Stem cell secretome- and extracellular vesicle-based therapies for retinal pathologies

Table 2 Studies of cell-free secretome and extracellular vesicle therapies for retinal and optic nerve disorders

Ref.	Indication/model and evidence level	Product/source	Preparation and characterization	Route and dose (source-reported metric)	Comparator	Sample size and follow-up	Main endpoints and findings	Major limitations/design reporting
Sun et al[14]	Diabetic retinopathy; high-fat diet/STZ Sprague-Dawley rats and high-glucose RPE cells. Level 2; direct acellular evidence	Human umbilical-cord MSC-derived sEVs; NEDD4 identified as bioactive cargo	Conditioned medium; differential centrifugation followed by ultracentrifugation and 0.22-μm filtration. TEM, NTA and western blot; CD9, CD63, HSP70, Alix and TSG101 positive; Calnexin negative; mean diameter 171.3 ± 71.7 nm	Intravitreal; single dose of 1 × 106 particles in 3 μL at 12 weeks after STZ (particle-number dose)	PBS; HFL1-derived sEVs; normal control	Endpoint-dependent: Retinal thickness n = 6/group; biochemical/qPCR analyses n = 3/group. Animals evaluated 20 weeks after STZ, approximately 8 weeks after treatment	Retinal morphology/thickness, apoptosis, oxidative stress, NEDD4/PTEN/AKT/NRF2 signaling. MSC-sEVs reduced oxidative injury and apoptosis	Rodent metabolic model; single administration; relatively small and endpoint-dependent sample sizes; random allocation reported; blinding not clearly reported
Zhang et al[15]	Retinitis pigmentosa; rd10 mice, with complementary 661W/BV2 experiments. Level 2; direct acellular evidence	Human umbilical-cord MSC-derived EVs; miR-146a implicated in Nr4a3 regulation	EVs enriched by differential ultracentrifugation. CD63/CD81 characterization and particle-size analysis; 30-200-nm fraction predominated	Intravitreal; 1 × 1010 EVs in 1 μL/eye at P14 and P21 (particle-number dose)	Normal saline; WT mice	Outcome-specific group sizes; optomotor analysis included approximately n = 5-7/group. Functional/morphological assessment at P27-P28	Optomotor response, ERG, photoreceptor survival, retinal morphology, microglial/Müller-cell/macrophage activation, NF-κB and miR-146a/Nr4a3 signaling. EVs preserved photoreceptors and retinal function	Genetic mouse model; short developmental follow-up; endpoint-dependent sample sizes; no long-term assessment; blinding/randomization not consistently reported
Bian et al[24]	Inherited retinal degeneration; Royal College of Surgeons rats. Level 2; direct acellular evidence	Mouse neural progenitor cell-derived exosome preparation	Differential centrifugation plus sucrose-gradient density centrifugation. HSP70, CD63, CD9 and CD81 positive; TEM; NTA; approximately 50-180 nm, mean ~98 nm	Subretinal; 1 μL/eye containing approximately 1.09 × 109 vesicles (particle-number dose)	Vehicle and untreated controls	At least 3 biological samples/experiment; assessments through 28 days	OKR, scotopic ERG, outer nuclear layer preservation, photoreceptor apoptosis and microglial uptake/activation. Functional and structural preservation reported	Small experimental samples; rodent inherited-degeneration model; invasive subretinal delivery; follow-up ≤ 28 days; randomization/blinding incompletely described
Cui et al[25]	Optic-nerve crush in rats. Level 2; direct acellular evidence	Rat bone-marrow MSC-derived exosomes	Exosomes isolated from MSC-conditioned medium; TEM and western blot characterization; approximately 93-nm peak size; CD63/TSG101 positive and GM130 negative	Intravitreal; 3 × 109 vesicles in 5 μL, single administration	PBS/vehicle	NR for consolidated total sample size; approximately 30-day follow-up	RGC survival, retinal histology, apoptosis, inflammatory cytokines, Bcl-2/Bax/caspase-3 and PI3K/AKT signaling. Increased RGC survival and reduced inflammatory/apoptotic signaling	Acute traumatic optic-nerve model; sample size not consistently reported across endpoints; single administration; limited follow-up; randomization/blinding not clearly reported
Zhang et al[26]	Oxygen-induced retinopathy and laser-induced choroidal neovascularization in mice. Level 2; direct engineered-acellular evidence	Human MSC-derived exosomes engineered with miR-205	Differential ultracentrifugation; TEM, NTA and western blot; predominantly 100-150 nm; CD63, HSP70 and TSG101 positive. miR-205 loaded by electroporation	Intravitreal; administered at P12 in the OIR model and immediately after laser injury in the CNV model; injected particle-number/volume dose NR	PBS, unloaded exosomes and free miR-205	Principal in vivo analyses n = 5/group. OIR endpoint P17; CNV endpoint 7 days	Retinal/choroidal neovascularization, Evans-blue leakage, OCT, FFA and VEGFA/ANGPT2 signaling. Exo-miR-205 reduced pathological angiogenesis and leakage	Acute neovascularization models; small groups; very short follow-up; injected vesicle dose not clearly reported; authors noted lack of in vivo confirmation of uptake by retinal endothelial cells
Usategui-Martín et al[28]	Spontaneous degeneration of ex vivo porcine neuroretinal explants. Level 1; direct acellular secretome evidence	Human bone-marrow MSC secretome/conditioned co-culture medium; not a purified EV preparation	BMSCs characterized phenotypically; secretome consisted of medium obtained from NR-BMSC co-culture; no fractionation into soluble and EV components	Ex-vivo medium exposure; 0.75 mL conditioned medium plus 0.75 mL culture medium (conditioned-medium volume; not an isolated EV dose)	Fresh NR; monocultured NR; NR co-cultured with viable BMSCs	8 porcine eyes; 32 explants; n = 8 per experimental condition; 72 hours	Retinal morphology, RGC/photoreceptor markers, TUNEL, apoptosis, necroptosis, autophagy, oxidative-stress and inflammatory gene expression. Secretome attenuated several degeneration-associated changes	Ex vivo rather than in vivo model; short 72-hour exposure; secretome was not fractionated or compositionally standardized; cannot attribute effects specifically to EVs or soluble factors; selected analyses performed by a masked investigator
Zhang et al[29]	STZ-induced diabetic retinopathy in C57BL/6 mice and high-glucose microglial experiments. Level 2; direct acellular evidence	Mouse bone-marrow MSC-derived exosomes; let-7b-5p manipulated using agomir/antagomir strategies	BMSC-derived exosome preparation with vesicle morphology assessment; let-7b-5p cargo experimentally manipulated and quantified	Intravitreal; 1 μL of an exosome preparation reported at 5 μg/μL every 3 days for 6 weeks (protein-concentration metric)	Unmodified BMSC-Exo and let-7b-5p-modified exosome groups; control/PBS conditions	24 mice reported; 6 normal controls and 18 diabetic mice; n = 6 in diabetic treatment groups; 6-week intervention	Retinal histology/apoptosis, microglial activation, inflammatory cytokines and TLR4/ATF4 signaling. let-7b-5p-enriched exosomes reduced inflammatory and retinal injury markers	Single STZ mouse model; frequent repeated intravitreal injections; predominantly histological/molecular endpoints; no long-term post-treatment follow-up. Random allocation and blinded operators/assessors were reported
Chen et al[35]	Diabetic retinopathy; high-fat diet/STZ Sprague-Dawley rats plus cellular experiments. Level 2; direct acellular evidence	Human umbilical-cord MSC-derived sEVs carrying miR-22-3p	TEM and NTA; peak size approximately 159 nm and concentration ~2.9 × 1011 particles/mL; CD63, CD81 and TSG101 positive; Calnexin negative	Intravitreal; single dose of 1.5 × 109 sEVs in 5 μL after 12 weeks of diabetes (particle-number dose)	PBS-treated diabetic rats and normal controls	Consolidated animal NR; animals sacrificed 4 weeks after injection	Retinal morphology, blood-retinal-barrier leakage, microglial activation, inflammatory cytokines and NLRP3 inflammasome signaling. sEV-miR-22-3p attenuated inflammatory injury and barrier dysfunction	Rodent DR model; single administration; 4-week follow-up; no clinical functional endpoint; random allocation reported, blinding not clearly stated
Sun et al[37]	Diabetic retinal neurodegeneration in db/db mice, with additional STZ/genetic mechanistic experiments. Level 2; direct engineered-acellular evidence	MSC-derived EVs engineered for photoreceptor targeting with MH42 and enriched with USP25	EVs isolated and characterized by TEM, particle analysis and EV-marker profiling; engineered by cargo enrichment and surface targeting	Intravitreal; 1 μL containing 106, 107, or 108 particles; 107 particles were selected for repeated administration every 4 weeks (particle-number dose)	Unmodified MEVs, MEV-USP25, targeted and scrambled-control preparations, and disease controls	Animal sample size varied by endpoint; several quantified experiments used n = 3/group. Treatment followed to approximately 24 weeks	ERG, retinal morphology, ONL/photoreceptor survival, TUNEL, opsin expression and USP25/CRYAA-related ubiquitination. Targeted USP25 delivery improved structural and functional retinal outcomes	Complex engineered product; mouse models only; multiple comparator constructs and endpoint-specific sample sizes; repeated injections; no clinical validation. Data collection/analysis reported as blinded
Guo et al[38]	MNU-induced retinal degeneration and Pde6βrd1/rd1 mice. Level 2; direct engineered-acellular evidence	Photoreceptor-derived EVs (non-stem-cell source) engineered with MH42 targeting peptide and Cul7 siRNA	Ultracentrifugation; TEM/NTA and EV-marker analysis; CD9, CD63, Alix and TSG101 positive; Calnexin negative. siCul7 loaded by electroporation	Intravitreal; 1 μL at 1 × 107 particles/μL (particle-number concentration)	Targeted control-siRNA EVs and non-targeted siCul7-EVs	Sample size reported separately for each experiment/figure; no overall number of mice provided; 2-week follow-up	Photoreceptor targeting, retinal structure/function, Cul7/GPX4 signaling and ferroptosis. Targeted siCul7 delivery reduced photoreceptor degeneration	Non-stem-cell EV source and therefore supportive mainly as an engineered-delivery proof of concept; two mouse models; short 2-week follow-up; endpoint-dependent sample sizes
Tang et al[39]	Dry-AMD-related oxidative injury; ARPE-19 cells and NaIO3-induced retinal degeneration in rats. Level 1-2; direct acellular evidence	Adipose-derived MSC exosomes	MSC exosome preparation characterized in the original study; detailed characterization parameters should be interpreted according to the authors’ reported “exosome” terminology	Intravitreal in the in vivo model; exact administered dose NR	Oxidant/NaIO3 controls; mechanistic Nrf2 inhibition with ML385	Consolidated animal sample size and exact follow-up NR	LDH, ROS, SOD, RPE/photoreceptor morphology, Bcl-2/Bax and Nrf2/Keap1/HO-1 signaling. Exosome pretreatment reduced oxidative retinal damage	Oxidative/toxin model rather than spontaneous human AMD; pre-administration design; exact dose, consolidated sample size, and follow-up NR; no human evidence
Zhang et al[41]	Large and long-standing idiopathic macular holes; uncontrolled human case series. Level 3; exploratory human evidence, with a direct acellular MSC-Exo subgroup	Human umbilical-cord MSC-derived exosome preparation; 5/7 patients received MSC-Exos and 2/7 received viable MSCs	MSC-Exos isolated from cultured MSC supernatants using sequential ultracentrifugation	Intravitreal administration following pars plana vitrectomy and internal limiting membrane peeling, with intraocular tamponade; exact MSC-Exo dose NR	No untreated concurrent control; viable-MSC recipients did not constitute a randomized comparator	Seven patients overall; five MSC-Exo recipients and two viable-MSC recipients; exact follow-up NR	Six of seven macular holes closed and BCVA improved in five patients overall. One patient receiving a higher MSC-Exo dose developed an inflammatory reaction. Aggregate outcomes cannot be attributed exclusively to the MSC-Exo subgroup	Very small, uncontrolled, mixed-intervention case series; concomitant PPV, ILM peeling, and tamponade; two patients received viable MSCs; aggregate outcomes prevent attribution of independent efficacy to MSC-Exos; no randomized confirmatory study

Evidence levels were used as a review-specific descriptive framework: (1) Level 1, in vitro/ex vivo mechanistic evidence; (2) Level 2, in vivo animal evidence; (3) Level 3, early uncontrolled human feasibility/efficacy evidence; and (4) Level 4, controlled clinical efficacy evidence. “Direct acellular evidence” indicates administration or testing of a secretome, conditioned-medium, extracellular vesicle (EV), small EV, or engineered-EV preparation rather than viable-cell transplantation. This classification is conceptual and is not intended as a validated clinical grading system. Dose metrics are reproduced in the units reported by the primary source. Particle-number doses (“particles” or “vesicles”, as reported), protein-mass or protein-concentration doses (μg or μg/μL), and conditioned-medium volumes are analytically distinct and should not be directly compared. Particle counts are not assumed to represent purified EV numbers unless supported by the source methodology. No particle-to-protein or other cross-unit conversion was performed. NR indicates information not reported or not sufficiently specified in the primary source. STZ: Streptozotocin; RPE: Retinal pigment epithelium; MSC: Mesenchymal stem cell; sEVs: Small extracellular vesicle; NEDD4: Neuron-expressed developmental downregulation 4; TEM: Transmission electron microscopy; NTA: Nanoparticle tracking analysis; HSP70: Heat shock protein 70; TSG101: Tumor susceptibility gene 101; PBS: Phosphate buffered saline; qPCR: Quantitative polymerase chain reaction; AKT: Protein kinase B; NRF2: Nuclear factor erythroid 2-related factor 2; ERG: Electroretinography; NF-κB: Nuclear factor kappa B; OKR: Optokinetic response; RGC: Retinal ganglion cell; PI3K: Phosphatidyl-inositol-3-kinase; OIR: Oxygen-induced retinopathy; CNV: Choroidal neovascularization; OCT: Optical coherence tomography; FFA: Fluorescein angiography; VEGFA: Vascular endothelial growth factor A; ANGPT2: Angiopoietin 2; BMSC: Bone marrow mesenchymal stem cell; EV: Extracellular vesicle; BMSC-Exo: Bone marrow mesenchymal stem cell-derived exosome; TLR4: Toll-like receptor 4; ATF4: Activating transcription factor 4; DR: Diabetic retinopathy; ONL: Outer nuclear layer; siRNA: Small interfering RNA; GPX4: Glutathione peroxidase 4; AMD: Age-related macular degeneration; LDH: Lactate dehydrogenase; ROS: Reactive oxygen species; SOD: Superoxide dismutase; HO-1: Heme oxygenase-1; MSC-Exo: Mesenchymal stem cell-derived exosome; BCVA: Best-corrected visual acuity; PPV: Pars plana vitrectomy; ILM: Internal limiting membrane.