Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

High efficiency non-viral transfection of retinal and iris pigment epithelial cells with pigment epithelium-derived factor

Gene Therapy volume 17, pages 181–189 (2010)Cite this article

Abstract

Transplantation of pigment epithelial cells in patients with age-related macular degeneration and Parkinson's disease has the potential to improve functional rehabilitation. Genetic modification of cells before transplantation may allow the delivery of neuroprotective factors to achieve functional improvement. As transplantation of cells modified using viral vectors is complicated by the possible dissemination of viral particles and severe immune reactions, we have explored non-viral methods to insert genetic material in pigment epithelial cells. Using lipofection or nucleofection ARPE-19 cells, freshly isolated and primary retinal and iris pigment epithelial (IPE) cells were transfected with plasmids encoding green fluorescent protein (GFP) and with three plasmids encoding recombinant pigment epithelium-derived factor (PEDF) and GFP. Transfection efficiency was evaluated by fluorescence microscopy and stability of protein expression by immunoblotting. Pigment epithelial cells were successfully transfected with plasmid encoding GFP. Expression of GFP in ARPE-19 was transient, but was observed for up to 1 year in IPE cells. Analysis of pigment epithelial cells transfected with PEDF plasmids revealed that PEDF fusion proteins were successfully expressed and functionally active. In conclusion, efficient transfer of genetic information in pigment epithelial cells can be achieved using non-viral transfection protocols.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Stover NP, Watts RL . Spheramine for treatment of Parkinson's disease. Neurotherapeutics 2008; 5: 252–259.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Stover NP, Bakay RA, Subramanian T, Raiser CD, Cornfeldt ML, Schweikert AW et al. Intrastriatal implantation of human retinal pigment epithelial cells attached to microcarriers in advanced Parkinson disease. Arch Neurol 2005; 62: 1833–1837.

    Article  PubMed  Google Scholar 

  3. Watts RL, Raiser CD, Stover NP, Cornfeldt ML, Schweikert AW, Allen RC et al. Stereotaxic intrastriatal implantation of human retinal pigment epithelial (hRPE) cells attached to gelatin microcarriers: a potential new cell therapy for Parkinson's disease. J Neural Transm Suppl 2003; 65: 215–227.

    Article  Google Scholar 

  4. Binder S, Krebs I, Hilgers RD, Abri A, Stolba U, Assadoulina A et al. Outcome of transplantation of autologous retinal pigment epithelium in age-related macular degeneration: a prospective trial. Invest Ophthalmol Vis Sci 2004; 45: 4151–4160.

    Article  PubMed  Google Scholar 

  5. Maaijwee K, Missotten T, Mulder P, van Meurs JC . Influence of intraoperative course on visual outcome after an RPE-choroid translocation. Invest Ophthalmol Vis Sci 2008; 49: 758–761.

    Article  PubMed  Google Scholar 

  6. Maaijwee K, Heimann H, Missotten T, Mulder P, Joussen A, van MJ . Retinal pigment epithelium and choroid translocation in patients with exudative age-related macular degeneration: long-term results. Graefes Arch Clin Exp Ophthalmol 2007; 245: 1681–1689.

    Article  PubMed  Google Scholar 

  7. Aisenbrey S, Lafaut BA, Szurman P, Hilgers RD, Esser P, Walter P et al. Iris pigment epithelial translocation in the treatment of exudative macular degeneration: a 3-year follow-up. Arch Ophthalmol 2006; 124: 183–188.

    Article  PubMed  Google Scholar 

  8. Thumann G, Aisenbrey S, Schraermeyer U, Lafaut B, Esser P, Walter P et al. Transplantation of autologous iris pigment epithelium after removal of choroidal neovascular membranes. Arch Ophthalmol 2000; 118: 1350–1355.

    Article  CAS  PubMed  Google Scholar 

  9. Binder S, Stanzel VB, Krebs I, Glittenberg C . Transplantation of the RPE in AMD. 2007; 26: 516–554.

  10. Bok D, Yasumura D, Matthes MT, Ruiz A, Duncan JL, Chappelow AV et al. Effects of adeno-associated virus-vectored ciliary neurotrophic factor on retinal structure and function in mice with a P216L rds/peripherin mutation. Exp Eye Res 2002; 74: 719–735.

    Article  CAS  PubMed  Google Scholar 

  11. Faktorovich EG, Steinberg RH, Yasumura D, Matthes MT, Lavail MM . Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor. Nature 1990; 347: 83–86.

    Article  CAS  PubMed  Google Scholar 

  12. Semkova I, Kreppel F, Welsandt G, Luther T, Kozlowski J, Janicki H et al. Autologous transplantation of genetically modified iris pigment epithelial cells: a promising concept for the treatment of age-related macular degeneration and other disorders of the eye. Proc Natl Acad Sci USA 2002; 99: 13090–13095.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Georgievska B, Kirik D, Rosenblad C, Lundberg C, Bjorklund A . Neuroprotection in the rat Parkinson model by intrastriatal GDNF gene transfer using a lentiviral vector. Neuroreport 2002; 13: 75–82.

    Article  CAS  PubMed  Google Scholar 

  14. Ye M, Wang XJ, Zhang YH, Lu GQ, Liang L, Xu JY et al. Transplantation of bone marrow stromal cells containing the neurturin gene in rat model of Parkinson's disease. Brain Res 2007; 1142: 206–216.

    Article  CAS  PubMed  Google Scholar 

  15. Robertson RP . Medical progress: Islet transplantation as a treatment for diabetes - A work in progress. N Engl J Med 2004; 350: 694–705.

    Article  CAS  PubMed  Google Scholar 

  16. Chen NK, Wong JS, Kee IH, Lai SH, Thng CH, Ng WH et al. Nonvirally modified autologous primary hepatocytes correct diabetes and prevent target organ injury in a large preclinical model. PLoS ONE 2008; 3: e1734.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Abe T, Tomita H, Ohashi T, Yamada K, Takeda Y, Akaishi K et al. Characterization of iris pigment epithelial cell for auto cell transplantation. Cell Transplant 1999; 8: 501–510.

    Article  CAS  PubMed  Google Scholar 

  18. Thumann G, Schraermeyer U, Bartz-Schmidt KU, Heimann K . Descemet′s membrane as membranous support in RPE/IPE transplantation. Curr Eye Res 1997; 16: 1236–1238.

    Article  CAS  PubMed  Google Scholar 

  19. Thumann G, Bartz-Schmidt KU, Heimann K, Schraermeyer U . Phagocytosis of rod outer segments by human iris pigment epithelial cells in vitro. Graefes Arch Clin Exp Ophthalmol 1998; 236: 753–757.

    Article  CAS  PubMed  Google Scholar 

  20. Thumann G, Hueber A, Dinslage S, Schaefer F, Yasukawa T, Kirchhof B et al. Characteristics of iris and retinal pigment epithelial cells cultured on collagen type I membranes. Curr Eye Res 2006; 31: 241–249.

    Article  CAS  PubMed  Google Scholar 

  21. Abe T, Yoshida M, Tomita H, Kano T, Sato M, Wada Y et al. Auto iris pigment epithelial cell transplantation in patients with age- related macular degeneration: short-term results. Tohoku J Exp Med 2000; 191: 7–20.

    Article  CAS  PubMed  Google Scholar 

  22. Crafoord S, Geng L, Seregard S, Algvere PV . Photoreceptor survival in transplantation of autologous iris pigment epithelial cells to the subretinal space. Acta Ophthalmol Scand 2002; 80: 387–394.

    Article  PubMed  Google Scholar 

  23. Lappas A, Foerster AM, Weinberger AW, Coburger S, Schrage NF, Kirchhof B . Translocation of iris pigment epithelium in patients with exudative age-related macular degeneration: long-term results. Graefes Arch Clin Exp Ophthalmol 2004; 242: 638–647.

    Article  PubMed  Google Scholar 

  24. Thumann G, Bartz-Schmidt KU, El Bakri H, Schraermeyer U, Spee C, Cui JZ et al. Transplantation of autologous iris pigment epithelium to the subretinal space in rabbits. Transplantation 1999; 68: 195–201.

    Article  CAS  PubMed  Google Scholar 

  25. Thumann G, Aisenbrey S, Schraermeyer U, Lafaut B, Esser P, Walter P et al. Transplantation of autologous iris pigment epithelium after removal of choroidal neovascular membranes. 2000; 118: 1350–1355.

  26. Arnhold S, Semkova I, Andressen C, Lenartz D, Meissner G, Sturm V et al. Iris pigment epithelial cells: a possible cell source for the future treatment of neurodegenerative diseases. Exp Neurol 2004; 187: 410–417.

    Article  CAS  PubMed  Google Scholar 

  27. Abe T . Regeneration of the retina using pigment epithelial cell transplantation]. Nippon Ganka Gakkai Zasshi 2002; 106: 778–803.

    CAS  PubMed  Google Scholar 

  28. Thumann G, Kirchhof B . Transplantation of iris pigment epithelium]. Ophthalmologe 2004; 101: 882–885.

    Article  CAS  PubMed  Google Scholar 

  29. Rebersek M, Faurie C, Kanduser M, Corovic S, Teissie J, Rols MP et al. Electroporator with automatic change of electric field direction improves gene electrotransfer in-vitro. Biomed Eng Online 2007; 6: 25.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Mutlu GM, Machado-Aranda D, Norton JE, Bellmeyer A, Urich D, Zhou R et al. Electroporation-mediated gene transfer of the Na+, K+-ATPase rescues endotoxin-induced lung injury. Am J Respir Crit Care Med 2007; 176: 582–590.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zeitelhofer M, Vessey JP, Xie Y, Tubing F, Thomas S, Kiebler M et al. High-efficiency transfection of mammalian neurons via nucleofection. Nat Protoc 2007; 2: 1692–1704.

    Article  CAS  PubMed  Google Scholar 

  32. Barbu A, Welsh N . Lipofection of insulin-producing RINm5F cells: methodological improvements. J Liposome Res 2007; 17: 49–62.

    Article  CAS  PubMed  Google Scholar 

  33. Lorenz P, Harnack U, Morgenstern R . Efficient gene transfer into murine embryonic stem cells by nucleofection. Biotechnol Lett 2004; 26: 1589–1592.

    Article  CAS  PubMed  Google Scholar 

  34. Kachi S, Oshima Y, Esumi N, Kachi M, Rogers B, Zack DJ et al. Nonviral ocular gene transfer. Gene Ther 2005; 12: 843–851.

    Article  CAS  PubMed  Google Scholar 

  35. Leung KW, Liu MG, Xu XM, Seiler MJ, Barnstable CJ, Tombran-Tink J . Expression of ZnT and ZIP zinc transporters in the human RPE and their regulation by neurotrophic factors. Invest Ophthalmol Vis Sci 2008; 49: 1221–1231.

    Article  PubMed  Google Scholar 

  36. Acland GM, Aguirre GD, Ray J, Zhang Q, Aleman TS, Cideciyan AV et al. Gene therapy restores vision in a canine model of childhood blindness. Nat Genet 2001; 28: 92–95.

    CAS  PubMed  Google Scholar 

  37. Rolling F, Le MG, Stieger K, Smith AJ, Weber M, Deschamps JY et al. Gene therapeutic prospects in early onset of severe retinal dystrophy: restoration of vision in RPE65 Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium. Bull Mem Acad R Med Belg 2006; 161: 497–508.

    CAS  PubMed  Google Scholar 

  38. Nusinowitz S, Ridder III WH, Pang JJ, Chang B, Noorwez SM, Kaushal S et al. Cortical visual function in the rd12 mouse model of Leber Congenital Amarousis (LCA) after gene replacement therapy to restore retinal function. Vision Res 2006; 46: 3926–3934.

    Article  CAS  PubMed  Google Scholar 

  39. Pang JJ, Chang B, Kumar A, Nusinowitz S, Noorwez SM, Li J et al. Gene therapy restores vision-dependent behavior as well as retinal structure and function in a mouse model of RPE65 Leber congenital amaurosis. Mol Ther 2006; 13: 565–572.

    Article  CAS  PubMed  Google Scholar 

  40. Yoshioka Y, Abe T, Wakusawa R, Moriya T, Mochizuki S, Saigo Y et al. Recombinant AAV-transduced iris pigment epithelial cell transplantation may transfer vector to native RPE but suppress systemic dissemination. Invest Ophthalmol Vis Sci 2006; 47: 745–752.

    Article  PubMed  Google Scholar 

  41. Worgall S, Sondhi D, Hackett NR, Kosofsky B, Kekatpure MV, Neyzi N et al. Treatment of late infantile neuronal ceroid lipofuscinosis by CNS administration of a serotype 2 adeno-associated virus expressing CLN2 cDNA. Hum Gene Ther 2008; 19: 463–474.

    Article  CAS  PubMed  Google Scholar 

  42. Farjo R, Skaggs J, Quiambao AB, Cooper MJ, Naash MI . Efficient non-viral ocular gene transfer with compacted DNA nanoparticles. PLoS ONE 2006; 1: e38.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Thumann G, Viethen A, Gaebler A, Walter P, Kaempf S, Johnen S et al. The in vitro and in vivo behaviour of retinal pigment epithelial cells cultured on ultrathin collagen membranes. Biomaterials 2009; 30: 287–294.

    Article  CAS  PubMed  Google Scholar 

  44. Stöcker M, Pardo A, Hetzel C, Reutelingsperger C, Fischer R, Barth S . Eukaryotic expression and secretion of EGFP-labeled annexin A5. Protein Expr Purif 2008; 58: 325–331.

    Article  PubMed  Google Scholar 

  45. Stöcker M, Tur MK, Sasse S, Krussmann A, Barth S, Engert A . Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells. Protein Expr Purif 2003; 28: 211–219.

    Article  PubMed  Google Scholar 

  46. Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25: 402–408.

    Article  CAS  PubMed  Google Scholar 

  47. Pfaffl MW . A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29: e45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Laemmli UK . Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 1970; 227: 680–685.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant from the Interdisciplinary Center for Clinical Research ‘Biomat.’ within the faculty of Medicine at the RWTH Aachen University. The authors thank Angela Bleck (Fraunhofer IME, Department of Pharmaceutical Product Development, Forckenbeckstraße 6, 52074 Aachen, Germany) for technical assistance and Ria Lindt for initial work in this project.

Author information

Authors and Affiliations

  1. IZKF ‘Biomat.’, RWTH Aachen University, Aachen, Germany

    G Thumann, M Stöcker, C Maltusch, A K Salz & S Johnen

  2. Department of Ophthalmology, RWTH Aachen University, Aachen, Germany

    G Thumann & P Walter

  3. Department of Experimental Medicine and Immunotherapy, Helmholtz-Institute for Applied Medical Engineering, Aachen, Germany

    S Barth

Authors
  1. G Thumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M Stöcker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C Maltusch
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A K Salz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S Barth
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. P Walter
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. S Johnen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G Thumann.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Thumann, G., Stöcker, M., Maltusch, C. et al. High efficiency non-viral transfection of retinal and iris pigment epithelial cells with pigment epithelium-derived factor. Gene Ther 17, 181–189 (2010). https://doi.org/10.1038/gt.2009.124

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2009.124

Keywords

This article is cited by

Search

Advanced search

Quick links