Purpose To investigate the development of embryonic stem cells (ESC)in the subretinal space． Methods ESC were cultivated in suspension for 4 days till they developed into cell aggregates,i.e.embryonic body(EB).ESC as well as EB combined with or without RA were respectively transplanted into vitreous cavity and subretina1 space in SD rats,and the subretinal transplanted eyes,transient ischemia-reperfusion injuries were made by ligating the ophthalmic artery for 40 seconds before the transplantation .The experimental eyes were enucleated for histological and immunohistochemical assays after 14～28 d. Results The EB was found to develope into photoreceptors induced by RA in the subretinal space under an ischemia-reperfusion condition，and EB transplantation without RA induction induced multiple differentiations in the subretinal space.The single injection of RA without EB induced hyperplasia of the neural retinal cells.ESC transplanted into vitreous cavity rapidly proliferated and developed into atypical hyperplastic mass. Conclusion EB derived from ESC can differentiate into photoreceptors induced by RA in the host subretinal space under an ischemia-reperfusion condition. (Chin J Ocul Fundus Dis，2000，16：213-284)
Purpose To investigate the characteristics of intraocular growth of mice embryonic stem cells (ESC) in nude mice. Methods The undifferentiated murine ESC in vitro were transplanted into the eyes of nude mice.Mophological and immunohistochemical examinations were implemented. Results Two to three days after transplantation,yellowish-white granules and masses were seen inside the anterior chamber and vitreous cavity and enlarged gradually.Morphological examination showed that there were undifferentiated cells and differentiated cells in anterior chamber and vitreous cavity.The morphology and alignment of some differentiated cells were similar to those of the retina of nude mice.The cells were highly positive in NSE staining. Conclusion The transplanted ESC could grow in the eyes of nude mice and differentiate into neurons and retina-like structure. (Chin J Ocul Fundus Dis，2000，16：213-284)
OBJECTIVE: To investigate the characteristic and phenotype of ectomesenchymal stem cells of human fetal facial processes and the procedure of spontaneous differentiation to smooth muscle cells. METHODS: The primary ectomesenchymal cells of E 50 human fetal facial processes were isolated by 2.5 g/L trypsin and cultured with DMEM/F 12 with 10(-6) U/L leukemia inhibitor factor(LIF). The morphology and growth rate were observed by inverted microscop. After being withdrawn LIF, the characteristic of cells were identified by immunohistochemistry and RT-PCR. Ultrastructure was observed by transmission electron microscope. RESULTS: The cultured cells displayed monolayer growth and were fibroblast-like with 2-4 processes. The cells were stainely positived for anti-human natural killer cell marker-1, Vimentin, S-100, neuron specific enolase, myoglobin and VIII factor, but negatively for glial fibrillary acidic protein, neural fiblament, alpha-SMA and cytokeratin in immunohistochemistry. Two days after being withdrawn the LIF, cells expressed alpha-SMA in protein and mRNA levels. The cells were rich in muscular filament-like structure and dense bodies under transmission electron microscope. CONCLUSION: Cultured cells are undifferentiated ectomesenchymal stem cells. The cells have the potential for differentiating spontaneously to smooth muscle cell.
ObjectiveTo investigate the impact of L-Phenylalanine on the efficiency of retinal pigment epithelial (RPE) cell derivation from human embryonic stem cells (hESCs) and explore the underlying mechanisms. MethodsH1 hESCs were routinely cultured with mTeSR medium and divided into control and experimental groups. When cells reached over-confluence, spontaneous differentiation was triggered using 10% KSR differentiation medium without bFGF. L-Phenylalanine (0.2 mmol/L) was supplemented in the experimental group from the 3rd week. The expression of RPE markers and Wnt signaling components in the two groups was detected by Real time-RCR, Western blot and Flow cytometry analyses. Purified hESC-RPE cells and PBS were injected into the subretinal space of sodium iodine-induced retinal degeneration rats separately. Retinal function was assessed by ERG 6 weeks after the transplantation. ResultsOn the 7th week, much more pigment cell clumps appeared in the experimental group compared to the control group. Within these areas there were monolayer hexagonal RPE cells full of pigment granules. The experimental group showed significantly higher expression of Pax6, MITF, Tyrosinase, RPE65, Wnt3a, Lef1 and Tcf7 genes than the control group (P < 0.01). Higher expression level of MITF and RPE65 proteins and higher percentage of RPE65 (+) cells (P < 0.01) were detected in the experimental group. 6 weeks after sub-retinal transplantation of hESC-RPE cells, the amplitudes of a-b wave in the transplanted eyes were significantly higher than those in the control eyes (P < 0.01) at the stimulus intensity of 3.0 cd·s/m2. ConclusionsL-Phenylalanine effectively promoted the differentiation of embryonic stem cells into retinal pigment epithelial cells, and its impacts on the Wnt/β-catenin signaling pathway may partially explain the underlying mechanisms. Subretinal transplantation of hESC-RPE remarkably improved the retinal functions of retinal degenerative animal models.
Retinal degeneration mainly include age-related macular degeneration, retinitispigmentosa and Stargardt’s disease. Although its expression is slightly different, its pathogenesis is photoreceptor cells and/or retinal pigment epithelial (RPE) cel1 damage or degeneration. Because of the 1ack of self-repairing and renewal of retinal photoreceptor cells and RPE cells, cell replacement therapy is one of the most effective methods for treating such diseases.The stem cells currently used for the treatment of retinal degeneration include embryonicstem cells (ESC) and various adult stem cells, such as retinal stem cells (RSC), induced pluripotent stem cells (iPSC). and mesenchyma1 stem cells (MSC). Understanding the currentbasic and clinical application progress of ESC, iPSC, RSC, MSC can provide a new idea for the treatment of retinal degeneration.
观察受体鼠妊娠和胚胎着床情况,并检测胚胎移植时小鼠子宫内膜中白血病抑制因子(Lif)表达水平,探讨超排卵对小鼠胚胎着床潜能的影响。方法:建立超排周期胚胎和自然周期胚胎移植小鼠模型,比较妊娠率、胚胎着床率的差异及其与Lif蛋白的表达水平之间的关系。结果:超排卵周期受体组的妊娠率(20.00%)和胚胎着床率(8.33%)显著低于自然周期组的妊娠率(55.00%)和胚胎着床率(35.00%)(P＜0.05)。自然周期胚胎和超排周期胚胎受体组内膜中Lif蛋白的表达水平相似(P＞0.05),妊娠受体组Lif蛋白的表达水平显著高于未孕受体组(P＜0.05),但单胎妊娠和多胎妊娠受体组内膜中Lif蛋白的表达水平相似(P＞0.05)。结论:超排卵可能降低胚胎的着床潜能,Lif蛋白的表达水平与胚胎着床有关,但与着床胚胎的数目无比例关系。
Objective To investigate the possibility of commitment differentiation of embryonic stem cells induced by the medium of cultured retinal neurons of SD rats. Methods The medium from cultured retinal neurons of SD rats were collected, sterilized and mixed with DMEM medium according to 2∶3 proportion, ES cells were cultured with these mixed medium and were observed under the phase contrast microscope daily, the induced cells were identified by NF immunohistochemistry methods. Results The ES cells cultured with these mixed medium can differentiate into neuron-like structure, and the induced cells were positive in NF immunofluorescence staining. Conclusion The medium from cultured retinal neurons of SD rats can induce ES cells commitment differentiation into neuron-like structure. (Chin J Ocul Fundus Dis, 2002, 18: 134-136)
Objective To study the culture and purification of the fetal mouse liver mesenchymal stem cells(MSCs) in vitro and to investigate their differentiation potential and the composite ability with true bone ceramic(TBC). Methods The single cell suspension of MSCs was primarily cultured and passaged, which was prepared from the fetal mouse liver; the flow cytometry was applied to detectCD29, CD34, CD44 and CD45. The osteogenic differentiation was induced in chemical inducing system; the osteogenic induction potency was tested. The purified fetal mouse liver MSCs were compounded with TBC covered with collagen type Ⅰ in vitro and the cell attachment and proliferation to the TBC were observed. Results The primary MSCs of fetal mouse liver were easy to culture in vitro. They proliferated well and were easy to subcultured. The proliferation ability of primary and passaged MSCs was similar. Flow cytometric analysis showed the positive results for CD29, CD44 and the negative results for CD34, CD45. After 7 days of induction, the MSCs expressed collagen type I and alkaline phosphatase(ALP) highly. After 14 days of induction, the fixed quantity of ALP increased significantly. After 28 days of induction, calcium accumulation was observed by Von Kossa’s staining. Many liver MSCs attached to the surface of TBC. Conclusion The MSCs of the fetalmouse liver can be obtained, subcultured and purified easily. After culturing in chemical inducing system, the MSCs of fetal mouse liver can be successfully induced to osteoblast-like cells, attach to the surface of TBC and proliferate well.
ObjectiveTo analyze the electro-clinical characteristics and surgical outcome of low-grade developmental tumors in temporal lobe. MethodsThe onset age, seizure duration, seizure types, electroencephalogram and surgical outcome of 49 patients with low-grade developmental tumor of temporal lobe were analyzed retrospectively. ResultsTwo groups of the seizure types were divided. The first group was spasm, the other was focal onset. There were 12 cases in spasm group, with an average onset age of (1.00±0.59) years. The discharge was extensive and multi-brain-area locaded, especially in the temporal montages and the ipsilateral posterior montages. There were 37 cases in second group, with an average onset age of (8.90±8.84) years, mainly including autonomic seizure, tonic seizure and automotor seizure. In this group, the discharge was mainly recorded in the temporal montages, which could spread to the frontal montages and less locaded in posterior montages. The difference of onset age between the two groups was statistically significant (P<0.01). The average follow-up of spasm group was (2.80±1.57) years, and the surgical outcome of all patients in this group were all Engel I (100.00%, 12/12). The focal onset group was followed up for an average of (6.50±4.78) years, and the rate of Engel I was 91.80% (34/37). There was no significant difference between the two groups (P>0.05). ConclusionsFor low-grade developmental tumors in temporal lobe, there are two seizure types, including spasm and focal onset. The onset age of spasm is earlier, while patients with focal onset mostly start at childhood or older, rare in infancy. Surgery has a good effect on the treatment of temporal lobe developmental tumor epilepsy.