Mitochondrial transfer between BMSCs and Müller promotes mitochondrial fusion and suppresses gliosis in degenerative retina

Summary Mitochondrial dysfunction and Müller cells gliosis are significant pathological characteristics of retinal degeneration (RD) and causing blinding. Stem cell therapy is a promising treatment for RD, the recently accepted therapeutic mechanism is cell fusion induced materials transfer. However, whether materials including mitochondrial transfer between grafted stem cells and recipient’s cells contribute to suppressing gliosis and mechanism are unclear. In present study, we demonstrated that bone marrow mesenchymal stem cells (BMSCs) transferred mitochondria to Müller cells by cell fusion and tunneling nanotubes. BMSCs-derived mitochondria (BMSCs-mito) were integrated into mitochondrial network of Müller cells, improving mitochondrial function, reducing oxidative stress and gliosis, which protected visual function partially in the degenerative rat retina. RNA sequencing analysis revealed that BMSCs-mito increased mitochondrial DNA (mtDNA) content and facilitated mitochondrial fusion in damaged Müller cells. It suggests that mitochondrial transfer from BMSCs remodels Müller cells metabolism and suppresses gliosis; thus, delaying the degenerative progression of RD.


Figure S2 .
Figure S2.The influence of Rotenone-Induced Model of mitochondrial damage to Müller cells or BMSCs, related to Figure 1.(A) White phase of Müller cells treated under different rotenone concentrations from 0μΜ to 25μM.

Figure S3 .
Figure S3.Ways of mitochondria transfer occur during direct co-culture of BMSCs and Müller-rot cells, related to Figure 1.(A) Ways of mitochondria transfer: cell fusion; Müller cells labelled by cell trace (blue); BMSCs labelled by Mito-GFP (green).Yellow arrows pointed to the transferred mitochondria.(B) Ways of mitochondria transfer: TNT (tunnelling nanotubes) after direct co-culture 24h between BMSCs with Müller-rot cells; Müller cells labelled by Mitot-RFP (magenta), BMSCs labelled by Mito-GFP (green).Yellow dotted lines showed the transfer.Yellow arrows pointed to the transferred mitochondria.(C) The representative images about mitochondria transfer between BMSCs and Müller cells (C1) or Müller-rot cells (C2) after direct coculture for 24 h.Yellow arrows pointed to the transferred mitochondria in Müller cells, showing the typical mitochondrial transfer ways as the figure A, B showed.(D) Effect of rotenone treatment on the ratio of mitochondria transfer between BMSCs and MG.n≥23.Data are presented as the mean ± standard deviation (SD), ****P < 0.0001 (T-test for D).Scale bars: 20μm (A, B). 100μm (C).

Figure S5 .
Figure S5.Intact images of Western blotting, related to Figure 4. (A) The images of protein bands of GFAP.(B) The images of protein bands the reference protein GAPDH.

Figure S6 .
Figure S6.The Müller cells in the retinas of RCS rats exhibit mitochondrial dysfunction and morphological abnormalities, related to Figure 5 and STAR Methods.(A-D) The representative analysis process of flow cytometry for mitochondrial membrane potential in retinal Müller cells of RCS rats or the normal groups RDY at 7 weeks after birth.A: single cells; B: live cells; C: CD29 + cells, which labelled the Müller cells; D: Flow cytometry analysis of mitochondrial membrane potential (MMP) with JC-1 in Müller cells, Q1 represents JC-1 aggregates: normal mitochondria, Q2 represents JC-1 monomers: MMP decrease.Number below the Qn is the ratio of this part cells.(E) Flow cytometry analysis of mitochondrial membrane potential (MMP) with JC-1 in Müller cells of RCS Figure S7.Puzzle pictures of whole retina of RCS rat after 6 weeks of subretinal transplantation of BMSCs-mito, related to Figure 5. (A) Puzzle pictures of whole retina of RCS rat after 6 weeks of subretinal transplantation of BMSCs-mito.Transplant area retinal had detachment.No tumor formation.Merge pictures of DAPI (blue), white phase.Black area is RPE cells layer.(B) Transplant area retinal showed some detachment.Ganglion cell layers (GCL), inner nuclear layers (INL), outer nuclear layers (ONL), yellow dashed line area is subretinal space (SRS).(C) Opposite of the transplant area retinal showed little detachment.Scale bars: 500μm (A), 150μm (B, C).

Figure S9 .
Figure S9.The pictures of PCA, DEGs, Volcano plot, and Cluster heat map of RCS+mito groups comparing to RCS+PBS groups in vivo, related to Figure 6.(A) The principal components analysis (PCA) pictures between RCS+mito and RCS+PBS.(B) The differentially expressed genes in different groups.FC＞1.2 and P＜0.05.Red is up, blue is down.

Figure S10 .
Figure S10.The pictures of PCA, DEGs, Volcano plot and Cluster heat map of Müller-rot+mito groups compared to Müller-rot groups in vitro, related to Figure 7.

Table S1 .
Summary of primary and secondary antibodies, related to STAR Methods.

Table S2 .
Primer sequences, related to STAR Methods.