Abstract
Purpose :
Retinal ganglion cells (RGCs) death due to glaucoma and other optic nerve diseases represents a crucial challenge. The emerging approach of small extracellular vesicles (sEVs)-based RGC protection demands more comprehensive investigation. Hence, we determined the effects of subpopulations of sEVs derived from a human embryonic reporter stem cell line on RGC survival and neurite outgrowth in vitro.
Methods :
H9-derived human embryonic stem cell line engineered with tdTomato and mouse THY1.2 knocked into the POU4F2 locus (BRN3B-H9) was cultured in mTeSR1 medium on Geltrex. Conditioned medium (CM) was collected at 48h and used to isolate sEVs by differential ultracentrifugation. sEVs were subsequently fractionated by density-gradient ultracentrifugation and characterized using Nanosight and Western Blot. Mixed primary mouse retinal cell cultures (N=3) were treated with different sEV fractions (3x109 sEVs per 1.25x105 cells) for 3 days. Beta III-tubulin staining was used to count RGCs and for morphometric analysis.
Results :
200mL CM produced ~2.32E+11 crude sEVs (size=148.7±43.1nm, N=3) after ultracentrifugation, while buoyant density-based fractionation showed variability in the number, size, and expression of sEVs markers across fractions 1 (BRIX=4) to 30 (BRIX=56). CD63 expression was detected from fractions 8-30, while CD9 expression was observed in all fractions. Crude sEV-treated retinal cultures showed ~62.5% increase in RGCs count than untreated cells (p=0.003). The highest RGCs survival was reported for combined fractions 21-30 (63.2%, p=0.0001) followed by fraction 30 (62.7%, p=0.006). Morphometric analysis of RGCs treated with crude sEVs showed significant improvement in neurite growth and was slightly higher in combined fractions 21-30, and fraction 30 alone for neuron count (52.5±9.2 vs ±57.5±0.7 & 64.0±13.5), primary neurites (63.5±12.1 vs 59.5±3.5 & 71.6±20.6), secondary neurites (61±11.3 vs 115.5±20.5 & 128.6±6.4) and tertiary neurites (8±5.6 vs 21.5±9.2 & 21.6±5.0), axon count (19.5±0.7 vs 27.0±7.1 & 22.3±8.1), and neurite length (250.2±58.8 µm vs 337.4±59.8 µm & 458.5±130.2 µm), respectively.
Conclusions :
The characterization of sEVs subpopulations derived from human stem cells offers a precise platform to design effective therapeutic strategies for enhanced RGC survival and neurite growth.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.