Olfactory ensheathing cell-derived extracellular vesicles protect the degenerated retinal pigment epithelium by delivering phagocytosis-related proteins

Abstract

Aim: Olfactory ensheathing cells (OECs) are commonly utilized in cell-based therapies for neurodegenerative diseases and central nervous system injuries, due to their unique properties of suppressing gliosis and providing neuroprotection. Small extracellular vesicles (sEVs) have emerged as promising therapeutic agents. This study aimed to investigate OEC-sEVs’ effect on retinal degeneration.

Methods: Proteomics analysis identified differentially expressed proteins between OEC-sEVs and OECs. We conducted subretinal injection of OEC-sEVs and assessed their protective mechanisms in degenerative retina. Real-time quantitative PCR and Western blot were used to detect the expression of phagocytosis-related molecules in retinal pigment epithelium (RPE) cells.

Results: Proteomics analysis revealed that proteins enriched in OEC-sEVs were associated with phagocytosis. Following injection into the subretinal spaces of Royal College of Surgeons (RCS) rats, OEC-sEVs were internalized by RPE cells, leading to the transient upregulation of tyrosine-protein kinase Mer (MerTK), activation of the downstream phosphoinositide 3-kinase/Akt signaling pathway, and restoration of the phagocytic activity of RPE cells towards the photoreceptor outer segments. This delayed photoreceptor degeneration, thereby improving retinal function in RCS rats. In the ARPE-19 cell line treated with the specific MerTK inhibitor UNC2881, enhanced phagocytosis of fluorescent particles and porcine photoreceptor outer segments was observed following OEC-sEV internalization.

Conclusion: OEC-sEVs were abundant in proteins associated with phagocytosis, which may partially compensate for the phagocytic deficit of the RPE cells observed in a retinitis pigmentosa rat model via the MerTK-related signaling pathway.

Keywords

Olfactory ensheathing cells / small extracellular vesicles / retinal pigment epithelium / retinitis pigmentosa / MerTK

References

[1] Hartong DT,Berson EL,Dryja TP. Retinitis pigmentosa Lancet 2006 368 1795 809

[2] Karuntu JS,Almushattat H,Nguyen XT.et al. Syndromic retinitis pigmentosa Prog Retin Eye Res 2025 107 101324

[3] Mazzoni F,Safa H,Finnemann SC. Understanding photoreceptor outer segment phagocytosis: use and utility of RPE cells in culture Exp Eye Res 2014 126 51 60 PMC4145030

[4] Bok D. The retinal pigment epithelium: a versatile partner in vision J Cell Sci Suppl 1993 17 189 95

[5] Strauss O. The retinal pigment epithelium in visual function Physiol Rev 2005 85 845 81

[6] Young RW. The renewal of photoreceptor cell outer segments J Cell Biol 1967 33 61 72 PMC2107286

[7] LaVail MM. Rod outer segment disk shedding in rat retina: relationship to cyclic lighting Science 1976 194 1071 4

[8] Yang S,Yu F,Yang M.et al. CYLD maintains retinal homeostasis by deubiquitinating ENKD1 and promoting the phagocytosis of photoreceptor outer segments Adv Sci 2024 11 e2404067 PMC11615780

[9] Parmeggiani F. Clinics, epidemiology and genetics of retinitis pigmentosa Curr Genomics 2011 12 236 7 PMC3131730

[10] Hosseini Shabanan S,Seyedmirzaei H,Barnea A,Hanaei S,Rezaei N. Stem cell transplantation as a progressing treatment for retinitis pigmentosa Cell Tissue Res 2022 387 177 205

[11] Musarella MA,Macdonald IM. Current concepts in the treatment of retinitis pigmentosa J Ophthalmol 2011 2011 753547 PMC2964907

[12] Zarbin M. Cell-based therapy for degenerative retinal disease Trends Mol Med 2016 22 115 34

[13] Kurzawa-Akanbi M,Tzoumas N,Corral-Serrano JC.et al. Pluripotent stem cell-derived models of retinal disease: elucidating pathogenesis, evaluating novel treatments, and estimating toxicity Prog Retin Eye Res 2024 100 101248

[14] Yu Y,Li L,Lin S,Hu J. Update of application of olfactory ensheathing cells and stem cells/exosomes in the treatment of retinal disorders Stem Cell Res Ther 2022 13 11 PMC8751324

[15] Li Y,Field PM,Raisman G. Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells Science 1997 277 2000 2

[16] Wewetzer K,Kern N,Ebel C,Radtke C,Brandes G. Phagocytosis of O4+ axonal fragments in vitro by p75- neonatal rat olfactory ensheathing cells Glia 2005 49 577 87

[17] Su Z,Chen J,Qiu Y.et al. Olfactory ensheathing cells: the primary innate immunocytes in the olfactory pathway to engulf apoptotic olfactory nerve debris Glia 2013 61 490 503

[18] Li Y,Zou T,Xue L,Yin ZQ,Huo S,Xu H. TGF-β1 enhances phagocytic removal of neuron debris and neuronal survival by olfactory ensheathing cells via integrin/MFG-E8 signaling pathway Mol Cell Neurosci 2017 85 45 56

[19] Jiang Y,Guo J,Tang X,Wang X,Hao D,Yang H. The immunological roles of olfactory ensheathing cells in the treatment of spinal cord injury Front Immunol 2022 13 881162 PMC9163387

[20] Lan YX,Yang P,Zeng Z.et al. Gene and protein expression profiles of olfactory ensheathing cells from olfactory bulb versus olfactory mucosa Neural Regen Res 2022 17 440 9 PMC8463967

[21] Li Y,Sauvé Y,Li D,Lund RD,Raisman G. Transplanted olfactory ensheathing cells promote regeneration of cut adult rat optic nerve axons J Neurosci 2003 23 7783 8 PMC6740610

[22] Andrews MR,Stelzner DJ. Evaluation of olfactory ensheathing and schwann cells after implantation into a dorsal injury of adult rat spinal cord J Neurotrauma 2007 24 1773 92

[23] Zhang L,Zhuang X,Chen Y,Xia H. Intravenous transplantation of olfactory bulb ensheathing cells for a spinal cord hemisection injury rat model Cell Transplant 2019 28 1585 602 PMC6923555

[24] Panni P,Ferguson IA,Beacham I,Mackay-Sim A,Ekberg JA,St John JA. Phagocytosis of bacteria by olfactory ensheathing cells and Schwann cells Neurosci Lett 2013 539 65 70

[25] Colombo M,Raposo G,Théry C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles Annu Rev Cell Dev Biol 2014 30 255 89

[26] Luo R,Liu M,Tan T.et al. Emerging significance and therapeutic potential of extracellular vesicles Int J Biol Sci 2021 17 2476 86 PMC8315015

[27] Kusuma GD,Barabadi M,Tan JL,Morton DAV,Frith JE,Lim R. To protect and to preserve: novel preservation strategies for extracellular vesicles Front Pharmacol 2018 9 1199 PMC6215815

[28] Zha X. Exosome-based therapy for spinal cord injury: a narrative review Adv Technol Neurosci 2025 2 128 34

[29] Samanta S,Rajasingh S,Drosos N,Zhou Z,Dawn B,Rajasingh J. Exosomes: new molecular targets of diseases Acta Pharmacol Sin 2018 39 501 13 PMC5888687

[30] Massoumi H,Amin S,Soleimani M.et al. Extracellular-vesicle-based therapeutics in neuro-ophthalmic disorders Int J Mol Sci 2023 24 9006 PMC10219002

[31] Yang M,Peng GH. The molecular mechanism of human stem cell-derived extracellular vesicles in retinal repair and regeneration Stem Cell Res Ther 2023 14 84 PMC10100447

[32] Habiba UE,Greene DL,Ahmad K,Shamim S,Khan N,Umer A. Safety of intravenous mesenchymal stem cell therapy: a meta-analysis of randomized controlled trials Regen Med Rep 2025 2 83 99

[33] Fan H,Chen Z,Tang HB.et al. Exosomes derived from olfactory ensheathing cells provided neuroprotection for spinal cord injury by switching the phenotype of macrophages/microglia Bioeng Transl Med 2022 7 e10287 PMC9115713

[34] Xia B,Gao J,Li S.et al. Extracellular vesicles derived from olfactory ensheathing cells promote peripheral nerve regeneration in rats Front Cell Neurosci 2019 13 548 PMC6908849

[35] Tu YK,Hsueh YH. Extracellular vesicles isolated from human olfactory ensheathing cells enhance the viability of neural progenitor cells Neurol Res 2020 42 959 67

[36] Tu YK,Hsueh YH,Huang HC. Human olfactory ensheathing cell-derived extracellular cesicles: miRNA profile and neuroprotective effect Curr Neurovasc Res 2021 18 395 408 PMC8972267

[37] Finnemann SC,Bonilha VL,Marmorstein AD,Rodriguez-Boulan E. Phagocytosis of rod outer segments by retinal pigment epithelial cells requires alpha(v)beta5 integrin for binding but not for internalization Proc Natl Acad Sci U S A 1997 94 12932 7 PMC24241

[38] Finnemann SC,Silverstein RL. Differential roles of CD36 and alphavbeta5 integrin in photoreceptor phagocytosis by the retinal pigment epithelium J Exp Med 2001 194 1289 98 PMC2195987

[39] Vollrath D,Feng W,Duncan JL.et al. Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk Proc Natl Acad Sci U S A 2001 98 12584 9 PMC60097

[40] Feng W,Yasumura D,Matthes MT,LaVail MM,Vollrath D. Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells J Biol Chem 2002 277 17016 22

[41] Lemke G,Rothlin CV. Immunobiology of the TAM receptors Nat Rev Immunol 2008 8 327 36 PMC2856445

[42] Mantero G,Francia S,Galluzzi F.et al. Phagocytosis by retinal pigment epithelium and microglia does not affect vision restoration by P3HT nanoparticles in Retinitis pigmentosa Cell Death Dis 2026 17 295 PMC13039545

[43] Smith AJ,Schlichtenbrede FC,Tschernutter M,Bainbridge JW,Thrasher AJ,Ali RR. AAV-Mediated gene transfer slows photoreceptor loss in the RCS rat model of retinitis pigmentosa Mol Ther 2003 8 188 95

[44] Lew DS,Mazzoni F,Finnemann SC. Microglia inhibition delays retinal degeneration due to MerTK phagocytosis receptor deficiency Front Immunol 2020 11 1463 PMC7381113

[45] Dowling JE,Sidman RL. Inherited retinal dystrophy in the rat J Cell Biol 1962 14 73 109 PMC2106090

[46] Carvalho LA,Teng J,Fleming RL.et al. Olfactory ensheathing cells: a Trojan horse for glioma gene therapy J Natl Cancer Inst 2019 111 283 91 PMC6410949

[47] Sun X,Tan Z,Huang X.et al. Direct neuronal reprogramming of olfactory ensheathing cells for CNS repair Cell Death Dis 2019 10 646 PMC6733847

[48] Zhang W,McIver AL,Stashko MA.et al. Discovery of Mer specific tyrosine kinase inhibitors for the treatment and prevention of thrombosis J Med Chem 2013 56 9693 700 PMC3962266

[49] Lu T,Chen L,Mansour AG.et al. Cbl-b is upregulated and plays a negative role in activated human NK cells J Immunol 2021 206 677 85 PMC8184061

[50] Molday RS,Hicks D,Molday L. Peripherin. A rim-specific membrane protein of rod outer segment discs Invest Ophthalmol Vis Sci 1987 28 50 61

[51] Bian B,Zhao C,He X.et al. Exosomes derived from neural progenitor cells preserve photoreceptors during retinal degeneration by inactivating microglia J Extracell Vesicles 2020 9 1748931 PMC7191912

[52] Van Deun J,Mestdagh P,Agostinis P.et al. ; EV-TRACK Consortium. EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research Nat Methods 2017 14 228 32

[53] A L,Qu L,He J.et al. Exosomes derived from IFNγ-stimulated mesenchymal stem cells protect photoreceptors in RCS rats by restoring immune homeostasis through tsRNAs Cell Commun Signal 2024 22 543 PMC11562488

[54] Jensen EC. Quantitative analysis of histological staining and fluorescence using ImageJ Anat Rec 2013 296 378 81

[55] Ma J,Chen T,Wu S.et al. iProX: an integrated proteome resource Nucleic Acids Res 2019 47 D1211 7 PMC6323926

[56] Chen T,Ma J,Liu Y.et al. iProX in 2021: connecting proteomics data sharing with big data Nucleic Acids Res 2022 50 D1522 7 PMC8728291

[57] Nakamura N,Rabouille C,Watson R.et al. Characterization of a cis-Golgi matrix protein, GM130 J Cell Biol 1995 131 1715 26 PMC2120691

[58] Prasad D,Rothlin CV,Burrola P.et al. TAM receptor function in the retinal pigment epithelium Mol Cell Neurosci 2006 33 96 108

[59] Fairn GD,Grinstein S. How nascent phagosomes mature to become phagolysosomes Trends Immunol 2012 33 397 405

[60] Nandrot EF,Anand M,Almeida D,Atabai K,Sheppard D,Finnemann SC. Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis Proc Natl Acad Sci U S A 2007 104 12005 10 PMC1924559

[61] Yang S,Zhou J,Li D. Functions and diseases of the retinal pigment epithelium Front Pharmacol 2021 12 727870 PMC8355697

[62] Jauregui-Lozano J,Hall H,Stanhope SC,Bakhle K,Marlin MM,Weake VM. The Clock:Cycle complex is a major transcriptional regulator of Drosophila photoreceptors that protects the eye from retinal degeneration and oxidative stress PLoS Genet 2022 18 e1010021 PMC8830735

[63] Valiente-Soriano FJ,Salinas-Navarro M,Di Pierdomenico J.et al. Tracing the retina to analyze the integrity and phagocytic capacity of the retinal pigment epithelium Sci Rep 2020 10 7273 PMC7190639

[64] Gao X,Li S,Yang Y.et al. A novel magnetic responsive miR-26a@SPIONs-OECs for spinal cord injury: triggering neural regeneration program and orienting axon guidance in inhibitory astrocytic environment Adv Sci 2023 10 e2304487 PMC10646239

[65] Chen Z,Fan H,Chen ZY.et al. OECs prevented neuronal cells from apoptosis partially through exosome-derived BDNF J Mol Neurosci 2022 72 2497 506

[66] Gal A,Li Y,Thompson DA.et al. Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa Nat Genet 2000 26 270 1

[67] Nandrot E,Dufour EM,Provost AC.et al. Homozygous deletion in the coding sequence of the c-mer gene in RCS rats unravels general mechanisms of physiological cell adhesion and apoptosis Neurobiol Dis 2000 7 586 99

[68] D’Cruz PM,Yasumura D,Weir J.et al. Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat Hum Mol Genet 2000 9 645 51

[69] Nandrot EF,Silva KE,Scelfo C,Finnemann SC. Retinal pigment epithelial cells use a MerTK-dependent mechanism to limit the phagocytic particle binding activity of αvβ5 integrin Biol Cell 2012 104 326 41 PMC3577091

[70] Steinberg RH,Wood I,Hogan MJ. Pigment epithelial ensheathment and phagocytosis of extrafoveal cones in human retina Philos Trans R Soc Lond B Biol Sci 1977 277 459 74

[71] Mao Y,Finnemann SC. Essential diurnal Rac1 activation during retinal phagocytosis requires αvβ5 integrin but not tyrosine kinases focal adhesion kinase or Mer tyrosine kinase Mol Biol Cell 2012 23 1104 14 PMC3302737

[72] Umapathy A,Torten G,Paniagua AE.et al. Spatiotemporal live-cell analysis of photoreceptor outer segment membrane ingestion by the retinal pigment epithelium reveals actin-regulated scission J Neurosci 2023 43 2653 64 PMC10089248

[73] Storm T,Burgoyne T,Futter CE. Membrane trafficking in the retinal pigment epithelium at a glance J Cell Sci 2020 133 jcs238279

[74] Gibbs D,Kitamoto J,Williams DS. Abnormal phagocytosis by retinal pigmented epithelium that lacks myosin VIIa, the Usher syndrome 1B protein Proc Natl Acad Sci U S A 2003 100 6481 6 PMC164472

[75] Haines LA,Baeckler AA,Schofield SJ.et al. Non-specific particle formation during extracellular vesicle labelling with the lipophilic membrane dye PKH26 J Extracell Vesicles 2025 14 e70079 PMC12087298

[76] Su X,Zhang S,Zhang T.et al. Glycan-anchored fluorescence labeling of milk-derived extracellular vesicles for investigating their cellular uptake and intracellular fate Small Methods 2025 9 e2401996

[77] Andronico LA,Jiang Y,Jung SR,Fujimoto BS,Vojtech L,Chiu DT. Sizing extracellular vesicles using membrane dyes and a single molecule-sensitive flow analyzer Anal Chem 2021 93 5897 905 PMC10243643

[78] Strohmeier K,Hofmann M,Hauser F.et al. CRISPR/Cas9 genome editing vs. over-expression for fluorescent extracellular vesicle-labeling: a quantitative analysis Int J Mol Sci 2021 23 282 PMC8745383

[79] Bertolio M,Li Q,Mowry FE.et al. Glutamatergic regulation of miRNA-containing intraluminal vesicle trafficking and extracellular vesicle secretion from cortical neurons J Extracell Vesicles 2025 14 e70100 PMC12120566

[80] Ablonczy Z,Dahrouj M,Tang PH.et al. Human retinal pigment epithelium cells as functional models for the RPE in vivo Invest Ophthalmol Vis Sci 2011 52 8614 20 PMC3208409

[81] Puddu A,Ravera S,Panfoli I,Bertola N,Maggi D. High glucose impairs expression and activation of MerTK in ARPE-19 cells Int J Mol Sci 2022 23 1144 PMC8835591

[82] Ramirez MI,Amorim MG,Gadelha C.et al. Technical challenges of working with extracellular vesicles Nanoscale 2018 10 881 906

[83] Patel DB,Santoro M,Born LJ,Fisher JP,Jay SM. Towards rationally designed biomanufacturing of therapeutic extracellular vesicles: impact of the bioproduction microenvironment Biotechnol Adv 2018 36 2051 9 PMC6250573