The effect of platelet gel-containing culture medium on the viability and expression of PAX6 and RPE65 genes in human retinal pigment epithelial cells

Document Type : Original Article

Authors

1 Department of Hematology, School of Medicine, Tarbiyat Modares University, Tehran, Iran

2 Ocular Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3 Department of Physiology, School of Medicine, Shahed University, Tehran, Iran

Abstract

Background and Objective: Degradation of human retinal pigment epithelium (hRPE) during some disorders such as aging is associated with severe impairment of ocular optical receptor function. Cellular and tissue transplantation of RPE layer is one of the treatment options that has been considered in recent years. The use of platelet gel has shown beneficial effects on the growth of various cultured cells.This study was conducted to investigate the effects of three different concentrations of platelet gel on the viability and the expression of PAX6 and RPE65 genes of cultivated RPE cells.
Materials and Methods: hRPE cells extracted from the human eye were cultured in plates containing platelet gel (PL) of different concentrations (10%, 20%, and 30%). Cells viability was tested by MTT test and the expression RPE65 and PAX6 genes were analyzed by using real time PCR. The number of expression changes of each gene in different treatments compared to the control and according to the internal GAPDH gene was calculated according to the 2-∆∆Ct method and the results were compared with each other in a bar chart.
Results: After 7 days, cultivated hRPE cells in culture media containing 30% PL showed a high rate of viability in comparison with those cultured in 10% PL (P<0.05). RPE65 gene expressions in cell cultures treated with 20% and 30% PL were higher than controls (P<0.05). However, there was no significant difference in the expression of PAX6 gene between the cell cultures treated with 20% and 30% platelet gels.
Conclusion: our results showed that using platelet gels in hRPE cell cultures can improve cell viability and identity, which can be promising for optimizing future clinical cell therapy strategies.

Keywords

References

  1. Mitchell P, Liew G, Gopinath B, Wong TY. Age-related macular degeneration. Lancet 2018;392(10153):1147-1159.
  2. Binder S, Stolba U, Krebs I, Kellner L, Jahn C, Feichtinger H, et al. Transplantation of autologous retinal pigment epithelium in eyes with foveal neovascularization resulting from age-related macular degeneration: A pilot study. American Journal of Ophthalmology 2002;133(2):215-25.
  3. Varey AH, Rennel ES, Qiu Y, Bevan HS, Perrin RM, Raffy S, et al. VEGF 165 b, An antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy. British Journal of Cancer 2008;98(8):1366-79.
  4. Arnhold S, Heiduschka P, Klein H, Absenger Y, Basnaoglu S, Kreppel F, et al. Adenovirally transduced bone marrow stromal cells differentiate into pigment epithelial cells and induce rescue effects in RCS rats. Investigative Ophthalmology and Visual Science 2006;47(9):4121-9.
  5. Atmaca-Sonmez P, Li Y, Yamauchi Y, Schanie CL, Ildstad ST, Kaplan HJ, et al. Systemically transferred hematopoietic stem cells home to the subretinal space and express RPE-65 in a mouse model of retinal pigment epithelium damage. Experimental Eye Research 2006;83(5):1295-302.
  6. Bates DO, Cui TG, Doughty JM, Winkler M, Sugiono M, Shields JD, et al. VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, is down-regulated in renal cell carcinoma. Cancer Research 2002;62(14):4123-31.
  7. Sussane Binder BVS, Ilse Krebs, Carl Glittenberg. Transplantation of the RPE in AMD. Progress in Retinal and Eye Research 2007;26:516-54.
  8. Rashid A, Bhatia SK, Mazzitello KI, Chrenek MA, Zhang Q, Boatright JH, et al. RPE Cell and Sheet Properties in Normal and Diseased Eyes. Advances in Experimental Medicine and Biology 2016;854:757-63.
  9. Gheorghe A, Mahdi L, Musat O. Age-related macular degeneration. Romanian Journal of Ophthalmology 2015;59(2):74-7.
  10. Sharma R, Khristov V, Rising A, Jha BS, Dejene R, Hotaling N, et al. Clinical-grade stem cell-derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs. Science Translational Medicine 2019;11(475).
  11. Carl Sheridan,Yamini Krishna, Rachel Williams, Sharon Mason, David Wong,Heinrich Heimann,David Kent &Ian Grierson . Transplantation in the treatment of age-related macular degeneration: past, present and future directions. Expert Review of Ophthalmology 2007;2:497-511
  12. Schaumberg DA, Christen WG, Buring JE, Glynn RJ, Rifai N, Ridker PM. High-sensitivity C-reactive protein, other markers of inflammation, and the incidence of macular degeneration in women. Archives of ophthalmology (Chicago, Ill : 1960) 2007;125(3):300-5.
  13. Ramaswamy Reddy SH, Reddy R, Babu NC, Ashok GN. Stem-cell therapy and platelet-rich plasma in regenerative medicines: A review on pros and cons of the technologies. Journal of oral and maxillofacial pathology : JOMFP 2018;22(3):367-74.
  14. Chong RS, Osborne A, Conceicao R, Martin KR. Platelet-Derived Growth Factor Preserves Retinal Synapses in a Rat Model of Ocular Hypertension. Investigative ophthalmology & visual science 2016;57(3):842-52.
  15. Del Pino-Sedeño T, Trujillo-Martín MM, Andia I, et al. Platelet-rich plasma for the treatment of diabetic foot ulcers: A meta-analysis. Wound Repair Regen 2019;27(2):170-182.
  16. Harrison P, Cramer EM. Platelet alpha-granules. Blood reviews. 1993;7(1):52-62.
  17. Gruber R, Karreth F, Frommlet F, Fischer MB, Watzek G. Platelets are mitogenic for periosteum-derived cells. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 2003;21(5):941-8.
  18. Kinzebach S, Dietz L, Kluter H, Thierse HJ, Bieback K. Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells. BMC cell biology 2013;14:48.
  19. Piccin A, Rebulla P, Pupella S, Tagnin M, Marano G, Di Pierro AM, et al. Impressive tissue regeneration of severe oral mucositis post stem cell transplantation using cord blood platelet gel. Transfusion 2017;57(9):2220-4.
  20. Li S, Goldowitz D, Swanson DJ. The requirement of pax6 for postnatal eye development: evidence from experimental mouse chimeras. Investigative ophthalmology & visual science 2007;48(7):3292-300.
  21. Fernald RD. Eyes: variety, development and evolution. Brain, behavior and evolution 2004;64(3):141-7.
  22. McUsic AC, Lamba DA, Reh TA. Guiding the morphogenesis of dissociated newborn mouse retinal cells and hES cell-derived retinal cells by soft lithography-patterned microchannel PLGA scaffolds. Biomaterials 2012;33(5):1396-405.
  23. Fronk AH, Vargis E. Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations. Journal of tissue engineering 2016;7:2041731416650838.
  24. Selvaggi TA, Walker RE, Fleisher TA. Development of antibodies to fetal calf serum with arthus-like reactions in human immunodeficiency virus-infected patients given syngeneic lymphocyte infusions. Blood 1997;89(3):776-9.
  25. Doerr HW, Cinatl J, Sturmer M, Rabenau HF. Prions and orthopedic surgery. Infection 2003;31(3):163-71.
  26. Martineau I, Lacoste E, Gagnon G. Effects of calcium and thrombin on growth factor release from platelet concentrates: kinetics and regulation of endothelial cell proliferation. Biomaterials 2004;25(18):4489-502.
  27. Haynesworth SE, Kadiyala S, Liang L, Bruder SP. Mitogenic Sttimulation of Human Mesenchymal Stem Cells By Platelet Releasate Suggests a Mechanism for Enhancement of Bone repair by Platelet Concentrate. Transactions of the 48th Annual Meeting, Orthopaedic Research Society 272002.
Daneshvar Medicine
Volume 28, Issue 4 - Serial Number 149
September and October 2020
Pages 1-9

Files

Share

How to cite

Statistics