Abstract
Objectives and results
Mesenchymal stromal cells (MSCs) are potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and have significant expansion capability. These characteristics make them strong candidates for delivering genes and restoring organ systems function. However, as other primary cells, MSCs are difficult to transfect. In order to standardize a simple protocol for transfection of MSCs, we conducted a series of experiments and achieved a protocol that does not require the use of viral particles or specific expensive equipment.
Conclusion
MSCs transfection at early passages using a ratio lipid/DNA of 3.0 µL/µg with Lipofectamine 3000® yields good transfection efficiencies for human MSCs (up to 26%) and is rapid, simple, and safe.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Amorin B, Alegretti AP, Valim V, Pezzi A, Laureano AM, da Silva MAL et al (2014) Mesenchymal stem cell therapy and acute graft-versus-host disease: a review. Hum Cell 27(4):137–150. https://doi.org/10.1007/s13577-014-0095-x
Brgles M, Šantak M, Halassy B, Forcic D, Tomašić J (2012) Influence of charge ratio of liposome/DNA complexes on their size after extrusion and transfection efficiency. Int J Nanomed 7:393–401. https://doi.org/10.2147/IJN.S27471
dos Santos F, Andrade PZ, Abecasis MM, Gimble JM, Chase LG, Campbell AM et al (2011) Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions. Tissue Eng C 17(12):1201–1210. https://doi.org/10.1089/ten.tec.2011.0255
Doucet C, Ernou I, Zhang Y, Llense J-R, Begot L, Holy X, Lataillade J-J (2005) Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications. J Cell Physiol 205(2):228–236. https://doi.org/10.1002/jcp.20391
Fiume G, Di Rienzo C, Marchetti L, Pozzi D, Caracciolo G, Cardarelli F (2016) Single-cell real-time imaging of transgene expression upon lipofection. Biochem Biophys Res Commun 474(1):8–14. https://doi.org/10.1016/j.bbrc.2016.03.088
Gresch O, Engel FB, Nesic D, Tran TT, England HM, Hickman ES et al (2004) New non-viral method for gene transfer into primary cells. Methods 33(2):151–163. https://doi.org/10.1016/j.ymeth.2003.11.009
Han Z, Jing Y, Zhang S, Liu Y, Shi Y, Wei L (2012) The role of immunosuppression of mesenchymal stem cells in tissue repair and tumor growth. Cell Biosci 2:8. https://doi.org/10.1186/2045-3701-2-8
Kim A, Yu HY, Heo J, Song M, Shin J-H, Lim J et al (2016) Mesenchymal stem cells protect against the tissue fibrosis of ketamine-induced cystitis in rat bladder. Sci Rep 6:30881. https://doi.org/10.1038/srep30881
Liu Q, Su R-C, Yi W-J, Zheng L-T, Lu S-S, Zhao Z-G (2017) pH and reduction dual-responsive dipeptide cationic lipids with α–tocopherol hydrophobic tail for efficient gene delivery. Eur J Med Chem 129:1–11. https://doi.org/10.1016/j.ejmech.2017.02.010
Lukashev AN, Zamyatnin AA (2016) Viral vectors for gene therapy: current state and clinical perspectives. Biochemistry 81(7):700–708. https://doi.org/10.1134/S0006297916070063
Madeira C, Mendes RD, Ribeiro SC, Boura JS, Aires-Barros MR, da Silva CL, Cabral JMS (2010) Nonviral gene delivery to mesenchymal stem cells using cationic liposomes for gene and cell therapy. J Biomed Biotechnol. https://doi.org/10.1155/2010/735349
Manzini S, Viiri LE, Marttila S, Aalto-Setälä K (2015) A comparative view on easy to deploy non-integrating methods for patient-specific iPSC production. Stem Cell Rev Rep 11(6):900–908. https://doi.org/10.1007/s12015-015-9619-3
Matte U, Baldo G, Giugliani R (2011) Non viral gene transfer approaches for lysosomal storage disorders. In: Yuan X (ed) Non-viral gene therapy. InTech, Rijeka
Mohammadi Z, Afshari JT, Keramati MR, Alamdari DH, Ganjibakhsh M, Zarmehri AM et al (2015) Differentiation of adipocytes and osteocytes from human adipose and placental mesenchymal stem cells. Iran J Basic Med Sci 18(3):259–266
Rao S, Morales AA, Pearse DD (2015) The comparative utility of viromer RED and lipofectamine for transient gene introduction into glial cells. Biomed Res Int. https://doi.org/10.1155/2015/458624
Schenborn ET, Oler J (2000) Liposome-mediated transfection of mammalian cells. Methods Mol Biol 130:155–164
Uchibori R, Tsukahara T, Ohmine K, Ozawa K (2014) Cancer gene therapy using mesenchymal stem cells. Int J Hematol 99(4):377–382. https://doi.org/10.1007/s12185-014-1537-7
Wang W, Xu X, Li Z, Lendlein A, Ma N (2014) Genetic engineering of mesenchymal stem cells by non-viral gene delivery. Clin Hemorheol Microcirc 58(1):19–48. https://doi.org/10.3233/CH-141883
Wang Y, Shang S, Li C (2015) Comparison of different kinds of nonviral vectors for gene delivery to human periodontal ligament stem cells. J Dental Sci 10(4):414–422. https://doi.org/10.1016/j.jds.2015.02.002
Wasungu L, Hoekstra D (2006) Cationic lipids, lipoplexes and intracellular delivery of genes. J Control Release 116(2):255–264. https://doi.org/10.1016/j.jconrel.2006.06.024
Zeitelhofer M, Vessey JP, Xie Y, Tübing F, Thomas S, Kiebler M, Dahm R (2007) High-efficiency transfection of mammalian neurons via nucleofection. Nat Protoc 2(7):1692–1704. https://doi.org/10.1038/nprot.2007.226
Acknowledgements
The authors thank FIPE/HCPA and CNPq for financial support and also Conselho Nacional de Desenvolvimento Científico e Tecnológico (Grant No. 470888/2014-8)
Supplementary information
Supplementary Figure 1—Plasticity of MSCs at passage 9 grown under our conditioned media for differentiation into (A) osteoblasts (in Alizarin Red staining), (B) adipocytes (in Oil Red O staining), and (C) chondrocytes (in Alcian Blue staining). 20× magnification. Size bars = 50 µm.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Rights and permissions
About this article
Cite this article
de Carvalho, T.G., Pellenz, F.M., Laureano, A. et al. A simple protocol for transfecting human mesenchymal stem cells. Biotechnol Lett 40, 617–622 (2018). https://doi.org/10.1007/s10529-018-2505-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-018-2505-8