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 After being withdrawn LIF, the characteristic of cells were identified by immunohistochemistry and RT-PCR.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 Two days after being withdrawn the LIF, cells expressed alpha-SMA in protein and mRNA levels.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.
To investigate the processes of retinal vascular development of human fe-
tus. METHODS:Eighty-six specimens of retinas of human fetus from 13th to 38th gestational week were studied by immunohistochemieal method(ABC). RESULTS:The spindle cells of mensenchymal origin was firstly found to migrate into the retina from optic disc at 12--13 weeks ,and spreaded subsequently toward the periphery of the retina. At the same time ,the consequential processes of differentiation,proliferation,canalilization and remodelling developed into vascular plexus in ganglion cell layer (GCL). Vascular buds formed in GCL in 26th gestational week extended toward nerve fiber layer (NFL)and inner nuclear layer(INL)and developed into capillary plexuses in NFL and inner and outer margin of INL respectively. CONCLUSIONS.. Four vascular layers could be distinguished in the ratina of full term fetuses,and these layers were formed through two principal developmental processes.
（Chin J Ocul Fundus Dis,1996,12: 88- 90）
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)
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)
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 evaluate the clinical results of repair of bone defect by embryonic bone transplantation. METHODS From January 1994 to June 1999, 148 cases of bone defect were repaired by embryonic bone transplantation following alcohol treatment, there were 63 cases with bone cyst, 42 cases with fibrous dysplasia of bone, 26 cases with giant cell tumor of bone, and 17 cases with enchondroma among them. The maximal bone defect was 3.5 cm x 10.0 cm, while the minimal defect was 0.5 cm x 1.0 cm. RESULTS All of those bone defect with benign tumor were bone union used by embryonic bone transplantation after 3 months to 1 year of operation, the average healing course was 6.2 months, followed up 1 to 6 years, averaged 14 months, no tumor recurrence and no obvious local or system response were observed. CONCLUSION Embryonic bone can be used as a good repairing material of postoperative bone defect of benign tumors, the clinical results are satisfactory.
Objective To observe the expression of miR-204 and 211 human embryonic stem cells (hESCs) differentiated into retinal pigment epithelial (RPE) cells. Methods RPE cells were derived from hESCs by natural differentiation method, and were identified. miRNA expression profiles and real-time polymerase chain reaction (RT-PCR) of miR-204 and 211 were generated from the following groups: hESCs, hESCs-derived cells containing pigmented foci, hESCs-derived RPE cells and human fetal RPE (hfRPE) cells. Results miRNA-204 was continuously upregulated throughout the entire differentiation process of hESCs to RPE cells. It increased 5.026 times in hESCs-derived cells containing pigmented foci compared to hfRPE cells; it was increased 3.337 times in hESCs-derived RPE cells compared to hESCs-derived cells containing pigmented foci; it increased 13.574 times in hfRPE cells compared to hESCs-derived RPE cells. miR-211 does not change during differentiation from hESC to RPE, but it increased 44.333 times in hESCderived RPE cells compared to hfRPE cells. miR-211 was the biggest difference in the miRNA expression pattern. In four cell types of hESCs, hESCs-derived cells containing pigmented foci, hESCs-derived RPE cells and hfRPE cells, RT-PCR showed the levels of miR-204 were 91.81plusmn;4.43, 2263.09plusmn;206.39, 5996.80plusmn;235.42, and 171676.45plusmn;999.82 respectively. miR-204 was significantly increased during the whole course (t=18.22, 20.66, 279.38;P＜0.001). The levels of miR-211 were 2.23plusmn;0.31, 129.33plusmn;3.75, 125.7592plusmn;4.78, and 16682.00plusmn;352.97 respectively. miR-211 was significantly increased from hESCs to cells containing pigmented foci and from hESCs-derived RPE cells to hfRPE (t=58.58, 81.24; P＜0.001). Conclusion There is a continuous change of miR-204 and 211 in differentiation of RPE cells from hESCs.
Objective To explore an optional condition to induce mouse embryonic stem cell(ESC) to differentiate into endothelial cells so as to provide seedcells for tissue engineered vascular. Methods The embryos from one pregnant 12.5days mouse was harvested to culture the mouse embryonic fibroblasts(MEF). The ESC was reanimated by common method, and used to cultured into embryoid body(EB) in vitro. The EB which was used to induce into endothelial cells was divided into two groups. The EB was cultured in the EB medium with 3ng/ml transforming growth factor β1, 50 ng/ml vascular endothelial cell growth factor and 1 μmol/L potent and selective inhibitor of activin receptorlike kinase receptors in experimental group. The EB was cultured in the EB medium in the control group. After 14 days, RTPCR and immunohistochemistry were used to detect vWF and CD34, to analyze the morphology and type of the differentiated cells fromESC. Results The primary MEF had a high proliferation activity. At the 3rdday, the fusion rate of MEF was about 90% with a fusiform shape. The cells was fusiform shape and arranged compactly with fullness of nucleus and 2-3 entoblasts. The 3rd5th generations EB was polygonal with fullness of cytoplasm and 3-4 entoblasts. ESC could maintain undifferentiated state, and the cells unit lookedlike bird nest with smooth margin; the cells was small at size and b refractivity with high rate of nuclein and rapid proliferation. At 3 days of dropculture, EB can seen grossly and at 3 days of suspension, large and transparent EBformed. EB was spread radiately with an intensive adhesion at the 2nd day. In experimental group, many round cells was differentiated around EB from the 4thday to the 7th day, and form tubular structures from the 10th day to the 14th day. The vWF and CD34 were expressed. In control group, EB could not form tubularstructures, and the vWF and CD34 were not expressed. Conclusion ESC can differentiate into endothelial cells under some conditions, and form vessellike structure under condition culture, which can provide sources of seed cells for tissue engineered vessel.
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.
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.
