Improvement Of Proliferation And Metabolism Abilities Of RPE Cells Isolated From RCS Rats By Mitochondrial Transferred From Neural Stem Cells

Objective:Recent years, the incidence of age-related macular degeneration(AMD) increased gradually, and the key pathological changes of AMD is the degeneration, necrosis and loss of retinal pigment epithelium cells(RPE cells). RPE cells are localized in the outer layer of the retina, and the main functions of RPE cells include the formation of blood retinal barrier, secretion of neurotropic factors, phagocytosis of photoreceptor outer segment, absorption of visible light and anti-oxidative stress. The normal functions of RPE cells depend on sufficient energy supply, during the metabolism of RPE cells,a large number of reactive oxygen species(ROS) were produced. The mitochondrion is the key organelle of energy metabolism and ROS abolishing in RPE cells. Recent study demonstrated that mitochondrial dysfunction of RPE cells is closely related to the occurrence of AMD.As a therapy of AMD, the development of stem cell transplantation has brought new hope to the repair and regeneration of RPE cells. The mechanism of stem cell transplantation is considered to be: short-term neurotropic effect(stander by effect) and long term cell replacement effect. Recently, it demonstrated that mesenchymal stem cells can transfer mitochondria to improve the function of the receptor cells. As AMD and other retinal degeneration diseases are closely related to mitochondrial dysfunction in RPE cells. There is no evidence that mitochondria transfer was also involved in the therapeutic effect of grafted stem cells in the retina. It was mentioned that the RPE cells(ARPE19 cell line) could form TNT structure with each other and could transfer mitochondrion by TNT.In this study, we will explore whether mouse neural stem cells could form TNT with RPE cells, and whether m NSC could transfer mitochondrion to RPE cells through TNT. Furthermore, we wondered if the mitochondrial transfer has influence on the function of the RPE cell.Methods:Part I: The transfer of mitochondrion from mNSC to RPE cells through TNT1. After 24 h co-culture, the confocal laser scanning microscope(CLSM) was used to observe the communication of mNSC and the ARPE19 cells.2. After 24 h co-culture of the mNSC and the primary RPE cells of LE rats, the CLSM was used to observe the TNT formation and the mitochondria transfer between cells.Part II: The function improvement of RPE cells by transferred mitochondrion from m NSC.1. Sorting LE-RPE out of co-culture groups of LE-RPE/mNSC by FACS, the concentration of ATP/ADP/AMP in LE-RPE was evaluated with High Performance Liquid Chromatography(HPLC), the level of ROS and the proliferation of LE-RPE cells was tested by 2’,7’-dichlorofluorescin diacetate(DCFH-DA) and propidium iodide(PI) with FACS.2. HPLC was used to evaluate the concentration of ATP/ADP/AMP in LE-RPE and RCS-RPE, FACS was used to test the level of ROS and the proliferation of LE-RPE and RCS-RPE.3. Sorting RCS-RPE from LE-RPE/mNSC by FACS, the concentration of ATP/ADP/AMP in RCS-RPE was evaluated with HPLC, the level of ROS and the proliferation of LE-RPE cells was tested by FACS.Results:Part I: The transfer of mitochondrion from mNSC to RPE cells through TNT.1. After 24 h co-culture, the mNSC can successfully transfer mitochondrion to ARPE19 cells or primarily isolated LE-RPE cells through TNT.Part II: Cell proliferation and mitochondrial function of RPE cells were markedly improved by transferred mitochondrion from m NSC1. There is no significant difference in energy metabolism and ROS level between LE-RPE cells which co-cultured with mNSC and LE-RPE cells cultured alone. While cell proliferation of LE-RPE cells when co-cultured with mNSC was significantly increased.2. Energy metabolism and cell proliferation of RCS-RPE cells were significantly decreased compared to that of LE-RPE cells, while ROS of RCS-RPE cells were markedly increased than that of LE-RPE cells.3 There is no significant difference in the energy metabolism between RCS-RPE cells when co-cultured with mNSC and RCS-RPE cells cultured alone, however ROS of RCS-RPE cells co-cultured with m NSC was markedly decreased while cell proliferation was significantly increased compared to that of RCS-RPE cells cultured alone.Conclusion:1. TNT were observed formation between mNSC and RPE cell lines(ARPE19) or primary LE-RPE cells, and through which mitochondrion were transferred from m NSC to RPE cells.2. Cell proliferation and mitochondrial function were significantly improved in the RCS-RPE cells by mitochondrion transfer from mNSC.