Int J Stem Cells.  2023 May;16(2):244-249. 10.15283/ijsc22041.

Regenerative Effect of Adipose Derived Mesenchymal Stem Cells on Ganglion Cells in the Hypoxic Organotypic Retina Culture

• Dov MB
• Krief B
• Benhamou M
• Klein A
• Schwartz S
• Loewenstein A
• Barak A
• Barzelay A

• KMID: 2542836
• DOI: http://doi.org/10.15283/ijsc22041

Abstract

Background and Objectives
To examine whether ischemic retinal ganglion cells (RGCs) will be salvaged from cell death by human adipose-derived mesenchymal stem cells (ADSCs) in an organotypic retina model.
Methods and Results
Deprived of arterial oxygen supply, whole mice retinas were cultured as an ex vivo organotypic cultures on an insert membrane in a 24-well plate. The therapeutic potential of ADSCs was examined by co-culture with organotypic retinas. ADSCs were seeded on top of the RGCs allowing direct contact, or at the bottom of the well, sharing the same culture media and allowing a paracrine activity. The number of surviving RGCs was assessed using Brn3a staining and confocal microscopy. Cytokine secretion of ADSCs to medium was analyzed by cytokine array. When co-cultured with ADSCs, the number of surviving RGCs was similarly significantly higher in both treatment groups compared to controls. Analysis of ADSCs cytokines secretion profile, showed secretion of anti-apoptotic and pro-proliferative cytokines (threshold＞1.4). Transplantation of ADSCs in a co-culture system with organotypic ischemic retinas resulted in RGCs recovery. Since there was no advantage to direct contact of ADSCs with RGCs, the beneficial effect seen may be related to paracrine activity of ADSCs.
Conclusions
These data correlated with secretion profile of ADSCs’ anti-apoptotic and pro-proliferative cytokines.

Keyword

Ischemia; Retina; Adipose-derived mesenchymal stem cells; Ganglion cells; Optic nerve; Hypoxia

Figure

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  Fig. 1 Schematic diagram of the study. Two co-culture techniques of organotypic retinas with ADSCs. (A) ADSCs seeded on 24-well-plate sharing the same culture media with the organotypic retina, for studying the cells’ paracrine effect. (B) ADSCs seeded on top of the retina with direct contact with RGCs for studying the cell’s direct contact effect. Blue is the tissue culture 24-well-plate. Black is the insert. Red is the retina. Green are ADSCs.

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  Fig. 2 Time depended effect of hypoxia on RGCs viability. Retinas were extracted and cultured as organotypic culture for different periods of times. Viability of RGCs was evaluated by staining for Brn3a. Repre-sentative pictures of retinas stained for Brn3a at (A) T0, (B) T24 hours, (C) T48 hours, (D) T72 hours. (E) Summary of time-dependent effect of hypoxic conditions on viability of RGCs. Bar=100 μm. Data are represented as mean±SEM. The experiment was conducted on 2 to 5 retinas per time point.

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  Fig. 3 Representative picture of Brn3a staining at 48 hours. (A) Control organotypic retinas cultured in same conditions without ADSCs. (B) Retinas treated with ADSCs seeded with direct contact on top of the RGCs. (C) Retinas treated with ADSCs seeded on tissue culture plate below the insert. Graphic summary of ADSCs regenerative potential at 48 hours. (D) Retinas treated with ADSCs on top of the retina and below the retina exhibited significantly more viable Brn3a+RGCs as compared to controls of retinas cultured for 48 hours with no treatment. Bar=100 μm. Data are represented as mean± SEM.

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  Fig. 4 Cytokine secretion profile of ADSCs conditioned medium. Medium containing secreted proteins of ADSCs obtained from 2 donors was analyzed for the 20 most abundant cytokines using RayBioⓇ Human Cytokine Antibody Array. Threshold for signifi-cant change was set at >1.4.

Reference

References

1. Olivares-González L, Martínez-Fernández de la Cámara C, Hervás D, Marín MP, Lahoz A, Millán JM, Rodrigo R. 2016; cGMP-phosphodiesterase inhibition prevents hypoxia-induced cell death activation in porcine retinal explants. PLoS One. 11:e0166717. DOI: 10.1371/journal.pone.0166717. PMID: 27861632. PMCID: PMC5115799. PMID: cf42187bb8aa4a45ae87ea861036b353.

2. Mead B, Tomarev S. 2016; Evaluating retinal ganglion cell loss and dysfunction. Exp Eye Res. 151:96–106. DOI: 10.1016/j.exer.2016.08.006. PMID: 27523467. PMCID: PMC5045805.

3. Klemm P, Hurst J, Dias Blak M, Herrmann T, Melchinger M, Bartz-Schmidt KU, Zeck G, Schultheiss M, Spitzer MS, Schnichels S. 2019; Hypothermia protects retinal ganglion cells against hypoxia-induced cell death in a retina organ culture model. Clin Exp Ophthalmol. 47:1043–1054. DOI: 10.1111/ceo.13565. PMID: 31152487.

4. Li N, Li XR, Yuan JQ. 2009; Effects of bone-marrow mesenchymal stem cells transplanted into vitreous cavity of rat injured by ischemia/reperfusion. Graefes Arch Clin Exp Ophthalmol. 247:503–514. DOI: 10.1007/s00417-008-1009-y. PMID: 19084985.

5. Baraniak PR, McDevitt TC. 2010; Stem cell paracrine actions and tissue regeneration. Regen Med. 5:121–143. DOI: 10.2217/rme.09.74. PMID: 20017699. PMCID: PMC2833273.

6. Barzelay A, Levy R, Kohn E, Sella M, Shani N, Meilik B, Entin-Meer M, Gur E, Loewenstein A, Barak A. 2015; Power-assisted liposuction versus tissue resection for the isolation of adipose tissue-derived mesenchymal stem cells: phenotype, senescence, and multipotency at advanced passages. Aesthet Surg J. 35:NP230–NP240. DOI: 10.1093/asj/sjv055. PMID: 26319084.

7. Barzelay A, Weisthal Algor S, Niztan A, Katz S, Benhamou M, Nakdimon I, Azmon N, Gozlan S, Mezad-Koursh D, Neudorfer M, Goldstein M, Meilik B, Loewenstein A, Barak A. 2018; Adipose-derived mesenchymal stem cells migrate and rescue RPE in the setting of oxidative stress. Stem Cells Int. 2018:9682856. DOI: 10.1155/2018/9682856. PMID: 30651740. PMCID: PMC6311721. PMID: 5e93dd85401b4290929c3bb39a1a050d.

8. Müller B. 2019; Organotypic culture of adult mouse retina. Methods Mol Biol. 1940:181–191. DOI: 10.1007/978-1-4939-9086-3_13. PMID: 30788826.

9. Wang F. 2006; Culture of animal cells: a manual of basic technique, fifth edition. In Vitro Cell Dev Biol Anim. 42:169. DOI: 10.1290/BR090501.1.

10. Nadal-Nicolás FM, Jiménez-López M, Sobrado-Calvo P, Nieto-López L, Cánovas-Martínez I, Salinas-Navarro M, Vidal-Sanz M, Agudo M. 2009; Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Invest Ophthalmol Vis Sci. 50:3860–3868. DOI: 10.1167/iovs.08-3267. PMID: 19264888.

11. Boudreau N, Sympson CJ, Werb Z, Bissell MJ. 1995; Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science. 267:891–893. DOI: 10.1126/science.7531366. PMID: 7531366. PMCID: PMC3004777.

12. Tejima E, Guo S, Murata Y, Arai K, Lok J, van Leyen K, Rosell A, Wang X, Lo EH. 2009; Neuroprotective effects of overexpressing tissue inhibitor of metalloproteinase TIMP-1. J Neurotrauma. 26:1935–1941. DOI: 10.1089/neu.2009.0959. PMID: 19469687. PMCID: PMC2822804.

13. Zhang P, Li J, Qi Y, Tang X, Duan J, Liu L, Wu Z, Liang J, Li J, Wang X, Zeng G, Liu H. 2016; Tissue inhibitor of matrix metalloproteinases-1 knockdown suppresses the prolifera-tion of human adipose-derived stem cells. Stem Cells Int. 2016:4761507. DOI: 10.1155/2016/4761507. PMID: 27239203. PMCID: PMC4863124. PMID: 69e3683105534e469b7513d5b969e040.

14. Brown SM, Lark KK. 2019; Re: Biousse et al.: Management of acute retinal ischemia: follow the guidelines! (Ophthal-mology. 2018;125:1597-1607). Ophthalmology. 126:e36–e37. DOI: 10.1016/j.ophtha.2018.11.032. PMID: 31005195.

15. Chen CS, Lee AW. 2008; Management of acute central retinal artery occlusion. Nat Clin Pract Neurol. 4:376–383. DOI: 10.1038/ncpneuro0811. PMID: 18542123.

16. Gimble JM, Katz AJ, Bunnell BA. 2007; Adipose-derived stem cells for regenerative medicine. Circ Res. 100:1249–1260. DOI: 10.1161/01.RES.0000265074.83288.09. PMID: 17495232. PMCID: PMC5679280.

17. Shimamura M, Nakagami H, Osako MK, Kurinami H, Koriyama H, Zhengda P, Tomioka H, Tenma A, Wakayama K, Morishita R. 2014; OPG/RANKL/RANK axis is a critical inflammatory signaling system in ischemic brain in mice. Proc Natl Acad Sci U S A. 111:8191–8196. DOI: 10.1073/pnas.1400544111. PMID: 24847069. PMCID: PMC4050556.

18. Wei X, Zhang Z, Zeng HH, Wang XF, Zhan W, Qiao N, Chang Z, Liu L, Fan C, Yang Z, Li X, Yang Y, Liu H. 2020. Regeneration of functional retinal ganglion cells by neuronal identity reprogramming. bioRxiv 203497 [Preprint]. Available from: https://doi.org/10.1101/2020.07.16.203497. cited 2020 Jul 16. DOI: 10.1101/2020.07.16.203497.

19. Feng Y, Zhu M, Dangelmajer S, Lee YM, Wijesekera O, Castellanos CX, Denduluri A, Chaichana KL, Li Q, Zhang H, Levchenko A, Guerrero-Cazares H, Quiñones-Hinojosa A. 2014; Hypoxia-cultured human adipose-derived mesenchymal stem cells are non-oncogenic and have enhanced viability, motility, and tropism to brain cancer. Cell Death Dis. 5:e1567. Erratum in: Cell Death Dis 2015;6:e1797. DOI: 10.1038/cddis.2015.176. PMID: 26111059. PMCID: PMC4669846.