The Induction Of Retinal Pigment Epithelial-like Cells Differentiated From Mesenchymal Stem Cells In Vitro

ObjectiveThe retinal pigment epithelium (RPE) is a vital tissue for the maintenance of photoreceptor function. Amongst the many important functions of the RPE, it has the ability to phagocytosis and digest the rod outer segments (ROS) which is shed on a daily basis. The malfunction of RPE leads to retinal diseases, such as retinitis pigmentosa(RP), Best's disease, Stargardt's disease,Leber's congenital amaurosis and age-related macular degeneration. For the abnormal lity of RPE,human RPE transplantation is the highlight nowadays. However, the ability to obtain an adequate source of autologous RPE and that homologo us cells still is an unsettled problem.The development of tissue engineering, especially stem cell engineering provide a new approach.The research in the fields of stem cell, tissue engineer ing, neural development,inducement and differentiation modulation provide a opportunity to rehabilitate and treat these diseases. mesenchymal stem cell (MSCs) is widely studied. Previous studies have shown that these progenitor cells express pluripotential. They were shown to differentiate into osteoblasts, cartilages, skeletal muscle cells, endothelium, cardiac muscle cells, hepatocyt, and neuroectoderm both in vitro and in vivo. In this study we cultured RPE in vitro and non-contact induce the MSCs to be pigment epithelial-like cells and observe their physiological properties. It can establish found to treat retina 1 disease by providing seed-cells. Methods1. Establishing indirect co-culture system of HRPE and MSC in vitro(1) HRPE culture and lablingHRPE cells were cultured from human donor eyes for keratoplasty after death (Peking University Eye Center,Peking, China).To collect the HRPE cells by the trypsin in sterile circumstance.RPE cell origin was confirmed by positive cytokeration immunocytochemical analysis.(2) MSC culture and lablingMSC isolation and culture by density gradient centrifugalization and adher ence sieving from human bone. MSC were tested by immunocytochemical analysis.(3) Establishing indirect co-culture system of HRPE and MSCIndirect co-culture was established using millicell culture plate inserts with 0.4 urn pore (Millipore, Oxford, UK). A cell suspension containing HRPE cells was added to the upper membrane surface. Inserts were positioned in the cult ure wells, which pre-seeded with MSCs, with 1:1 mix of growth medium for the two cell types used. We observing the change of cells morphology by microscope.2. Differentiation protocols(1) Pigment epithelial-like cells Immunocytochemical analysisPerforming the cytokeration immunocytochemical analysis on pigment epithet lial-like cells.(2) Reverse transcriptase-PCR was used to examine expression of RPE65 and Mertk①Extracting the total RNA: the total RNA of three cells was extracted using the TRIZOL reagent.②Primer design:According RPE65 and Mertk gene order which NCBI published,I designed primers by Primer 3 software internet.③PCR:After adding different primer,I take 25μl PCR product to analysis by gel imaging system. 3. Assay of pigment epithelial-like cells Phagocytosis(1) Double Fluorescent Vital Assay of PhagocytosisROS was isolated from human donor eyes by a discontinuous sucrose gradient in a dark room according to the method of Papermaster et al.MSCs and rod outer segments (ROS) were labled by sulforhodamine (SR) (Invitrogen cor, California, U.S.A.) and fluorescein isothiocyanate (FITC) (Sigma-aldrich, Saint Louis, U.S.A.) respectively. Phagocytosis was observe by Double Fluorescent Vital Assay according to Margaret.Briefly, under fluorescence microscopy, ingested ROS was shown by yellow fluorescence, which was a mixture of red (SR labled) and green (FITC labled), ROS just bounding to MSCs surface only revealed green.(2) Assay of pigment epithelial-like cells Phagocytosis by TEMHRPE were incubated with ROS for 24 hours,removed liquid.The sample were fixed by 2.5% glutaric dialdehyde for transmission electron microsco pe(TEM).Result1. Establishing indirect co-culture system of HRPE and MSC in vitro(1) HRPE culture and lablingThe just digested HRPE cells were round,and contained large quantity of pigments, but those pigments in the RPE cells decreased along with the cell division, Phase-contrast examination of living cells were performed with a photomicro scope. RPE cell origin was confirmed by positive cytokeration immunocytochemical analysis.(2) Bone MSCs isolation, culture and lablingAfter culturring for primary MSC 24 hours, the cultured cells were characteriz ed by spindle-shaped appearance and prolifered rapidly. MSCs cytoplasm were stained positively by CD44 monoclonal antibody.MSCs were positive in nucleus with P63 monoclonal antibody.(3) indirect co-culture system of HRPE and MSC in vitro protocolsHRPE were characterized by spindle-shaped appearance,filled with pigm ents.MSCs were characterized by spindle-shaped appearance and prolifered rapidly,after 7 days,part of MSCs of spindle-shaped appearance changed to polygon-shaped gradually, filled with typical pigments;After 10 days ,more and more MSCs showed the same change. The differentiated rate were 45±5.12%.2. Differentiation protocols(1) Immunocytochemical analysisThrough microscope, I observed that MSCs were differentiated pigment epithelial-like cells.Differentiated pigment epithelial-like cells showed in green fluorescence,which indicated the cell were positive stain for pan- cytokeratin.(2) Reverse transcriptase-PCR was used to examine expression of RPE65 and Mertk.I detect RPE65 and Mertk mRNA expression of pigment epithelial-like cells by RT-PCR, the result show RPE and pigment epithelial-like cells have mRNA expression,but MSCs have no expression.3. Assay of pigment epithelial-like cells phagocytosis(1) Double Fluorescent Vital Assay of PhagocytosisAfter added HRPE cells' supernatant for 90 min, ROS were found in pigment epithelial-like cells surface , the phagocytosis reach the saturation. MSC can' t adhere and phagocytosis ROS.(2) Assay of pigment epithelial-like cells Phagocytosis by TEMIt can indicate the pigment epithelial-like cells' ultramicrostructure by TEM. which have Phagolysosome including ROS.Conclusion1.MSC can be differentiated epithelial-like cells in indirect co-culture system of HRPE.2.These MSCs-converted pigment epithelial cells showed some of the charac teristics of epithelioid cell. 3.These MSCs-converted pigment epithelial cells showed some of the characteristics of HRPE.4.These MSCs-converted pigment epithelial cells have the function of phagocytosis ROS.