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)
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.
Objective To review the latest development of the research on the selfrenwal signaling pathway and culture system in vitro of the embryonic stem cells(ESCs). Methods The recent articlesabout the selfrenewal signaling pathway and culture system in vitro of the ESCs were extensively reviewed. Results Understanding of the molecular mechanism of the selfrenewalin vitro and pluripotency of the ESCs was considered important for developing improved methods of deriving, culturing and differentiating these cells into the cells that could be successfully used in the clinical practice. Conclusion A further research is needed to elucidate the selfrenewal signaling pathway and the pluripotency of the ESCs and the culture systemin vitro forthe human ESCs remains to be further improved and developed.
Diabetic retinopathy is a serious complication of diabetes and is the leading cause of blindness in people with diabetes. At present, there are many views on the pathogenesis of diabetic retinopathy, including the changes of retinal microenvironment caused by high glucose, the formation of advanced glycation end products, oxidative stress injury, inflammatory reaction and angiogenesis factor. These mechanisms produce a common pathway that leads to retinal degeneration and microvascular injury in the retina. In recent years, cell regeneration therapy plays an increasingly important role in the process of repairing diseases. Different types of stem cells have neurological and vascular protection for the retina, but the focus of the target is different. It has been reported that stem cells can regulate the retinal microenvironment and protect the retinal nerve cells by paracrine production, and can also reduce immune damage through potential immunoregulation, and can also differentiate into damaged cells by regenerative function. Combined with the above characteristics, stem cells show the potential for the repair of diabetic retinopathy, this stem cell-based regenerative therapy for clinical application provides a pre-based evident. However, in the process of stem cell transplantation, homogeneity of stem cells, cell delivery, effective homing and transplantation to damaged tissue is still a problem of cell therapy.
ObjectiveTo study the characteristics of the human umbilical cord perivascular cells (HUCPVC) isolated from human first trimester umbilical cord perivascular layer tissues and the differentiation into islet-like cell clusters in vitro. MethodsThe HUCPVC derived from human first trimester umbilical cord which was donated by the volunteers were isolated and subcultured. The surface markers such as stage-specific embryonic antigen 1 (SSEA-1), SSEA-3, SSEA-4, OCT-4, TRA-1-60, and TRA-1-81 were detected by immunohistochemical method. The first trimester HUCPVC were induced to embryoid bodies (EB)-like cell aggregations and islet-like cell clusters in vitro through a simple stepwise culture protocol (5 steps). The expressions of specific markers[α-fetoprotein (AFP), Nestin, and smooth muscle actin (SMA)] were measured by immunohistochemical method; and the ability of glucose-stimulated insulin secretion was analyzed. ResultsThe first trimester HUCPVC were successfully isolated and could be passaged steadily more than 10 generations, which expressed SSEA-3, SSEA-4, OCT-4, TRA-1-61, and TRA-1-81. The first trimester HUCPVC were successfully induced into EB-like cell aggregations and islet-like cell clusters. The EB-like cell aggregations could express markers of three germ lineages:AFP, Nestin, and SMA. The islet-like cell clusters could release insulin significantly in response to elevated concentrations of glucose in vitro (t=7.444, P=0.002). The insulin contents were (23.2±5.3) mU/L and (7.0±0.5) mU/L in high and low glucose media, respectively. ConclusionThe first trimester HUCPVC has the ability to differentiate into islet-like cell clusters which can secret insulin in vitro.
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 observe the effect of TGF-β receptor inhibitor Compound C on the directed differentiation of human embryonic stem cells (hESC) into retinal pigment epithelial (RPE) cells. MethodsH1 hESC were divided into control group and experimental group. When the hESC reached over confluence, the medium was changed to knockout serum replacement medium without bFGF to induce RPE differentiation. The experimental group was supplemented with 1 μmol/L TGF-β receptor inhibitor Compound C at the first six days of induction. Real-time PCR was carried out to examine the expression of paired-box gene 6 (PAX6), microphthalmia-associated transcription factor (MITF), cellular retinaldehyde blinding protein (CRALBP), and RPE65 in both groups at the 1, 3, 5 weeks of the induction process. hESC-derived RPE (hESC-RPE) cells were isolated mechanically and purified. Real-time PCR, Western blot and immunofluorescence were used to characterize the purified hESC-RPE cells. ResultsPigmented colonies were observed in experimental group at the 4 weeks of the induction process, while no pigmented colony could be detected in the control group. All the purified pigmented cells from experimental group showed polygons morphology. Experimental group showed significantly higher expression of RPE marker genes PAX6, MITF, CRALBP and RPE65 than the control group(P<0.05). Compared with the hESC and ARPE-19 cells line, purified hESC-RPE cells showed much higher expression of PAX6, MITF, CRALBP and RPE65(P<0.05).High expression level of PAX6 and RPE65 proteins were observed in hESC-RPE cells. Immunofluorescence verified the expression of PAX6 and ZO-1 in hESC-RPE cells. ConclusionTGF-β receptor inhibitor Compound C significantly improved the differentiation efficiency of hESC into RPE.
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)
The capacity of embryonic spinal cord tissue in the repair of injured structure of spinal cord has been noted for years. In order to investigate the embryonic spinal cord graft in the repair of motor function of injured spinal cord, the embryonic spinal cord tissue was transplanted to the hemisection cavity in spinal cord in adult rat. One hundred adult Wistar Rats were used to simulate the hemisectional injury of spinal cord by drilling 2-3 mm cavity in lumbar enlargement. Sixty rats were treated with rat embryonic spinal cord tissue grafting while the other forty were chosen as control. The outcome was evaluated according the combined behavioural score (CBS) and motor evoked potential (MEP) in the 1, 2, 4 and 12 weeks. The grafting group was superior to the control as assessed by CBS (P lt; 0.05), especially within 4 weeks. (P lt; 0.01). The restoration of the latent peak of early wave(P1, N1) was better in the grafting group, too. This suggested that embryonic spinal cord graft could improve the recovery of motor function of injured spinal cord in adult rat. The effect of the embryonic spinal cord tissue graft might be concerned with its secretion of several kinds of neurotrophic factors, nerve growth factor, nerve transmitted factor, or adjustment of hormone.
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)