Objective To induce embryonic stem cell (ESC) to differentiate into endothel ioid cells using a simple adhesive culture method, and to provide a new cells seed source for vascular tissue engineering or cell therapy. Methods SV129-derived ESC were seeded at 2 × 104/cm2 and maintained undifferentiated on ESC culture medium in the presence of 1 000 U/mL leukaemia inhibitory factor (LIF). Embryoid body (EB) formatted when ESC cultured in suspension in the lack of LIF. At 4 days, EB was transferred to 0.1% gelatin coated dish and cultured with medium supplementary of VEGFto be induced differentiation. The characteristics of differentiated cells were determined by immunohistochemistry staining, flow cytometry (FCM), 1, 1-dioctadecyl-3, 3, 3, 3-tetramethyl indocarbocyanine-labeled acetylated low density l ipoprotein (DiIAc- LDL) takeup test, and TEM detection. Results Differentiated cells were morphologically characterized as endothel ial cells. They could takeup DiI-Ac-LDL, be stained positive by Flk-1 and CD31. The CD31 positive cells reached above 90% when measured by FCM. Furthermore, Weibel-Palade bodies were detected and tight junctions were found when differentiated cells were examined by TEM. Conclusion Using a simple adhesive culture method and by suppl ied with VEGF alone, ESCs can be induced to differentiate into endothel ioid cells. The differentiation method is simple and economic, and can provide seed cells for vascular tissue engineering or cell-therapy.
Objective To investigate the effect of 1,25(OH)2VD3 on differentiation of embryonic stem cells (ESCs) into osteoblasts. Methods Osteoblasts were isolated and cultured from calvarium of 2-day-old Kunming white mice, embryoid bodies (EBs) were prepared with modified zur Nieden method. EBs were divided into 4 groups according to different mediums: group A, as the control group, in which EBs medium contained no leukemia inhibitory factor; group B, in which EBs medium contained supplements of Vitamin C (VC, 50 μg/mL) and β-glycerophosphate (β-GP, 50 mmol/L); group C, inwhich EBs medium was the same as that of group B and 5 × 104 osteoblasts of 3rd passage were seeded into each well; group D, in which the medium contained supplements of VC (50 μg/mL), β-GP (50 mmol/L) and 1,25(OH)2VD(4 × 10-9 mol/L), and 5 × 104 osteoblasts of 3rd passage were seeded into each well. The ALP activity was determined by ALP reagent kit every 5 days. The RQ-PCR was performed to measure the mRNA expressions of osteocalcin (OCN). Al izarin red S staining was performed to count the bone nodules. Results The expression of ALP witnessed no obvious change in each group within 5 days after adherence of EBs, but increased gradually after 5 days. The expression of ALP in group D reached the peak at 20 days. Red nodules with clear outl ine and different sizes were evident by microscope. Al izarin red S staining testified the number of bone noudles in groups A, B, C and D was 20 ± 8, 18 ± 5, 31 ± 1 and 50 ± 1, respectively, indicating significant differences between groups C, D and groups A, B (P lt; 0.05), no significant difference between group A and group B (P gt; 0.05), and a significant difference between group C and group D (P lt; 0.05). The result of RQ-PCR showed that the mRNA expressions of OCN in groups A, B, C and D was 10.18 ± 1.17, 20.29 ± 1.03, 18.84 ± 4.07 and 32.15 ± 5.23, respectively, indicating significant differences between groups C, D and groups A, B (P lt; 0.05), no significant difference between group A and group B (P gt; 0.05), and a significant difference between group C and group D (P lt; 0.05). Conclusion The combined action of 1,25(OH)2VD(4 × 10-9 mol/L), VC, and β-GP can effectively promote the differentiation of the ESCs-derived osteoblasts.
Objective To review the progress, methods and obstacles in the differentiation of embryonic stem cells(ESCs) into osteoblasts in vitro. Methods The recent literature concerned with the differentiation of ESCs into the osteoblasts was extensively reviewed and briefly summarized. Results ESCs was a good tool for derivation of obsteoblasts.Conclusion The study on the induction of ESCsinto the osteogenic lineage provides a model for analyzing the molecular processes of osteoblasts development in vivo and establishes the foundation for the use of ESCs in skeletal tissue repair.
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.
Objective To observe the effect of transplantation of embryonic stem cell(ES) on neurological functional recovery of injured spinal cord in adult mouse. Methods The ES cells were cultured and induced in vitro. Fifty C57/BL6J mice were made animal model of semicut mice of T9，10. The ES cellderived neural precursors cells were transplanted into the vertebral canalaround injured spinal cord semi-cut mice. Twenty-eight C57/BL6J mice were randomly divided into three groups: sham operation group(group A,n=9), operation／cell group (group B，n=10), and operation／DMEM group(group C,n=9). RT-PCR analysis, X-gal staining and immunofluorescence were used to observe the cells survival and differentiation in the spinal crod. BBB test was performed to study functional improvement. Results ES cells induced and cultured in vitro displayed clonal growth with circle or ovoid shape and had one or more nucleoli. RT-PCR result showed that the induced ES cells expressed mRNA of Nestin and microtubuleassociated protein, but did not express glial fibrillory acidic protein(GFAP). There was statistically significant difference in BBB scoring between group A and groups B, C after operation (P＜0.01). There was statistically significant difference in BBB scoring at 1, 2 and 4 weeks of operation(P＜0.01), but no statistically significant difference at 6 and 8 weeks of operation between groups B and C(P＞0.05). The X-gal staining results werepositive in group B and negative in groups A and C. The immunoflurescence resultshowed neurofilament green fluor and no expression of GFAP in injured spinal cord region. Conclusion After transplantation, ES cellderived cells can survive, transfer into the injury position, and differentiate into neurons, but spinal cord function has no obvious improvement.
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.
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.
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.