(W0930-02-07) Human Retinal Organoids Derived Extracellular Vesicles Loaded with Cannabinoids Attenuates Oxidative Stress Changes in ARPE-19 Cells

Purpose: Extracellular vesicles (EVs) are phospholipid bilayer-bound particles released by cells that aid in cell-to-cell communication, signaling, and extracellular matrix remodeling. EVs are gaining popularity as treatments, biomarkers, and drug delivery vehicles for ophthalmic applications. The properties of bioreactors cultured-3D human retinal organoids produced EVs and their effects on RPE disease models, however, are poorly understood. The ability to use retinal organoid EVs for diagnostic and therapeutic studies requires such a broad spectrum of characterization. This study for the first time compares retinal organoid EVs to human umbilical cord (hUC) MSCs EVs in terms of morphological, nanomechanical, molecular, and proteomic characterization and assesses the anti-oxidative stress effects of CBD loaded retinal organoid EVs.

Methods: The effective isolation of EVs from growing 3D retinal organoids generated from hiPSC and cultured in a novel PBS Vertical Wheel (PBS-VW) bioreactor is described in this study. Using AFM and proteomics methods, we evaluated and compared the biophysical, nanostructural, nanomechanical, molecular, and proteomic profile of hiPSC-differentiated retinal organoids EVs to human umbilical cord mesenchymal stem cells (hUCMSC) EVs. Cannabidiol was loaded into these EVs using a sonication technique, and the effects of cannabidiol on oxidative stress in ARPE-19 were examined using western blot and immunocytochemistry

Results: The average size of EV, according to nanoparticle tracking studies, is 100–250 nm. Exosome biogenesis genes were found to have high expression in late retinal organoids produced EVs ( >120 days) when gene expression was analyzed using qPCR. Exosomal markers Alix, CD63, Flotillin-2, HRS, and HSP70 were shown to be strongly expressed in late retinal organoids EVs (120 days) compared to early retinal organoids EVs. Protein profiling of retinal organoid EVs exhibited higher differential expression of retinal function-related proteins and EV biogenesis/marker proteins than hUCMSC EVs, suggesting that retinal organoid EVs may have a better therapeutic effect on retinal diseases. Anti-oxidative stress is a beneficial strategy for the prevention or treatment of AMD because oxidative damage is a critical element in the pathogenesis of age-related macular degeneration (AMD). Cannabidiol (CBD) was loaded into retinal organoids EVs employing sonication, resulting in an entrapment efficiency of approximately 92.3 percent and stability for several months at 4 °C. Atomic force microscopy revealed that retinal organoid EVs were softer and smoother than the MSC derived EVs. CBD EVs reduced the expression of proteins implicated in inflammation and apoptosis and improved mitochondrial parameters in H2O2-treated ARPE cells, according to western blotting and immunocytochemical analyses. Dichlorofluorescin diacetate (DCFDA) staining was used to determine the amounts of intracellular reactive oxygen species (ROS). In H2O2 treated ARPE-19 cells, CBD EVs, CBD, and EVs alone significantly reduced ROS levels.

Conclusion: This research adds to the knowledge of the characteristics of EVs released by retinal organoids and their potential as drug delivery vesicles in the treatment of ocular diseases. EV biogenesis markers are expressed at high levels in late retinal organoids compared to early retinal organoids, according to our findings. The protein cargo of late retinal organoids and hUCMSC-derived EVs was studied in this research. Due to the general unique culture system employed in the retinal maturation process, the retinal organoid EVs have a better therapeutic protein cargo profile, which could potentially be used in treating ocular disease

Acknowledgements: This research is partly funded by the National Institute on Minority Health and Health Disparities, Grant/Award Number: U54 MD007582, and NSF-CREST Center for Complex Materials Design for Multidimensional Additive Processing (CoManD), Grant/Award Number:1735968. Work was supported, in part, by grants from the National Institutes of Health (R01- EY032197 and U24 EY029891 to D.A. L, R01-NS125016 to Y.L.) P30 Vision Core grant to UCSF Dept of Ophthalmology (EY002162), the Research to Prevent Blindness (unrestricted grant to UCSF Dept of Ophthalmology) and Eagles fifth District Cancer Telethon−Cancer Research Fund and Jay and Deanie Stein Career Development Award for Cancer Research at Mayo Clinic Jacksonville, 2019 Benefactor Funded Champions for Hope Pancreatic Cancer to S.B.
The authors declare that they have no competing interests.


Retinal organoids differentiation and characterization. (A) Schematic illustration showing the differentiation of retinal organoids from hiPSC and timeline representation of the early (Day 30-120) and late organoids (Day 121-300) for exosome preparation. (B) Phase contrast microscopic images showing the morphology of retinal organoids at different time points of differentiation (B1, Day 2; B2, Day 25; B3, Day 45; B4, Day 200). Scale bar, 200 µm. (C) Confocal images of 90-day old retinal organoids (C1-8) stained for retinal progenitor cells (Pax6, C1; Vx2, C2), ganglion and amacrine cells (Brn3a, C3), pan photoreceptors (Crx, C4; Rcvrn, C5; Aipl1; C6), rod photoreceptor (NRL; C7), and cone photoreceptor (ARR3, C8). Image of 200-day old retinal organoids showing the staining of rhodopsin protein indicating the maturation of rod photoreceptor (Rho, C9). Scale bar, 50 µm.


2:Topographical and nanomechanical characteristics of EVs. (A) Representative AFM images of plain mica and APTES: DIPEA modified mica (1, 3) Height image (2) Peak force error image. Representative images of retinal organoid and hUCMSC EVs (5, 7) Height image 6, 8) Peak force error image. (Scale bar: 1µm; color bar for height image: -65 nm to 65 nm; color bar for peak force error image: -300 pN to 300 pN). Morphology quantification i) Average height ii) Average surface roughness. (Statistical significance: ****; p < 0.0001). A representative force-separation curve displaying adhesion value corresponding to exosomes derived from (B) Retinal organoid and (C) hUCMSC. Nanomechanical attributes displaying (D) Young’s modulus (E) Deformation (F) Adhesion. (****; p < 0.0001).
3:Figure 4. Biogenesis of exosome in human 3D retinal organoids. (A) qRT-PCR assay of early (Day 90) and late (Day 200) retinal organoids showing the expression of ESCRT-dependent (Alix, TSG101), ESCRT-independent (Synthenin1, Syndecan1), Rabs family of transport and membrane fusion and pan exosome targets (Rab27B, CD63, CD81, ADAM10). (ns, p>0.05; *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001). (B) Western blots of early and late retinal organoid EVs for exosome markers HRS, Calnexin, Alix, Caveolin 1, HSP 70, Flotillin-2 and CD63. (C) Densitometric analysis of western blots. ***represents p < 0.001.


Proteomics analysis comparing retinal organoid with hUCMSC EVs. (A) Venn diagram of total identified proteins in both EV groups. (B) GO analysis of Retinal EVs. (C) Venn diagram of retinal organoid EVs, Human Protein ATLAS of retinal tissue and overlapped proteins between them.