxygen in physiological concentrations on mesenchymal stem cells (MSC) cultures. The aim of the present work was to study the impact of physiological oxygen tensions on non-viral transfection of MSC from human Wharton jelly (WJ-MSC). Methods. WJ-MSC at passage 2 were cultivated for 48 hours in different gas mixtures: nitrogen-based (oxygen – 3 %, CO2–5 %, nitrogen – 93 %) (=101 % ? nr?) and argon-based (oxygen – 3 %, CO2–5 %, argon – 93 %), (=101 % ? nr?) while the control group was cultivated under standard conditions of CO2-incubator (ambient oxygen concentration, CO2–5 %). After 48 hours of cultivation the cells were transfected with polyplexes pEGFP-C1/PEI/ and pEGFP-C1/TurboFect, containing 3 μg of plasmid DNA. The cultures were in contact with complexes for 1 hour, at standard conditions of CO2 – incubator. Next, the media were changed, and WJ-MSC were cultivated for 48 hours in described gas mixtures, and CO2 – incubator. Transfection effectiveness was estimated by flow cytometry as the number of eGFP+ fluorescent cells, (BD FACSAria). Results. In the groups cultivated in gas mixtures containing 3 % of oxygen, the number of cells, synthesizing eGFP+ before and after the transfection procedure, was on average 2.58 times higher in the nitrogen-based mixture and 1.37 times higher in the argon-based mixture than in control groups from the CO2 –incubator. Conclusions. The cultivation of human WJ-MSC under physiological oxygen tensions allowed an increase in the percentage of transfected cells, and is promising to be used as a method for optimization of transfection.
Keywords: mesenchymal stem cells, Wharton jelly, non-viral transfection, transfection, hypoxia, argon
Full text: (PDF, in English)

References

[1] Taghizadeh RR, Cetrulo KJ, Cetrulo CL. Wharton's Jelly stem cells: future clinical applications. Placenta. 2011; 32(Suppl 4):S311-5.
[2] Hass R, Kasper C, Böhm S, Jacobs R. Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell Commun Signal. 2011; 9:12.
[3] Lund TC, Kobs A, Blazar BR, Tolar J. Mesenchymal stromal cells from donors varying widely in age are of equal cellular fitness after in vitro expansion under hypoxic conditions. Cytotherapy. 2010; 12(8): 971-81.
[4] Neri S. Genetic stability of mesenchymal stromal cells for regenerative medicine applications: a fundamental biosafety aspect. Int J Mol Sci. 2019; 20(10):2406.
[5] Estrada JC, Torres Y, Benguría A, Dopazo A, Roche E, Carrera-Quintanar L, Pérez RA, Enríquez JA, Torres R, Ramírez JC, Samper E, Bernad A. Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy. Cell Death Dis. 2013; 4(6):e691.
[6] Jiang T, Xu G, Wang Q, Yang L, Zheng L, Zhao J, Zhang X. In vitro expansion impaired the stemness of early passage mesenchymal stem cells for treatment of cartilage defects. Cell Death Dis. 2017; 8(6):e2851.
[7] He Y, Zhou S, Liu H, Shen B, Zhao H, Peng K, Wu X. Indoleamine 2, 3-dioxgenase transfected mesenchymal stem cells induce kidney allograft tolerance by increasing the production and function of regulatory T cells. Transplantation. 2015; 99(9):1829-38.
[8] Huang B, Qian J, Ma J, Huang Z, Shen Y, Chen X, Sun A, Ge J, Chen H. Myocardial transfection of hypoxia-inducible factor-1α and co-transplantation of mesenchymal stem cells enhance cardiac repair in rats with experimental myocardial infarction. Stem Cell Res Ther. 2014; 5(1): 22.
[9] Delyagina E, Schade A, Scharfenberg D, Skorska A, Lux C, Li W, Steinhoff G. Improved transfection in human mesenchymal stem cells: effective intracellular release of pDNA by magnetic polyplexes. Nanomedicine. 2014; 9(7):999.
[10] Papayannakos C, Daniel R. Understanding lentiviral vector chromatin targeting: working to reduce insertional mutagenic potential for gene therapy. Gene Ther. 2013; 20(6):581-8.
[11] Schaffert D, Wagner E. Gene therapy progress and prospects: Synthetic polymer-based systems. Gene Ther. 2008; 15(16):1131-8.
[12] Wang W, Xu X, Li Z, Lendlein A, Ma N. Genetic engineering of mesenchymal stem cells by non-viral gene delivery. Clin Hemorheol Microcirc. 2014; 58(1):19-48.
[13] Mas-Bargues C, Sanz-Ros J, Román-Domínguez A, Inglés M, Gimeno-Mallench L, El Alami M, Viña-Almunia J, Gambini J, Viña J, Borrás C. Relevance of oxygen concentration in stem cell culture for regenerative medicine. Int J Mol Sci. 2019; 20(5):1195.
[14] Estrada JC, Albo C, Benguría A, Dopazo A, López-Romero P, Carrera-Quintanar L, Roche E, Clemen-te EP, Enríquez JA, Bernad A, Samper E. Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis. Cell Death Differ. 2012; 19(5):743-55.
[15] Wang Z, Fang B, Tan Z, Zhang D, Ma H. Hypoxic preconditioning increases the protective effect of bone marrow mesenchymal stem cells on spinal cord ischemia/reperfusion injury. Mol Med Rep 2016; 13(3):1953-60.
[16] Liu H, Liu S, Li Y, Wang X, Xue W, Ge G, Luo X. The role of SDF-1-CXCR4/CXCR7 axis in the therapeutic effects of hypoxia-preconditioned mesenchymal stem cells for renal ischemia/reperfusion injury. PLoS One. 2012; 7(4):e34608.
[17] Chen L, Xu Y, Zhao J, Zhang Z, Yang R, Xie J, Liu X, Qi S. Conditioned medium from hypoxic bone marrow-derived mesenchymal stem cells enhances wound healing in mice. PLoS ONE. 2014; 9(4): e96161.
[18] Zhang J, Xiong L, Tang W, Tang L, Wang B. Hypoxic culture enhances the expansion of rat bone marrow-derived mesenchymal stem cells via the regulatory pathways of cell division and apoptosis. In Vitro Cell Dev Biol Animal. 2018; 54(9):666-76.
[19] Hu C, Li L. Preconditioning influences mesenchymal stem cell properties in vitro and in vivo. J Cell Mol Med. 2018; 22(3):1428-42.
[20] Zhang L, Yang J, Tian YM, Guo H, Zhang Y. Beneficial Effects of Hypoxic Preconditioning on Human Umbilical Cord Mesenchymal Stem Cells. Chin J Physiol. 2015;58(5):343-53.
[21] Shuvalova NS, Kordium VA. Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions. Biopolym Cell. 2015; 31(3): 233-9.
[22] Shuvalova NS, Kordium VA. Proliferation of Wharton jelly mesenchymal stem cells, derived by preserving the cells with reduced attachment rate, under various gas conditions. Biopolym Cell. 2015; 31(6):447-54.
[23] Lykhmus O, Koval L, Voytenko L, Uspenska K, Komisarenko S, Deryabina O, Shuvalova N, Kordium V, Usty-menko A, Kyryk V, Skok M. Intravenously injected mesenchymal stem cells penetrate the brain and treat inflammation-induced brain damage and memory impairment in mice. Front Pharmacol. 2019; 10: 355.
[24] Lykhmus O, Kalashnyk O, Koval L, Voytenko L, Uspenska K, Komisarenko S, Deryabina O, Shuvalova N, Kordium V, Ustymenko A, Kyryk V, Skok M. Mesenchymal Stem Cells or Interleukin-6 Improve Episodic Memory of Mice Lacking α7 Nicotinic Acetylcholine Receptors. Neuroscience. 2019; 413: 31-44.
[25] Maslova OO, Shpilova SP, Shuvalova NS, Derybia OG, Kordium VA. Spontaneous formation of spheroids in human umbilical cord matrix derived cells culture. Biol Stud. 2012: 6(2); 79-86.
[26] Shuvalova NS, Kordium VA. Morphological characteristics of mesenchymal stem cells from Wharton jelly, cultivated under physiological oxygen tensions, in various gas mixtures. Biopolym Cell. 2016; 32(4):262-70.
[27] Freshney RI. Animal cell culture: a practical approach. Oxford: "IRL Press Limited", 1986. 348 p.
[28] Kordyum VA, Toporova EK, Okunev OV, Pokholenko YA, Suchorada EM, Ruban TA, Andrienko VI, Irodov DM. Novel cell line with multiple markers, derivative of CHO-K1. Biopolym Cell. 2003; 19(3):252-6.
[29] Toporova O, Pokholenko I, Shuvalova N, Kyryk V, Deriabina O, Morgunov P, Irodov D, Kordium V. Genetic engineering of mesenchymal stem cells from umbilical cord Wharton's jelly. World Conference on Regenerative Medicine at: Leipzig, Germany, October 2015. Regenerative Medicine 2015; 10 (07):220.
[30] Madeira C, Mendes RD, Ribeiro SC, Boura JS, Aires-Barros MR, da Silva CL, Cabral JM. Nonviral gene delivery to mesenchymal stem cells using cationic liposomes for gene and cell therapy. J Biomed Biotechnol. 2010; 2010:735349.
[31] Jo J, Tabata Y. Non-viral gene transfection technologies for genetic engineering of stem cells. Eur J Pharm Biopharm. 2008; 68(1): 90-104.
[32] Nekanti U, Dastidar S, Venugopal P, Totey S, Ta M. Increased proliferation and analysis of differential gene expression in human Wharton's jelly-derived mesenchymal stromal cells under hypoxia. Int J Biol Sci. 2010; 6(5):499-512.
[33] Lavrentieva A, Majore I, Kasper C, Hass R. Effects of hypoxic culture conditions on umbilical cord-derived hu-man mesenchymal stem cells. Cell Commun Signal. 2010; 8:18.
[34] Brunner S, Fürtbauer E, Sauer T, Kursa M, Wagner E. Overcoming the nuclear barrier: cell cycle independent nonviral gene transfer with linear polyethylenimine or electroporation. Mol Ther. 2002; 5(1):80-6.
[35] Männistö M, Rönkkö S, Mättö M, Honkakoski P, Hyttinen M, Pelkonen J, Urtti A. The role of cell cycle on polyp-lex-mediated gene transfer into a retinal pigment epithelial cell line. J Gene Med. 2005; 7(4):466-76.
[36] King WJ, Kouris NA, Choi S, Ogle BM, Murphy WL. Environmental parameters influence non-viral transfection of human mesenchymal stem cells for tissue engineering applications. Cell Tissue Res. 2012; 347:689-99.
[37] Gheisari Y, Soleimani M, Azadmanesh K, Zeinali S. Multipotent mesenchymal stromal cells: optimization and comparison of five cationic polymer-based gene delivery methods. Cytotherapy. 2008;10(8):815-23.
[38] Boura JS, Santos FD, Gimble JM, Cardoso CM, Madeira C, Cabral JM, Silva CL. Direct head-to-head compari-son of cationic liposome-mediated gene delivery to mesenchymal stem/stromal cells of different human sources: a comprehensive study. Hum Gene Ther Methods. 2013; 24(1)38-48.