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BACKGROUND:Cdh1 has been shown to express in rat hippocampus and cortex in a large number. Moreover, in vitro test demonstrated that Cdh1 expression was higher in neurons than in neural stem cel s, which possibly associated with the differentiation of neural stem cel s into neurons. However, the effects of anaphase promoting complex Cdh1 on ischemic neuronal damage remain unclear. OBJECTIVE:To investigate the expression of Cdh1 and its downstream substrate in primary cultured neurons with oxygen-glucose deprivation. METHODS:Primary neurons from cortex of postnatal 24-hour rat pups were cultured in vitro, and identified by immunofluorescence staining. The oxygen-and glucose-deprived models were established by three gas incubator fil ed with nitrogen in sugar-free Earle’s solution. After 1 hour of hypoxia, reoxygenation was conducted. Real-time fluorescent quantitative PCR was used to detect the mRNA expression of Cdh1 and its downstream substrates Skp2, Cyclin B1 before hypoxia, 6 hours, 1, 3, 7 days after oxygen glucose deprivation. RESULTS AND CONCLUSION:After oxygen glucose deprivation, the expression of Cdh1 and Cyclin B1 in primary neurons was increased (P<0.05), while Skp2 expression was decreased (P<0.05). Above data indicated that Cdh1 expression in neurons increased after oxygen-glucose deprivation. It may degrade Skp2 and participate in hypoxic neuronal apoptosis by ubiquitination.
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BACKGROUND:Ten-eleven translocation (TET) family proteins are recently discovered DNA dioxygenases that convert methylcytosine to hydroxymethyl cytosine, which is essential for regulating cel proliferation and differentiation, but the expression pattern of TET family proteins in human dental pulp cel s is stil unclear. OBJECTIVE:To investigate the expression pattern of TET family proteins during the differentiation of human dental pulp cel s. METHODS:Cel ular distribution and expression of TET family proteins were determined by immunofluorescence in human dental pulp cel s that were cultured and isolated using digestion method. The protein levels of TETs during cel passage (P1-P7) were detected with western blot assay, and their potential changes during odontogenic induction (7 and 14 days) were confirmed using real-time quantitative PCR and western blot analyses at mRNA and protein levels, respectively. RESULTS AND CONCLUSION:Al TETs were expressed in the nucleus and the cytoplasm of human dental pulp cel s During serial cel passage, TET1 protein expression was increased until the 6th passage, TET2 significantly increased at the 2nd and 3rd passages and then decreased (P0.05). Both mRNA and protein expression levels of al TETs were elevated during odontogenic induction (P<0.05). These results indicated that TETs may contribute to cel differentiation of human dental pulp cel s.
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BACKGROUND:Bone morphogenetic protein 9 is proved to promote the osteogenic differentiation of various kinds of stem cel s, but whether it can induce the osteogenic differentiation of dental fol icle cel s in vitro is yet unclear. OBJECTIVE:To investigate whether bone morphogenetic protein 9 can induce the osteogenic differentiation of rat dental fol icle cel s in vitro. METHODS:Purified rat dental fol icle cel s at passage 3 were transfected with bone morphogenetic protein 9 adenovirus. Then, alkaline phosphatase activity, calcium deposition and expression of osteogenesis-related factors at mRNA and protein levels were detected in the dental fol icle cel s. RESULTS AND CONCLUSION:After transfection with bone morphogenetic protein 9, the dental fol icle cel s showed continuously enhanced alkaline phosphatase activities and obviously enhanced calcium deposition. Real-time PCR results demonstrated that the mRNA expressions of alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin and core binding factor were increased significantly. The western blot assay showed that the expression of osteopontin enhanced in the dental fol icle cel s after transfection with bone morphogenetic protein 9. In summary, bone morphogenetic protein 9 can induce the osteogenic differentiation of dental fol icle cel s.
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BACKGROUND:Hematopoietic microenvironment regulates hematopoietic stem cel s mainly through the cel-cel , cel-soluble regulator and cel-extracel uar matrix modes. OBJECTIVE:To review the molecular mechanism underlying regulation of hematopoietic stem cel s under the cel-cel , cel-soluble factors and cel-extracel ular matrix modes. METHODS:A computer-based online search of PubMed database was performed to search related articles with the key words of“Niche, HSC, VCAM-1, VLA-4, TPO, MPL, ECM, Integrin, N-cadherin, ANG-1, Tie2, VLA-5, Jagged-1, Notch, CXCL12, CXCR4, SCF, Kit, BMPs/TGF-β, TGF-βR, IFNα, IFNαR”in English. Irrelevant or repetitive studies as wel as old literature were excluded, and final y 80 articles were included in result analysis. RESULTS AND CONCLUSION:Cel s on the endosteal surface, vascular endothelial cel s, perivascular cel s, and some unknown or certain cel s or smal molecules in the bone marrow cavity constitute the specialized microenvironment for hematopoietic stem cel s. Hematopoietic stem cel s in the hematopoietic microenvironment remain a relatively steady state, which is the result of mutual contact of hematopoietic stem cel s and hematopoietic microenvironment under the regulation of some important molecules, such as Pten and osteopontin, via the pathway between cel-intercel ular adhesions, intercel ular soluble factors, and cel s and extracel ular matrix.
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BACKGROUND:With the development of stem cel culture technology, stem cel transplantation has been a research hotspot in the biological cytology. OBJECTIVE:To review the current situation and progress of basic research on stem cel transplantation for human refractory diseases. METHODS:A computer-based search of CNKI, VIP, Wanfang and PubMed databases was performed for articles related to animal experiments of stem cel transplantation published from January 2008 to October 2014. The key words were“induced pluripotent stem cel s, neural stem cel s, top-up between stem cel s, transplantation”in Chinese and English in the title and abstract. Papers published in authoritative journals or recently were preferred. Total y 113 articles were searched initial y, and only 50 articles were included in result analysis. RESULTS AND CONCLUSION:With the progress of stem cel culture technology, basic research on the stem cel transplantation for human diseases have been promoted and carried out, and these researches have achieved phased outcomes that provide conditions for more extensive and in-depth fundamental research. It is believed that with the development of basic research on stem cel transplantation to solve a series of difficult problems, stem cel transplantation wil bring hope for treatment of human disease, especial y for treatment of refractory disease.
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BACKGROUND:Stable and efficient labeling of dental pulp stem cel s in vitro is most important in tracer technique, which is also the basis of tooth regeneration in vivo. OBJECTIVE:To determine the optimal condition and method for transfection of stem cel s derived from rat dental pulp with green fluorescent protein infection by lentiviral vector and to determine whether green fluorescent protein-labeled dental pulp stem cel s maintain their stem cel properties. METHODS:Rat dental pulp stem cel s were obtained by modified enzyme digestion method, to identify the immune phenotype and differentiation potential. Dental pulp stem cel s were infected with green fluorescent protein by lentiviral vector for 24 and 48 hours at different multiplicity of infection (MOI) (5, 10, 25, 50 and 100). The infection efficiency and fluorescence intensity were analyzed by inverted fluorescent microscopy. The clonal and proliferation ability, cel cycle and the mineralization potential were compared before and after transfection. Based on those mentioned above, we could evaluate the influence of infection on their biological characteristics. RESULTS AND CONCLUSION:Flow cytometry results showed that rat dental pulp stem cel s expressed STRO-1 and CD146 but not CD34 or CD45. The dental pulp stem cel s could differentiate into osteoblasts and adipocytes when cultured in specific medium for each lineage differentiation. The highest efficiency of infection and strongest fluorescence expression appeared at 48 hours of infection and MOI 50. There were no significant differences in growth ability, cel colony formation rate and cel cycle before and after transfection (P>0.05). And the alkaline phosphatase expressed positively. Infection for 48 hours at MOI 50 is optimal for transfecting dental pulp stem cel s with green fluorescent protein by a lentiviral vector, thereby providing reliable tracer method for the study of rat dental pulp stem cel s in vivo.
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BACKGROUND:Animal studies have indicated ultrasound-mediated microbubbles can significantly enhance the effect of stem cel transplantation to treat ischemic diseases. But its mechanism is stil unknown. OBJECTIVE:To explore the mechanism of ultrasound-mediated microbubbles to significantly enhance the effect of stem cel transplantation in the treatment of ischemic diseases. METHODS:Bone marrow mesenchymal stem cel s and vascular endothelial cel s of rats were cultured in vitro, and then randomized to three groups:control group with no intervention, ultrasound group exposed to ultrasound at 1 MHz, 1 W/cm2 for 90 seconds, and ultrasound-mediated microbubble group treated with 5μL liposomes ultrasound microbubbles containing fluorocarbon gases (about 2×1011/L) and ultrasound exposure at 1 MHz, 1 W/cm2 for 90 seconds. RESULTS AND CONCLUSION:Compared to the control group, ultrasound-mediated microbubbles significantly increased expressions of vascular endothelial growth factor and stromal cel-derived factor 1 in the supernatant of vascular endothelial cel s (P0.05). These findings suggest that 1 W/cm2 ultrasound-mediated microbubbles can promote vascular endothelial growth factor and stromal cel-derived factor 1 secretion by vascular endothelia cel s, and meanwhile promote CXCR4 gene expression in bone marrow mesenchymal stem cel s. This may be the mechanism of the ultrasound-mediated microbubbles enhancing homing effect of transplanted stem cel s.
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BACKGROUND:Bone marrow mesenchymal stem cel s are the ideal cel s for tissue repair. Whether the ability of in vitro proliferation can be enhanced is a key factor to promote tissue repair. OBJECTIVE:To observe the effect of transforming growth factorβ1 on the proliferation of bone marrow mesenchymal stem cel s in vitro. METHODS:Blood samples were taken from the central artery of rabbits to prepare platelet-rich fibrin by centrifugation method which was then placed into fresh DMEM at 37℃for 7, 14, 21, 28 days to col ect exudates. The mass concentrations of transforming growth factor-β1 in the exudates of platelet-rich fibrin were detected. Rabbit bone marrow mesenchymal stem cel s were col ected and cultured in the conditioned medium made by the exudates of platelet-rich fibrin, and the proliferation of cel s was observed. RESULTS AND CONCLUSION:Concentration of transforming growth factorβ1 was increased with time increasing, increased fastest at 21-28 days, and peaked at 28 days. Under the same stimulus concentration, the proliferation of bone marrow mesenchymal stem cel s was reduced at 0-1 day, increased obviously at 1-2 days, and entered into a steady phase at 2-3 days. Under 150 ng/L transforming growth factorβ1, bone marrow mesenchymal stem cel s proliferated fastest. Experimental findings indicate that with the increase of time, the concentration of transforming growth factorβ1 in the exudates of platelet-rich fibrin increase gradual y, and the conditioned media containing different concentrations of transforming growth factorβ1 play different roles in promoting the proliferation of bone marrow mesenchymal stem cel s. Bone marrow mesenchymal stem cel s cultured in the conditioned medium containing 150 ng/L transforming growth factorβ1 for 2-3 days can proliferate fastest.
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BACKGROUND:Bone marrow mesenchymal stem cel s from chickens are important cel models for embryonic developmental biology, immunology and oncology research. However, it is difficult to keep bone marrow mesenchymal stem cel s with good undifferentiated potential in a large-scale expansion system. OBJECTIVE:To establish a culture system in vitro with laminin coating to expand bone marrow mesenchymal stem cel s from chickens. METHODS:Isolated bone marrow mesenchymal stem cel s from chickens were seeded in laminin-coated plates and traditional two-dimensional plates, respectively. After expansion in vitro, the morphological characteristics, expression of surface markers, expansion characteristics and adipogenic differentiation of bone marrow mesenchymal stem cel s in both conditions were analyzed and compared. RESULTS AND CONCLUSION:There were no statistical differences in the morphological characteristics and expression of surface markers of bone marrow mesenchymal stem cel s expanded by laminin-coated plates and traditional two-dimensional plates. But, the expansion characteristics and adipogenic differentiation of bone marrow mesenchymal stem cel s cultured in laminin-coated plates were better than those in traditional two-dimensional plates. Laminin culture system could quickly amplify out of a large number of chicken bone marrow mesenchymal stem cel s with better proliferation ability and undifferentiated performance. Al above results indicated that a more efficient expansion system with laminin coating is established.
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BACKGROUND:Mesenchymal stem cel s as potential seeded cel s have been widely used in tissue engineering and clinic therapy;thus, the precise, safe, effective isolation of mesenchymal stem cel s is the particular important premise to build culture system. OBJECTIVE:To review the methods of isolating mesenchymal stem cel s and to compare the merit and demerit of different methods, thereby providing theoretical basis for safe and high-effective isolation of mesenchymal stem cel s. METHODS:A computer-based online research of CNKI and PubMed databases was performed to col ect articles, which included reviews, clinical trials and experiments, published between 1965 and 2014 with the key words of“mesenchymal stem cel s (MSCs), isolation methods”in Chinese and English. A total of 52 articles were included according inclusion and exclusion criteria RESULTS AND CONCLUSION:(1) The whole bone marrow culture method can derive a mass of mesenchymal stem cel s, which need to be purified. (2) The density gradient centrifugation method which uses the media with the density of 1.073 g/mL can be used to harvest more purified cel s. (3) The tissue digestion method is suitable for digestion and isolation of adipose tissue and umbilical cord tissue. Type II col agenase digestion is better, but they are both limited by a high demand for operative techniques. (4) Immunomagnetic bead separation is appropriate to study the biological characteristics of a kind of subpopulation of mesenchymal stem cel s which express special surface markers. (5) The combination method is also an optimal way. (6) Some new methods limited by few dates require further studies.
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BACKGROUND:Coculture of bone marrow mesenchymal stem cel s and human umbilical vein endothelial cel s can improve both osteogenic and angiogenic outcomes and provide a promising strategy for bone tissue engineering and osteanagenesis. OBJECTIVE:To summarize recent researches and related progresses in coculture of human umbilical vein endothelial cel s and bone marrow mesenchymal stem cel s. METHODS:A computer-based online search of CNKI database from January 2000 to March 2012, PubMed database and Web of Knowledge database from January 1980 to March 2012, was performed with the keywords of“human umbilical vein endothelial cel s, bone mesenchymal stem cel s, coculture, tissue engineering”both in Chinese and English. A total of 135 articles were screened out, 103 of them were excluded due to unrelated study objective and repeated contents, and final y 32 articles were involved in further analysis. RESULTS AND CONCLUSION:At present, studies on coculture of bone marrow mesenchymal stem cel s and human umbilical vein endothelial cel s mainly focus on mimicking in vivo environments, the interactions between cel s, and the influence of different cel ratios and culture media. Most of these researches play important roles in bone tissue engineering and bone regeneration therapy, but the mechanism of action and concrete regulation in vivo between bone marrow mesenchymal stem cel s and human umbilical vein endothelial cel s stil need further research and analysis.
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BACKGROUND:With the development of the research, induced pluripotent stem cel s are applied to the build of disease model, drug screening, regenerative medicine, and many other research fields, and have made significant achievements, especial y in the study of nervous system diseases. OBJECTIVE:To summarize the recent development of induced pluripotent stem cel s and to raise problems and prospects based on the latest research in this field. METHODS:The first author searched the PubMed database for articles about the induced pluripotent stem cel s, including reviews, clinical research and basic research, published from January 2006 to September 2014. The keywords were“iPS, induced pluripotent stem cel”, and final y 60 articles were included in result analysis. RESULTS AND CONCLUSION:Induced pluripotent stem cel research continues to make breakthrough from its discovery by Yamanaka’s team in 2006 to winning Nobel Prize in 2012. Induced pluripotent stem cel research has broad prospects in the disease model construction, drug screening and regenerative medicine. Currently, problems such as reprogramming methods, cel stability, and clinical transformation stil need to be solved, and further researches are necessary.
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BACKGROUND:With the development of biochemistry and cel biology, fracture has being study deeper, blood supply has been known to be an important factor influencing the fracture healing. Endothelial progenitor cel s with good ability of angiogenesis wil have a good clinical prospect in fracture healing. OBJECTIVE:To review the recent research of endothelial progenitor cel s in fracture healing. METHODS:A computer-based online search of CNKI, Wanfang, PubMed databases was performed to col ect articles published between 1980 and 2014 with the key words“endothelial progenitor cel , fracture, neovascularization, angiogenesis”in Chinese and English. A total of 48 articles addressing endothelial progenitor cel for angiogenesis in fracture healing were included in result analysis. RESULTS AND CONCLUSION:Increasing evidence has shown that endothelial progenitor cel s have great ability of neovascularzition and angiogenesis. Endothelial progenitor cel s used in tissue engineering scaffolds can promote the survival rate of scaffolds in vivo, which is appropriate to a great part of delayed union and nonunion patients. However, the large-scale treatment with endothelial progenitor cel s stil has many problems, such as isolation, culture and amplification of endothelial progenitor cel s in vitro, the number of transplanted cel s and selection of scaffolds for transplanted cel s, which need further research.
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BACKGROUND:Studies have shown that bone loss can lead to a series of diseases, such as osteoporotic fractures, thus seeking to increase bone mass has become a goal of the majority of researchers. OBJECTIVE:To summarize the current studies of improving bone mass by using stem cel transplantation, hoping to the extensive application of stem cel transplantation in the clinical treatment of osteoporosis as early as possible. METHODS:A computer-based search of PubMed and CNKI was performed by the first author to retrieve articles relevant to stem cel therapy for osteoporosis published from January 1997 to October 2014. The keywords were“to improve bone mass, regenerative medicine, bone marrow mesenchymal stem cel transplantation, stem cel therapy”in Chinese and English, respectively, which appeared in the title, abstract or keywords. Articles published recently or in authoritative journals were preferred, and final y 28 articles were included in result analysis. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cel s which are isolated and cultured easily can proliferate rapidly and have multi-lineage differentiation potential. Studies have shown that the osteogenic differentiation of bone marrow mesenchymal stem cel s can real y improve bone mass, and obtain more achievements in the treatment of orthopedic disorders. This new cel therapy can help to accelerate bone healing and reduce treatment time, offering a new therapeutic choice for orthopedic surgery, plastic surgery, oral and maxil ofacial surgery, and therefore, it has broad application prospects.
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BACKGROUND:The development of tissue engineering techniques provides new methods and ideas for the repair and functional reconstruction of articular cartilage defects. OBJECTIVE:To summarize the research progress in the application of mesenchymal stem cel s, as seed cel s, in articular cartilage tissue engineering. METHODS:A computer-based retrieval was performed to search articles describing articular cartilage tissue engineering and mesenchymal stem cel s published between January 1st, 2000 and September 30th, 2014 in PubMed database with the key words of“articular cartilage defects;cartilage tissue engineering;mesenchymal stem cel s”in English. Seventy articles were retrieved initial y, and only 49 were included in further analysis. RESULTS AND CONCLUSION:The ability of the articular cartilage for defect self-repair is limited, and current clinical treatments cannot be satisfactory. Development of tissue engineering provides a new idea for problem-solving. In the selection of seed cel s, chondrocyte is limited to dedifferentiate, and embryonic stem cel is restricted by ethical, legal and other aspects. Autologous mesenchymal stem cel s, which are easy to be amplified and exhibit excel ent cartilage differentiation potential, have gained widespread attention. But there is stil some controversy on the current application of tissue engineering techniques for repair of articular cartilage defects, including a certain gap between the long-term effects and the clinical applications. So the effect of mesenchymal stem cel s on biological structure and mechanical function stil needs further studies.
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BACKGROUND:Chronic tendinopathy is a tendon disorder extremely common in athletes and in the general population with repetitive strain injuries of tendons. The pathogenesis of tendinopathy remains unclear and hence treatment of tendinopathy is usual y pal iative. OBJECTIVE:To investigate the of adipogenic and tenogenic ability of patel ar tendon-derived stem cel s isolated from chronic tendinopathy and healthy rats in vitro. METHODS:Tendon-derived stem cel s were isolated from patel ar tendons of chronic tendinopathy and healthy rats respectively. The tendon-derived stem cel s were cultured to the 3rd passage in complete culture medium, and cel morphology was observed. The cel s were divided into adipogenic induction group and control group. Cel s in the adipogenic induction group were cultured in adipogenic induction medium, while those in the control group cultured in complete culture medium. The ability of adipogenic differentiation between tendon-derived stem cel s isolated from the tendon of chronic tendinopathy and healthy rats in vitro was examined by oil red O staining and quantification assay. The mRNA expressions of C/EBPαand PPARγ2 were detected by real-time quantitative PCR. When 70%-80%cel s were confluent, the mRNA expressions of Col1a1, Scx, Tnmd and Dcn were also detected by real-time quantitative PCR. RESULTS AND CONCLUSION:At the third passage, slender spindle-shaped cel s were seen in both two groups, but there was a little change in the cel morphology in the chronic tendinopathy group. Lipid droplets were formed after the cel s were cultured in adipogenic induction medium for 21 days. This was not observed in the control group. We observed more oil red O-positive oil droplets in tendon-derived stem cel s from the tendons of chronic tendinopathy rats than healthy rats. The difference between them was statistical y significant (P=0.004). The results of real-time quantitative PCR showed that the mRNA expressions of C/EBPαand PPARγ2 in the tendon-derived stem cel s from the tendons of chronic tendinopathy rats were significantly higher than those in tendon-derived stem cel s from the tendons of healthy rats (P=0.004);the mRNA expressions of Col1a1, Scx, Tnmd and Dcn in the tendon-derived stem cel s from the tendons of chronic tendinopathy rats were significantly lower than those in tendon-derived stem cel s from the tendons of healthy rats (P=0.009). In conclusion, tendon-derived stem cel s from chronic tendinopathy rats showed a higher ability of adipogenic differentiation, but a lower capacity of tenogenic differentiation compared to tendon-derived stem cel s from healthy rats, which might contribute to better understand the pathogenesis of tendinopathy.
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BACKGROUND:It is important to produce and save a large amount of high-activity feeder cel s for the culture of human embryonic stem cel s. OBJECTIVE:To establish the optimal method for isolation and culture of Kunming mouse embryonic fibroblasts, and to evaluate the feasibility of preparing feeder layers for culture of human embryonic stem cel s. METHODS:Embryonic fibroblasts were isolated and cultured by different concentrations of trypsin from Kunming mouse fetuses in vitro. The biological characteristics and growth rule of mouse embryonic fibroblasts were investigated, and then the feeder layers for human embryonic stem cel s culture were produced. The growth of human embryonic stem cel s on the prepared feeder layer was tested. RESULTS AND CONCLUSION:The optimal fetal age for preparing Kunming mouse embryonic fibroblast feeder layer was 13.5 days. Kunming mouse embryonic fibroblasts at different concentrations grew wel with high purity and active proliferation by trypsin digestion method. There was no significant difference in the survival rate of cel s after cryopreservation for 2 weeks, 1 month, 3 months and 6 months. The cel s were proliferative from the second to fourth passage and declined sharply after the fifth passage. Human embryonic stem cel s which grew on Kunming mouse embryonic fibroblasts feeder layers were stil to remain the typical undifferentiated morphology and were strongly positive for alkaline phosphatase and periodic acid-Schiff after long-term subculture. The mouse embryonic fibroblasts can be used as the stable and high-quality feeder cel s for human embryonic stem cel s.
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BACKGROUND:To in vitro isolate neural stem cel s with high purity and uniform biological properties and to establish a complete set of neural stem cel culture system is the basis for neural stem cel research. OBJECTIVE:To establish an isolation and culture system for neural stem cel s from newborn mouse hippocampus, olfactory bulb and cortex and to analyze the biological properties of cel s. METHODS:Neural stem cel s were isolated from the hippocampus, olfactory bulb and cortex tissue of newborn Kunming mice by mechanical separation and trypsin digestion. Serum-free culture technology, mechanical pipetting and trypsin digestion were used for subculture of neural stem cel s. 10%fetal bovine serum was used to induce differentiation of neural stem cel s. Neural stem cel s and their differentiated products were identified by immunofluorescent staining of Nestin, CD133,β-TubulinIII, glial fibril ary acidic protein. RESULTS AND CONCLUSION:The neural stem cel obtained from newborn mouse hippocampus, olfactory bulb and cortex had the capacity of self-renewal and differentiation which were positive for Nestin and CD133. After induction with fetal bovine serum, neural stem cel could differentiation toβ-tubulinIII or glial fibril ary acidic protein positive cel s that were neurons and astrocytes. This experiment has successful y established the neural stem cel isolation, culture, identification and induction system, providing experimental basis for subsequent studies of neural stem cel s.
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BACKGROUND:Neuroblastoma is a common solid tumor in children. Pediatric tumors are little affected by environmental factors, but closely related to child development. The suspension method is an effective and reliable method to harvest neoplastic stem cel s. OBJECTIVE:To culture the cel clones of human neuroblastoma cel line SK-N-SH and to assess the biological properties of the cel clones. METHODS:Using the suspension method with no serum media, tumor cel clones were obtained. Immunofluorescence method was used to identify whether tumor cel clones exhibit stem cel properties. SK-N-SH neuroblastoma was labeled by luciferase, and tumor cel clones and tumor cel s were seeded onto the back of nude mice to monitor cel proliferative properties in nude mice using in vivo imaging. RESULTS AND CONCLUSION:Using the suspension culture method, SK-N-SH neuroblastoma cel s could successful y develop into cloning bal s. Under serum-free culture, cloning bal s were immunofluorescently used to detect molecular markers that showed strong positive expression. Cloning bal s subcutaneously implanted into nude mice showed the strong ability of self-renewal and differentiation as stem cel s. The cel clones cultured by the suspension method strongly expressed Nestin, but weakly expressed glial fibril ary acidic protein, neuron-specific tubulin, possessing stem cel characteristics and strong proliferation and metastasis in nude mice.
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BACKGROUND:The quantity and quality of seed cel s is a critical bottleneck of the development of vascular tissue engineering. To address this issue, stem cel-derived endothelial cel s have been a hot spot in this field due to their potential in providing the ideal seed cel s. OBJECTIVE:To elucidate the effect of vascular endothelial growth factor (VEGF) supplementation combined with hypoxic culture condition on the lineage-specific differentiation of embryonic stem cel s into endothelial cel s. METHODS:Serum-free medium mTeSR?1 was applied to cultivate H9 cel s in vitro. A conditioned medium containing 50μg/L vascular endothelial growth factor was utilized to induce H9 cel s to differentiate into endothelial cel s under the hypoxic culture condition (5%O2). The cel under normal condition (5%CO2) with or without vascular endothelial growth factor served as controls. The phenotype and function of human embryonic stem cel s-derived endothelial cel s were assayed by immunofluorescence staining, quantitative RT-PCR, and low-density lipoprotein uptake experiment. RESULTS AND CONCLUSION:Compared with the control group, the H9 cel s were induced to be differentiated into endothelial-like cel s more efficiently when they were cultivated under a conditioned medium with vascular endothelial growth factor supplementation under the hypoxic condition. These differentiated cel s not only expressed some important surface markers of endothelia cel s, including kdr, pecam, but also took in low-density lipoprotein to form microvessle-like structures. This culture system supports a synergy effect of vascular endothelial growth factor and hypoxic environment that can efficiently promotes the lineage-specific differentiation of embryonic stem cel s into endothelial cel s with good phenotype and functionality.