Mitochondrial transfer of mesenchymal stem cells effectively protects corneal epithelial cells from mitochondrial damage

Abstract

Recent studies have demonstrated that mesenchymal stem cells (MSCs) can donate mitochondria to airway epithelial cells and rescue mitochondrial damage in lung injury. We sought to determine whether MSCs could donate mitochondria and protect against oxidative stress-induced mitochondrial dysfunction in the cornea. Co-culturing of MSCs and corneal epithelial cells (CECs) indicated that the efficiency of mitochondrial transfer from MSCs to CECs was enhanced by Rotenone (Rot)-induced oxidative stress. The efficient mitochondrial transfer was associated with increased formation of tunneling nanotubes (TNTs) between MSCs and CECs, tubular connections that allowed direct intercellular communication. Separation of MSCs and CECs by a transwell culture system revealed no mitochiondrial transfer from MSCs to CECs and mitochondrial function was impaired when CECs were exposed to Rot challenge. CECs with or without mitochondrial transfer from MSCs displayed a distinct survival capacity and mitochondrial oxygen consumption rate. Mechanistically, increased filopodia outgrowth in CECs for TNT formation was associated with oxidative inflammation-activated NFκB/TNFαip2 signaling pathways that could be attenuated by reactive oxygen species scavenger N-acetylcysteine (NAC) treatment. Furthermore, MSCs grown on a decellularized porcine corneal scaffold were transplanted onto an alkali-injured eye in a rabbit model. Enhanced corneal wound healing was evident following healthy MSC scaffold transplantation. And transferred mitochondria was detected in corneal epithelium. In conclusion, mitochondrial transfer from MSCs provides novel protection for the cornea against oxidative stress-induced mitochondrial damage. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.

Figure 1

Intercellular mitochondrial transfer through TNTs during co-cultivation. (a) High yield of CECs and MSCs with molecular labeling. (i) MSCs labeled with Mito-GFP (green). (ii) CECs labeled with Violet. (b) Co-culturing of Violet-labeled CECs and mito-GFP-labeled MSCs for 24 h. Images show TNT formation and Mito-GFP-labeled mitochondria (arrow) were transferred from CECs to MSCs

Figure 2

Enhanced efficiency of mitochondrial transfer from MSCs to CECs when CECs were exposed to Rot treatment. (a) Mitochondrial transfer ratio (Violet-positive cells containing mito-GFP/total Violet cells) was measured. Compared with CECs under normal conditions, Rot-pretreated CECs (CEC(R) ) enhanced mitochondria transfer of MSCs when co-cultured (MSCs+CECs versus MSCs+CECs(R), *P<0.05, n=100). The enhanced mitochondrial transfer was attenuated when MSCs were pretreated with Rot (MSCs+CECs(R) versus MSCs(R)+CECs(R), *P<0.05, n=100). (b) (i) Mito-GFP-labeled MSCs and Violet-labeled CECs were co-cultured at 1:1 ratio. Representive images of intercellular mitochondrial transfer between CEC(R) and MSCs at 2 and 24 h of co-culture. (ii) Mitochondrial transfer ratio was measured over time from 2, 4, 6, 12, 24 to 48 h during co-culture of CECs(R) and MSCs. (c) (i) Rare membrane protrusions (MPs) were observed when CECs were in normal conditions without Rot treatment. (ii) Representative images showing a lot of MPs when CECs were pretreated with Rot. (iii) Quantitative analsyis of MP formation between CECs and Rot pretreated CECs (CECs(R) versus CECs, *P<0.05)

Figure 3

MSC-transferred mitochodria improve mitochondrial respiratory function of CECs. OCR of sorted CECs, CECs(R), CECs(R) co-cultured with MSCs (CECs(R)+MSCs) and CECs(R) co-cultured with MSCs in Transwell (CECs(R)+MSCs(T)) for 24 h were measured over time (mins) by extracellular flux analyzer. (a) Fifteen total OCR measurements were taken over 2 h: 3 basal respiration, 3 Oligomycin-sensitive respiration, 3 maximal respiratory capacity, and 6 non-mitochondrial respiration. The x axis in panel (a) describes the measurement (mins). (b) Basal mitochondral OCR of CECs from different groups. (c) ATP production of CECs from different groups. (d) Maximal respiration of CECs from different groups. (e) Rest respiration of CECs from different groups (^$P<0.05 versus CECs; *P<0.05, versus CECs(R); ^#P<0.05 versus CECs(R)+MSCs(T))

Figure 4

Rot-induced ROS activates NF-κB in CECs and enhances TNT formation via upregulation of TNFαip2. (a) Western blotting showing the level of p-IκB, TNFα and TNFαip2 in different groups. (b) p-IκB and TNFαip2 expression induced by Rot was strikingly decreased in hCECs with SC-514. (c) Quantification of p-IkB intensity. The levels of phosphorylation were normalized to β-Actin. Results were obtained from three independent experiments (*P<0.001 versus UT; ^#P<0.05 versus Rot). (d) hCECs were treated with Rot, Rot plus NAC or Rot plus SC-514 for 12 and 24 h; the number of Filiopodia outgrowth per cell was calculated (*P<0.001 versus UT; ^#P<0.05 versus Rot, n=100). (e) The proposed mechanism whereby Rot-induced ROS activates NF-κB and enhances TNT formation via upregulation of TNFαip2 in hCECs

Figure 5

MSC transplanation and mitochondrial transfer in vivo. (a) Images of MSC culture on acellular porcine cornea matrix. After 72 h culture, the inside surface of matrix was covered by MSCs. Representative images of (i) DAPI (4,6-diamidino-2-phenylindole), (ii) GFP and (iii) Merge. (b) Slit-lamp images of MSCs on Matrix (MSC Matrix), on Rot-pretreated MSC Matrix (MSC(R)+Matrix) and Matrix only group cornea. Transplantation of the above matrix was performed following Alkali-induced corneal injuries. Matrix was removed and corneal epithelial fluorescein staining was performed at 48 h after transplantation. (c) (i) The statistical data of the rest of corneal erosion after 48 h matrix transfer; mitochondria donation from MSCs promoted corneal epithelium healing but the healing effort significantly declined if MSCs were pretreated with Rot. (ii) Representative images of immunofluorescent staining of anti-GFP (green) and CK3 (red) performed on the corneal tissue of MSC Matrix groups. Mitochondria from MSCs (green) were detected within CECs (amplified). (*P<0.05; NS, not significant)