Molecular Mechanisms Of Glaucoma

National Eye Institute

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Abstract

Several approaches for RGC neuroprotection have been suggested and tested in animal models. We previously demonstrated that intravitreal injection of human bone marrow-derived mesenchymal stem cells (BMSCs), or small extracellular vesicles (sEVs) secreted by BMSCs, provided statistically significant RGC neuroprotection when compared to control samples. Our data also revealed that miRNAs play an important role in the observed RGC neuroprotection. The emerging approach of sEV-based RGC protection demands more comprehensive investigation. sEVs isolated using traditional methods represent a heterogeneous population of sEVs which are comprised of different subpopulations such as microvesicles, exosomes, and recently identified mitovesicles. Hence, we fractionated isolated sEVs using density gradient-based differential ultracentrifugation and compared their neuroprotective effects with crude sEVs on RGC survival and neurite outgrowth in vitro. A 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 48 hours 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 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. 200 mL CM produced 2.32x1011 crude sEVs (size=148.743.1 nm) 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 8-28, 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 (52.59.2 vs 57.50.7 and 64.013.5), primary neurites (63.512.1 vs 59.53.5 and 71.620.6), secondary neurites (6111.3 vs 115.520.5 and 128.66.4) and tertiary neurites (85.6 vs 21.59.2 and 21.65.0), axon count (19.50.7 vs 27.07.1 and 22.38.1), and neurite length (250.258.8 m vs 337.459.8 m and 458.5130.2 m), respectively. We concluded that 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. To investigate the candidate proteins involved in neuroprotection, we unveiled the protein composition of crude sEVs produced by undifferentiated BRN3B-H9, NCRM-1, BMSC, and human dermal fibroblast (HDF) cultures by mass spectrometry. Protein enrichment analysis demonstrated the existence of highly enriched proteins in sEVs from stem cells which are parts of different cellular structures including mitochondria, cytoskeleton and synapses. sEVs from BMSCs were enriched in proteins that were involved in cell adhesion, protein stabilization, protein folding, and cell-matrix adhesion. We also performed total RNA and miRNA-sequencing of crude sEVs derived from undifferentiated BRN3BH9, NCRM-1, and human dermal fibroblasts cultures. We demonstrated the existence of several unique protein-coding RNAs and lncRNA in BRN3B-H9 sEVs. Further in-depth analysis of total RNA and miRNA sequencing data as well as proteomics data of crude sEVs derived from the undifferentiated cultures of BRN3B-H9 and NCRM-1 cells, BMSCs and HDFs is pending. Since sEVs produced by BMSCs growing in 2D cultures provide statistically significant RGC neuroprotection when compared to control samples, we tried to find conditions that stimulate production of biologically active sEVs. Our results demonstrated that BMSCs produced 10 times more sEVs per ml of CM when grown in 3D cultures versus 2D cultures. sEVs from BMSCs grown in 3D cultures are now being tested in different model systems in vitro and in vivo.