Mitochondria Transfer Between Stem Cells And RPE Cells And Influence The Functions Of RPE Cells

Objective:Retinal degenerative diseases mainly include the retinitis pigmentosa(RP),the age related macular degeneration(AMD),Stargardt diseases and cone-rod dystrophy.The main common pathological change of these diseases is the degeneration and the loss of photoreceptor and/or retinal pigment epithelium(RPE)cells,and what is more important is that these cells all cannot regeneration.So far,retinal degenerative diseases are still cureless all around the world.The RPE cells are lying in the outer layer of retina,and the key functions of RPE cells include: forming the outer blood retinal barrier,phagocytosing the outer segment of photoreceptor,absorbing visible light,secreting growth or neurotropic factors,as well as anti-oxidative stress.Study reported that mitochondrion is the key organelle to abolish reactive oxygen species(ROS),and during the metabolism process lots of ROS were produced.Recently,study showed that mitochondrial dysfunction of RPE cells is related to the occurrence of AMD and diabetic retinopathy closely.Recently,cell transplantation therapy brought new light to the regeneration or repair of the degenerated or lost cells.The main mechanism of stem cell transplantation therapy to be: the cell replacement effect,the neurotropic effect(stander by effect),and the immune-regulatory effect.Stem cells therapy seems to holding great promise for the regenerative medicine.Rustom has reported findings of a straight-thin structure directly connecting untouched but near single cells over long distance,which can provide a way to facilitate the transfer of cell membrane or cell organs.This structure,named tunneling nanotube(TNT).Recent study reported that the mesenchymal stem cells affect the function of the receptor cells via the way of transferring mitochondria.But there is still no evidence about whether mitochondria transfer is involved in the therapeutic effect of the grafted stem cells in retina.In our study,we explored about whether stem cell and human embryonic stem cell-derived retinal pigment epithelium(hESC-RPE)cells could form TNT structure with RPE cells isolated from Royal college of surgeons(RCS)rat,and whether h ESC-RPE could transfer mitochondrion to RPE cells via TNT.Furthermore,we explored whether mitochondrial transfer has effects on the function of the RPE cell isolated from RCS rat.In order to detect whether mitochondria transfer via TNT structure to be a mechanism of stem cell therapy to cure retinal degenerative diseases.Methods:Part I: The transfer of mitochondrion between mouse neural stem cells(mNSC)and RPE cells isolated from RCS(RCS-RPE)rat through TNT and the influence of the function of RPE cells.1.After 24 h co-culture,use the confocal laser scanning microscope(CLSM)to observe the TNT formation and mitochondria transfer between mNSC and RPE cells isolated from RCS rat.2.After co-culture for 48 h,sorting RCS-RPE from RCS-RPE/mNSC(GFP)by FACS.3.Detect the level of ROS,the proliferation and apoptosis of RCS-RPE cells cultured alone and the sorted RCS-RPE cells.Part II: The transfer of mitochondrion between normal human retinal pigment epithelial cells(ARPE-19)and NaIO3 pretreated injured ARPE-19 cells through TNT and the influence of function of injured ARPE-19 cells.1.After 24 h co-culture,use the CLSM to observe the TNT formation and mitochondria transfer between normal ARPE-19 cells and Na IO3 pretreated ARPE-19 cells.2.After co-culture for 24 h,sorting ARPE-19 cells from ARPE-19(GFP)cells by FACS.3.Detect the level of ROS,the cell proliferation and cell apoptosis of NaIO3 pretreated ARPE-19 cells which then culture alone for 24 h,the sorted ARPE-19 cells,and Na IO3 pretreated ARPE-19 cells which co-cultured with normal ARPE-19 cells by Transwell.Part III: The transfer of mitochondrion between h ESC-RPE cells and ARPE-19 cells or RCS-RPE cells.1.After 24 h co-culture,use the CLSM to observe the TNT formation and the mitochondria transfer of h ESC-RPE cells and normal ARPE-19 cells.2.After 24 h co-culture,use the CLSM to observe the TNT formation and the mitochondria transfer between hESC-RPE cells and ARPE-19 cells pretreated by 200ug/ml Na IO3 for 24 h.3.After 24 h co-culture,use the CLSM to observe the TNT formation and the mitochondria transfer of hESC-RPE cells and ARPE-19 cells pretreated by 2000ug/ml Na IO3 for 24 h.4.After 24 h co-culture,use the CLSM to observe the TNT formation and the mitochondria transfer of hESC-RPE cells and RCS-RPE cells.Results:Part I: The transfer of mitochondrion between m NSC and RPE cells isolated from RCS rat through TNT and the influence of the function of RPE cells.1.TNT structure can be formed between mNSC and RCS-RPE,and mitochondrion can transfer from NSC to RCS-RPE cells through TNT.2.Mitochondrion transfer can influence the ROS,the cell proliferation and apoptosis of RCS-RPE cells.Part II: The transfer of mitochondrion between normal human retinal pigment epithelial cells(ARPE-19)and NaIO3 pretreated injured ARPE-19 cells through TNT and the influence of function of injured ARPE-19 cells.1.TNT structure can be detected formed between normal ARPE-19 cells and 200ug/ml Na IO3 pretreated injured ARPE-19 cells,and mitochondrion can transfer from normal ARPE-19 cells to injured ARPE-19 cells through TNT.2.Mitochondrion transfer can influence the ROS level,the cell proliferation and cell apoptosis of Na IO3 pretreated ARPE-19 cells.Part III: The formation of TNT structure and mitochondria transfer between h ESC-RPE cells and ARPE-19 cells or RPE cells isolated from RCS rat.1.TNT structure can form between hESC-RPE cells and ARPE-19 cells,and mitochondrion can be detected transferring from normal ARPE-19 cells to h ESC-RPE cells via TNT.2.TNT can form between h ESC-RPE cells and 200ug/ml NaIO3 pretreated ARPE-19 cells,and mitochondrion detected transfer from 200ug/ml Na IO3 pretreated ARPE-19 cells to hESC-RPE cells through TNT.3.TNT can form between hESC-RPE cells and 2000ug/ml Na IO3 pretreated ARPE-19 cells,and mitochondrion detected transfer from 2000ug/ml NaIO3 pretreated ARPE-19 cells to hESC-RPE cells through TNT.4.TNT can form between h ESC-RPE cells and RCS-RPE,and mitochondrion can be detected transfer from RCS-RPE to hESC-RPE cells through TNT.Conclusion:1.Mitochondrion can transfer from mNSC to RCS-RPE through TNT and can influence the ROS,the cell proliferation and apoptosis of RPE cells.2.Mitochondrion can transfer from normal ARPE-19 cells to injured ARPE-19 cells through TNT and influence the ROS,the cell proliferation and apoptosis of injured ARPE-19 cells.3.Mitochondrion can transfer from normal ARPE-19 cells,ARPE-19 cells pretreated by 200ug/ml?2000ug/ml Na IO3 through TNT to hESC-RPE cells.Mitochondrion detected transfer from RCS-RPE to h ESC-RPE cells through TNT.We think it perhaps because that mitochondria transfer from which are more mature to less mature ones.