Objective To isolate and purify the melanoma stem cells (MSC) in choroidal melanoma OCM-1 cells. Methods OCM-1 cells were resuscitated, and after cultured in standard Dubecco's modifided Eagle's medium (DMEM)/F12, they were cultured in serum-free medium (SFM). The cultured MSC were isolated and purified, and the positive rate of CD133, the specific markers of neurostem cells, was observed by flow cytometry (FCM). The 6th generation of the cells were stained by musashi-1 immunocytochemistry, and the rate of the positive cells was observed under the microscope. Results After the Adherent OCM-1 cells cultured in SFM, the number of the adherent number decreased obviously. The cells at the 6th generation grew as the suspended gobbets, which represented the typical grow manner of the stem cells. Positive CD133 could be found in the cells of different generations, which was 2.5％, 21.7％, and 57.8％ in the non-isolated OCM-1 cells, the 1st generation of isolated cells, and the 2nd generation cells, respectively. The positive rate of CD133 in the cells at the sixth generation was 79.8% with b positive expression of musashi-1. Conclusion MSC is in the human choroidal melanoma OCM-1 cells. The suspended stem cells may be purified by limited differentiation and serial passage in SFM. (Chin J Ocul Fundus Dis, 2007, 23: 87-90)
Objective To observe the survival of human umbilical cord derived mesenchymal stem cells (hUC-MSCs) after injection into the vitreous of rabbits,and the animal safety under those procedures.Methods Twentyseven pigmented rabbits were randomly divided into 3 groups (intravitreal injection 1 week group，2 weeks group and 4 weeks group)， each with 9 rabbits.For each animal the right eye was the experimental eye receiving hUCMSCs injection,while the left eye was the control eye receiving culture medium. The rabbit eyes were examined by slitlamp microscope, indirect ophthalmoscopy, fundus photography, fundus fluorescence angiography（FFA）and Tonopen tonometer before and after injection. hUCMSCs were labeled by CMDil in vitro, and their survival status was measured by confocal fluorescence microscopy, light microscope and transmission electron microscope at 4 weeks after injection. Results Four weeks after injection, a large number of the hUCMSCs were still alive in the vitreous cavity. The overall condition of those rabbits was good. The anterior segment and retina of experimental eyes were normal, without hyperfluorescence, hypofluorescence and leakage in the retina at 1,2 and 4 weeks after injection. There was no significant difference on IOP before and after injection at different time points (P＞0.05), and no obvious changes at cornea, anterior chamber angle,lens,retinal structure by.light microscope and transmission electron microscope examination.Conclusion hUC-MSCs can survive in the rabbit vitreous for four weeks；intravitreal injection of hUCMSCs was safe and feasible.
Objective To observe whether transforming growth factor-beta;2（TGF-beta;2）could promote the differentiation of retinal stem cells in rats cultured in vitro. Methods The retinal stem cells were separated from the embryonic ratsprime; eyes under the dissecting microscope, cultured, and subcultured. The cells were identified by nestin and Chx-10 immunofluorescence. The sixth generation of cells were induced and differentiated, immunofluorescent stained with anti-glial fibrillary acidic protein，anti-opsin, anti-b-tubulin, and anti-protein kinase C, and identified the final cells. Results The cultured cells after induced by TGF-beta;2 differentiated to the mature cells. The results of immunofluorescence showed that the differentiated cells induced by TGF-beta;2 were more than which induced by the embryonic bovine blood serum. Conclusion TGF-beta;2 may induce the retinal stem cell differentiating into retinal cells. The inductive and differentiating effect of TGF-beta;2 is ber than which of the blood serum. (Chin J Ocul Fundus Dis, 2007, 23: 104-107)
Objective To isolate neural stem cells (NSCs) from rabbit retina and brain, and induce differentiation of those NSCs using different culture media. Methods Single-cell suspensions of retina and cerebral cortex were prepared from rabbit embryo, cultured in 5 types of different media to isolate the NSCs by continual passages. After 3 passages, NSCs were induced to differentiation in 2 types of different media for 8 to 10 days. NSCs and inducedretinal cells were examined by immunofluorescence and flow cytometry for the expression pattern of some specific antigens.Results Immunofluorescence showed that NSCs from retina and brain, cultured in serumfree media, both expressed Nestin partially. Flow cytometry showed that Nestin positive cells were significantly decreased while the Rhodopsin and Thy1.1 positive cells were increased after induction. Compared with the combined induction of alltrans retinoid acid (ATRA) and serum, 5%FBS (fetal bovine serum) led to higher expression of Rhodopsin（P＜0.01），but lower expression of Thy1.1（P=0.01）.Conclusion Serumfree media with N2, EGF, bFGF, LIF is the best for NSCs purification. Both induciton media can induce NSCs to differentiate.Retina NSCs have higher potentials to differentiate into retinal neuroepithelial cells than brain NSCs.
Objective To investigate the viability and the characters of proliferation and differentiation of retinal stem cells (RSCs) after cryopreservation and anabiosis. Methods The RSCs of a Long Evans rat with the embryonic age of 17 days were separated and cultured in vitro. The third-passage RSCs in the cryopreservation liquid consisted of 80% Dulbecco modified Eagle medium (DMEM)/F12，10% bovine serum albumin (BSA)，10% dimethylsulfoxide (DMSO)，and basic fibroblast growth factor (bFGF) (20 ng/ml), were stored in liquid nitrogen. After 1, 2, 4, 8, 12, and 16 weeks of freezing period, these cells were thawed. The livability of the cells was counted and the differentiation was induced while the proliferation and characters of differentiation were detected by immunofluorescence. Results The effects of different durations of cryopreservation on the livability of RSCs did not differs much (Pgt;0.05). These cells were reculturd well and presented specific marker of RSCs. In addition, they also could be induced and differentiated into several types of retinal cells. Conclusion Cryopreservation and anabiosis of RSCs does not affect the cellular intrinsic characters of proliferation and differentiation. (Chin J Ocul Fundus Dis, 2007, 23: 94-97)
Objective To establish a culture method for human fetal retinal progenitor cells (RPC) in vitro. Methods Retinal neuroepithelium of 8-12-week human fetal were isolated and cultured in suspension and adherent methods. The passage cells were cultured and differentiated for 14 days with 5% fetal bovine serum without basic fibroblast growth factor (bFGF). The expressions of RPC and retinal final cells markers before and after the differentiation were detected by immunohistochemical analysis. Results The isolated cells cultured in suspension method congregated as the neurospheres and expressed the neuroectodermal marker nestin, but failed in passage and expansion; while the expression of nestin and serial passage were found in the cells cultured in adherent way. The differentiated passage cells expressed retinal final cells markers including glial fibrillary acid protein, beta;-tubulin and recoverin. Conclusions RPC derived from human fetal neural retina at the 8th12th week of gestation are capable of expansion and multipotentiality. (Chin J Ocul Fundus Dis, 2007, 23: 98-100)
Objective To observe the effects of subretinal transplantation of rat mesenchymal stem cells (rMSCs) on Sodium Iodate (SI)induced retinal degeneration. Methods One hundred and twenty BrownNorway (BN) rats were divided into three groups including SI injection group,rMSCs transplantation group and normal control group, each with 40 rats. The retinal degeneration was induced by caudal vein injection of SI. The retinal pigment epithelium（RPE）and neural retinal were evaluated by ocular fundus photograph, fluorescein fundus angiography (FFA)，electroretinogram (ERG) and histological approach, and TUNEL（terminal deoxynucleotidyl transferasemediated dUTP nick end labeling ）. CMDiIprelabeled primary rMSCs were transplanted into the subretinal space of SIinduced rats. The survival, integration, and differentiation of rMSCs were observed between 14 day to 60 day after the transplantation.Results The rat retinal function was gradually reduced after14 days of SI injection, with a timedependent manner. After the RPE cells were damaged，the outer segments of photoreceptors became disrupted and shortened until karyopyknosis. The nuclear morphology and positive TUNEL labeling indicated that the death of photoreceptor cells was apoptosis. After rMSCs transplantation, CMDiI labeled donor cells were observed to be scattered in the subretinal space and expressed RPE cell markers. Average amplitude of b wave and Ops (oscillation potential) in ERG improved 27.80%,59.38% respectively after rMSCs transplantation.Conclusions Transplanted rMSCs can survive in subretinal space and differentiate into RPE.
Objective To investigate the feasibility of differentiation of invitro induced rat bone marrowderived mesenchymal stem cells（rMSCs） into retinal pigment epithelial （RPE） cells.Methods The rMSCs from BrwonNorway (BN) rats were isolated and cultured by adherent screening method. RPE cells lysate made by repeated freezethawing was put into the rMSCs culture system to identify whether the induced cells could express characteristic label cytokeratin（CK）and S-100 simultaneously or not.Results The growth rate of rMSCs induced by RPE cells lysate was slower and protuberant burr surrounded the fusiform cells. The results of immunoblotting and double immunofluorescence showed that partial induced cells expressed CK and S-100 simultaneously. The result of flow cytometry indicated that 14.1% induced cells expressed CK and S-100 simultaneously.Conclusion Induced by RPE cells lysate, rMSCs can differentiate into RPE cells.