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Researches showed that stem cells can rescue damaged cells through mitochondrial transfer.This mode has been used to regenerative cell-based therapy.Retinal pigment degeneration is an eye disease of retinal pigment epithelial (RPE) cell apoptosis as pathogenetic mechanism.Whether stem cells can repair the target cells by above mechanism has not been clarified.
This study was to investigate the influence of mitochondrial transfer on the function of RPE cells.
The RPE cells of Long-Evans rats were isolated and cultured and the third generation of cells were used in sequential experiment.The cells were identified by detecting the expressions of RPE65 and Bestrophin proteins with immunofluorescence stain.Mouse neural stem cells (NSCs) (C17.2 strain) with green fluorescence protein (GFP) and without GFP were cultured.Mitotracker-green and Mitotracker-red staining were separately used to labeled the mitochondria of RPE cells and NSCs.RPE cells were co-cultured with NSCs, and wheat germ agglutinin (WGA) was used to mark the tunneling nanotubes (TNT) between the two kinds of cells, and then the mitochondrial migration in TNT was exhibited by the laser scanning confocal microscope.The proportion of RPE cells in different cycles was assayed after marked with propidium iodide (PI) by flow cytometry.The contents of ATP, ADP and AMP in RPE cells were detected by high performance liquid chromatography (HPLC).
The third-generation of RPE cells grew well with the RPE65- and Bestrophin-positive rate >85%.The Mitotracker-red- labeled rates of NSCs and RPE cells were no less than 95%.TNT structure was seen to appear the blue fluorescence between RPE cells and NSCs 24 hours after co-cultured and the red dye mitochondria from NSCs migrated toward red dye mitochondria from RPEs with the lapse of time.The RPE cell proportion reduced in G1phase and increased by 5% and 2% in the S phase and G2/M phase respectively after mitochondrial transfer than before (P=0.016, 0.114, 0.189). The contents of ATP, ADP and AMP in the RPE cells were (8.77±3.68), (2.76±0.92) and (1.07±0.65)μg/mg after cell co-culture, and those before co-culture were (11.29±2.29), (3.12±0.95) and (1.59±1.22)μg/mg, without significant differences between them (P=0.370, 0.668, 0.553).
NSCs can transfer normal mitochondria to co-cultured RPEs via TNT structure.Mitochondrial exchange might be one of therapeutic mechanisms of NSCs recuing damaged RPE cells.
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Contributor Information
Department of Ophthalmology, Key Laboratory of Visual Damage and Regeneration &
Restoration of Chongqing, Southwest Hospital, Third Military Medical University, Chongqing 400038, China