Objective To establish a culture system in vitro of fetal and adult human retinal neural cells provide a model for the basic research of retinal neural cells and the medicinal exploitation. Methods Fetal human retinas(10~13 weeks after conception) and adult human retinas(20~40 years old) were dissected, dissociated, and put into culture plate which was coated with polylysine or rat tail gel. Specific growth factor EGF、FGF、BDNF or NT-4 were added to the culture medium. BrdU incorporation, Tunnel assessment and immuno-histochemistry and immuno-fluorescent staining were applied to determine cells proliferation, apoptosis and identify the component of cultured cells. Results Fetal human retinal cells and adult human retinal cells survived for up to 100 and 180 days in vitro. The addition of EGF、FGF、BDNF or NT-4 promoted the survival of both fetal and adult retinal neurons and stimultated proliferation of fetal retinal cells. The neurons or the rate of ganglion cells was observed with higher percentage in the group with growth factor adding than the group without. Conclusion Fetal and adult human retinal cells can be maintained in vitro and the fetal cells also can be expanded, which are helpful to generate retinal neurons for basic research and drug exploitation. The exogenous growth factors added to the culture medium can promote survival, proliferation and differentiation of retinal cells in culture. (Chin J Ocul Fundus Dis, 2002, 18: 279-282)
In order to repair cartilage defect in joint with transplantation of cryopreserved homologous embryonic periosteum, 30 rabbits were used and divided into two groups. A 4 mm x 7 mm whole thickness cartilage defect was made in the patellar groove of femur of each rabbit. The homologous embryonic rabbit skull periosteum (ERSP), preserved in two-step freezing schedule, was transplanted onto the cartilage defect of joints of one group and autogenous periosteal graft was done in the joint defect of the other group. The knees were not immobilized, following operation and 16 weeks later, the newly formed tissue in the defects were assessed by gross observation, histochemical examination and biochemical analysis. The results showed that new hyaline-like cartilage was formed in the cryopreserved ERSP grafted knee, and had no significant difference from that of the knee receiving autogenous periosteal graft, but had significant difference from that of the fresh ERSP grafted knee and the non-grafted knee. Furthermore, the new hyaline-like cartilage had the biochemical characteristics of a fibrous cartilage. The conclusion was that this method might be feasible to repair articular cartilage defects.
Objective To investigate the possibility of ectomesenchymal stem cell of human embryo facial process in differentiating into osteoblasts.Methods Ectomesenchymal stem cells of human embryo facial process were isolated and cultured in mineralized promoting solution containing 10 mmol/L β-glycerophosphate, 100 μg/ml ascorbic acid and 10 nmol/L dexamethasone supplemented with 15% FBS. The morphological change was observed by phase contrast microscopy. The characteristics of cells was identified by immunohistochemistry assay. Alkaline phosphatase activity was tested and the form of mineralized nodules was tested with Von Kossa staining. The expression of osteocalcin was identified by RT-PCR.Results There were significant changes in the shape of the cells after 3 days cultured in mineralized promoting solution. The cells became larger and the shape changed from fibroblast-like to multilateral. The result for anticollogen typeⅠstaining was positive. The alkaline phosphatase activity increased. Mineralized nodules were formed aftercultured 25 days by Von Kossa staining. RT-PCR assay showed induced cells expressed osteocalcin.Conclusion Ectomesenchymal stem cells of humanembryo facial process can be induced to differentiate into osteoblasts by mineralized promoting solution.
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.
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.
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.
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)
There are over 8 million blind patients in China, 1/3 of them are suffered from retinal degeneration diseases. Stem cells transplantation can delay the photoreceptor cell degeneration or replace the dead photoreceptor cells, provides hopes for these patients. How to make enough seed cells is the major barrier for cell therapy. Good seed cells should be safe and with great pluripotency, and can be made from a wide range of sources, easy to be standardized and industrialized. Seed cells made from three-dimensional embryonic stem cells cultures can reach the above criteria, thus three-dimensional embryonic stem cell culture is a new strategy for making seed cells for cell treatment of blind diseases.
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.
ObjectiveTo investigate the effects of over expression of Mash-1 gene on the differentiation of embryonic stem cells (ESC) into neural cells in vitro. MethodsThe ESC of rats (CE3 cells) were transfected with MSCVMash- 1 (MSCV-Mash-1-CE3 group) or MSCV (MSCV-CE3 group). The expression of Mash-1 gene was detected by RT-PCR. After transfection, hanging-drop culture was used to form embryonic bodies, and then embryonic bodies were cultured with neural induction medium. The cell morphology was observed under inverted phase contrast microscopy at 7 and 21 days; the positive rates of neural stem cells marker protein (nestin) and neuron marker protein (β-tubulin Ⅲ) were measured by immunofluorescence staining after cell attachment; and the gene expressions of α-fetal protein (AFP), Brachyury, fibroblast growth factor 5 (FGF-5), Oct3/4, nestin, and β-tubulin Ⅲ were detected by real-time fluorescence quantitative PCR at 0, 1, 7, 14, and 21 days after culture. The CE3 cells were used as control (CE3 group). ResultsCompared with MSCV-CE3 and CE3 groups, the expression of Mash-1 gene in MSCV-Mash-1-CE3 group was significantly increased. At 7 and 21 days after neural induction cultured, cells in MSCV-Mash-1-CE3 group had axons growth and showed neural stem cell-like and neuron cell-like morphology (unipolar, bipolar, and multipolar neurons), but few cells had axons growth in MSCV-CE3 and CE3 groups. The positive rates of nestin at 7 days and β-tubulin Ⅲ at 21 days in MSCV-Mash-1-CE3 group were significantly higher than those in MSCV-CE3 and CE3 groups (P<0.05). Real-time fluorescence quatitative PCR results showed that the gene expression of Brachyury was significantly decreased after 1 day (P<0.05), and the gene expressions of FGF-5 and nestin were significantly increased after 1 day (P<0.05) in MSCV-Mash- 1-CE3 group when compared with CE3 and MSCV-CE3 groups; the gene expression of β-tubulin Ⅲ was significantly increased after 7 days (P<0.05). There was no significant difference in above indexes between CE3 and MSCV-CE3 groups (P>0.05). The expressions of AFP and Oct3/4 showed no significant difference among groups at each time point (P>0.05). ConclusionOver expression of Mash-1 gene can promote differentiation of ESC into neural cells in vitro.