Effective treatment of optic neuropathies by intraocular delivery of MSC-sEVs through augmenting the G-CSF-macrophage pathway

Abstract

Optic neuropathies, characterized by injury of retinal ganglion cell (RGC) axons of the optic nerve, cause incurable blindness worldwide. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) represent a promising "cell-free" therapy for regenerative medicine; however, the therapeutic effect on neural restoration fluctuates, and the underlying mechanism is poorly understood. Here, we illustrated that intraocular administration of MSC-sEVs promoted both RGC survival and axon regeneration in an optic nerve crush mouse model. Mechanistically, MSC-sEVs primarily targeted retinal mural cells to release high levels of colony-stimulating factor 3 (G-CSF) that recruited a neural restorative population of Ly6C^low monocytes/monocyte-derived macrophages (Mo/MΦ). Intravitreal administration of G-CSF, a clinically proven agent for treating neutropenia, or donor Ly6C^low Mo/MΦ markedly improved neurological outcomes in vivo. Together, our data define a unique mechanism of MSC-sEV-induced G-CSF-to-Ly6C^low Mo/MΦ signaling in repairing optic nerve injury and highlight local delivery of MSC-sEVs, G-CSF, and Ly6C^low Mo/MΦ as therapeutic paradigms for the treatment of optic neuropathies.

Keywords: G-CSF; MSC; axon regeneration; macrophage; small extracellular vesicle.

Fig. 1.

MSC-sEV treatment recruited myeloid cells and promoted axon regeneration and RGC survival. (A) Characterization of MSC-sEVs (Left: TEM, Middle: NTA, Right: WB). (B–D) Optic nerves and retinas were harvested from ONC mice at 2 wk after i.o. treatment with vehicle (PBS) or MSC-sEVs every 3 d (starting on day 0). The sham surgery group served as a negative control. (B) Representative images of retinal whole mounts with anti-RBPMS (white) to visualize RGCs. (Scale bar, 50 μm.) The frequency of viable RBPMS⁺ RGCs was normalized to that in healthy retinas (n = 5 mice/group). (C) Representative images of optic nerves from longitudinal sections immunostained with anti-GAP43 (green). The asterisks indicate the crush site (n = 6 nerves/group). (Scale bar, 200 μm.) (D) Quantification of GAP43⁺ regenerating axons at serial distances from the crush site. Statistical significance was determined by t test or two-way ANOVA and Sidak’s post hoc comparisons (Mean ± SEM, ***P < 0.001, ****P < 0.0001). (E–H) scRNA analysis of retinal cells harvested from ONC mice at 2 wk after i.o. treatment with vehicle (PBS) or MSC-sEVs every 3 d (started on day 0) (n = 6 mice/group). (E) UMAP visualization and the percentage of the retinal cells in 10 major clusters. (F) UMAP visualization of retinal cells and (G) percentage of different clusters in each group. (H) Network centrality analysis identified the strength of cell–cell communication for each cluster in the control and MSC-sEV groups.

Fig. 2.

Retinal infiltration of Mo/MΦ was required for RGC survival and axon regeneration. (A) Quantification of different immune cells in retinas from ONC mice at 2 wk after i.o. treatment with vehicle (PBS) or MSC-sEVs every 3 d (started on day 0) (n = 5 mice/group) using flow cytometry. (B–F) ONC mice received i.o. treatment with vehicle (PBS) or MSC-sEVs (started on day 0) after intraperitoneal (i.p.) injections of anti-Ly6g, anti-Gr-1, or isotype control (started on day −1) every 3 d. Retinal cells or optic nerves were harvested on day 14. (B) Representative flow cytometric contour map and quantification of Ly6c/Ly6g gated myeloid cells (n = 3 mice/group). (C) Representative images of retinal whole mounts immunostained with anti-RBPMS (white). (Scale bar, 50 μm.) (D) RGC survival from each group normalized to healthy retinas (n = 5 retinas/group). (E) Representative images of optic nerves from longitudinal sections immunostained with anti-GAP43 (green). (Scale bar, 200 μm.) and (F) Quantification of GAP43⁺ regenerating axons (n = 5 nerves/group). Statistical significance was determined by one-way or two-way ANOVA and Sidak’s post hoc comparisons. (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 3.

The Ly6C^low Mo/MΦ subset displayed restorative features. (A−F) scRNA analysis of retinal cells harvested at 2 wk after ONC from mice with i.o. vehicle (PBS) or MSC-sEVs treatment every 3 d (started on day 0). (A) UMAP visualization of the Mo/MΦ subclusters. (B) Violin plots showing the normalized and scaled Ccr2, Tmem119, and Cxcr2 expression levels in microglia and the two Mo/MΦ subclusters. (C) Network centrality analysis illustrated the strength of the CCC for each subclusters in MSC-sEV-treated group. (D) Circle plot showed the inferred outgoing communication patterns among the Mo/MΦ subsets and other cell clusters. Circle sizes are proportional to the number of cells in each cell group, and the edge width represents the communication probability. (E) Dotplot showing the distribution and scaled expression level of top differential expressed genes among the two Mo/MΦ subclusters and granulocytes. (F) Barplot showing the significantly enriched pathways that were up-regulated or down-regulated in the Mo/MΦ2 subcluster compared with the Mo/MΦ1. (G) ONC mice received i.o. treatment with MSC-sEVs (started on day 0) after i.p. injections of anti-Ly6g or isotype control (started on day −1) every 3 d. (Left) Histogram of the surface Ly6c expression of Mo/MΦ cells from the retinas. (Right) the percentages of Ly6c^low and Ly6c^high Mo/MΦ were quantified (n = 3 mice/group). Statistical significance was determined by two-way ANOVA and Sidak’s post hoc comparisons (**P < 0.01).

Fig. 4.

Intraocular administration of MSC-sEVs induced significant G-CSF release. (A–C) Representative image of retina whole mount from perfused ONC mice with i.o. MSC-sEVs treatment at 1 h (A), 6 h (B), and 12 h (C) showing myeloid cells (CD11b, green) and retinal vessels (CD31, red). (Scale bar, 50 µm.) (D–G) Retinas and plasmas were harvested from ONC mice at 1 d after treatment with i.o. MSC-sEVs or vehicle (PBS). (D) Volcano plot of the differentially expressed genes and (E) GSEA of enriched pathways in retinas. (F) Quantification of Csf3 transcripts by qPCR in retinas and (G) G-CSF protein by ELISA in plasma (n = 3 mice/group). (H) Ridge plot showing the distribution of the Csf3r gene in different immune cell clusters from the scRNA-seq dataset of retinas harvested at 2 wk from ONC mice with i.o. MSC-sEVs treatment every 3 d (started on day 0). Statistical significance was determined by t test and Sidak’s post hoc comparisons. (*P < 0.05, **P < 0.01).

Fig. 5.

Intraocular G-CSF promoted the recruitment of restorative Ly6C^low Mo/MΦ for axon regeneration. (A–C) ONC mice received treatment with i.o. MSC-sEVs (started on day 0) and i.p. injections of anti-G-CSF or isotype control (started on day −1) every 3 d. Cells or tissues were harvested on day 14. (A) Quantification of Ly6c/Ly6g gated myeloid cells in retinas. (B) Representative images and (C) quantification of GAP43⁺ (green) regenerating optic nerves (n = 4 nerves/group). (Scale bar, 200 μm.) (D and E) ONC mice received treatment with i.o. MSC-sEVs every 3 d or i.o. G-CSF every day for a total of 5 d (started on day 0) and retinal samples were harvested at 1 wk. (D) Representative flow cytometric contour map and quantification of Ly6c/Ly6g gated myeloid cells (n = 3 mice/group). (E) Arg1, Mrc1, and Igf1 transcripts were quantified by qPCR (n = 3). (F and G) ONC mice received treatment with i.o. MSC-sEVs every 3 d or i.o. G-CSF every day for a total of 5 d (started on day 0) and nerve samples were harvested at 2 wk. (F) Representative images and (G) quantification of GAP43⁺ (green) regenerating optic nerves. (Scale bar, 200 μm.) Statistical significance was determined by two-way ANOVA and Sidak’s post hoc comparisons. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 6.

Donor Ly6G^low Mo/MΦ cells promoted RGC survival and axon regeneration. (A) Schematic graph of the adoptive transfer in (B−E). A 2.5-μL cell (1 × 10⁵ cells/μL) suspension was i.o. injected to recipient mice on day 0, day 3, and day 6. Optic nerves and retinas were harvested at 2 wk. (B) Representative images of retinal whole mounts immunostained with anti-RBPMS (white) from each group of recipient mice. (Scale bar, 50 μm.) (C) RGC survival normalized to healthy retinas was quantified (n = 8 retinas/group). (D) Representative images of optic nerves from each group showing GAP43⁺ (green) regenerating axons. (Scale bar, 200 μm.) (E) Quantification of GAP43⁺ regenerating axons. (n = 4 nerves/group). Statistical significance was determined by one-way or two-way ANOVA and Sidak’s post hoc comparisons. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 7.

MSC-sEVs were primarily taken up by retinal mural cells. (A–C) Representative image of retina whole mount from perfused ONC mice 6 h after i.o. MSC-pHluorin-sEVs (white). Vessels were visualized by anti-CD31 immunostaining (red). (A) The Upper row shows the landscape of the retinal mural cells (NG2, green) on the vessels around the optic nerve head at low magnification. The white arrows indicated the distribution of the pHluorin signal along the blood vessels. (Scale bar, 100 µm.) The Middle and Bottom rows show the overlapping pHluorin signal (white) in retinal mural cells (NG2, green) surrounding the endothelial cells (CD31, red) of the vessels at high magnification zoomed in on the areas labeled by the cyan boxes. (Scale bar, 5 µm.) (B and C) Nonoverlapping (yellow arrows) pHluorin signal (white) in (B) astrocyte branches (GFAP, green) or (C) microglia (IBA1, green). (Scale bar, 10 µm.)

Fig. 8.

Mural cells engulfed MSC-sEVs to release G-CSF and recruit leukocytes. (A) Uptake of fluorescently labeled MSC-sEVs (PKH dye, green) by primary mural cells in vitro at 1 h. (Scale bar, 10 μm.) (B and C) Bulk-RNA-seq dataset of mural cells treated with MSC-sEVs or vehicle (PBS) for 12 h. (B) Volcano plot of differentially expressed genes and (C) GSEA of enriched KEGG pathways visualized by dotplot. (D) Quantification of migrated cells in the chemotaxis experiment (n = 3). (E−G) Mural cells were seeded on 6-well plates and treated with MSC-sEVs (4 × 10⁹/mL) or vehicle (PBS) for 12 h. (E) Retinas were harvested from ONC mice at 1 d after treatment with i.o. MSC-sEVs or vehicle (PBS). Heatmap of bulk RNA-seq dataset showing the significantly upregulated cytokine genes in MSC-sEV-stimulated mural cells or retinas. (F) Csf3 transcripts in primary mural cells were quantified by qPCR (n = 3). (G) The G-CSF protein level in mural cell-CM was quantified by ELISA (n = 3 mice/group). (H) Uptake of fluorescently labeled MSC-sEVs (green) by primary mural cells was largely eliminated by cotreatment with dynasore (20 μM, started at −1 h). (I) MSC-sEVs induced Csf3 upregulation was significantly blocked by dynasore (20 μM, started at −1 h) in primary mural cells at 12 h (n = 3). (J) Quantification of migrated BM-derived Ly6g^-Ly6c^low Mo/MΦ cells by flow cytometry in the chemotaxis experiment (n = 4). Statistical significance was determined by t test or one-way ANOVA and Sidak’s post hoc comparisons. (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 9.

Schematic graph of the therapeutic mechanism of MSC-sEVs for repairing optic nerve injury. MSC-sEVs delivered by i.o. injection are primarily taken up by retinal mural cells, inducing the release of the colony-stimulating factor G-CSF. This leads to the recruitment of a Ly6C^low Mo/MΦ population that significantly promotes RGC survival and axon regeneration.