ABSTRACT
Objective To investigate the effects of transplantatation human endothelial progenitor cells onneurological functional recovery from spinal cord injury model in rats,survival of transplanting cells and differentia-tion. Method The human endothelial progenitor cells were provided by Shanghai Developmental Biology Labora-tory.Forty SD rats were made for the animal model of spinal cord complete transection.Thirty survival SD rats wererandomly divided into three groups:sham operation gronp(group A, n = 10),operation/cell group(greup B, n =10) and operation/DMEM group (group C, n = 10).Suspension containing (hEPCs 6×106) was transplanted in-to the vertebral canal around injured spinal cord. In group C, equal volume of DMEM was injected insbead in the same way as in the group B. The BBB score was obtained 2,4,6,8 weeks after injection, Immunohistochemistry andin-situ hybridization were used to observe the cells survival and differentiation in the spinal cord. The BBB test wasperformed to study the functional improvement of cells. The SAS version. 1.3 software for statistics was to studyethology and functional improvement. The sum of ranks was checlced with Kruskal-Wallis Test and Nemenyi test.Results There were statistically significant differences in BBB scoring between group A and group B as well asgroup C after operation (P<0.05). The BBB score in group B was higher than that in group C after 2,4,6 and8 weeks,but lower than in group A. The hybridization in-situ and immunohistochemistry showed that transplantedcells survived for 8 weeks after transplantation and expressed specific characteristics for ancestral cell and differentiated into vascular endothelial cell (VEC). Conclusions After transplantation, hEPCs can survive, differenti-ate into vascular endothelial cell,and improve spinal cord function as compared with control group.
ABSTRACT
BACKGROUND: Embryonic stem cell (ESC) is a kind of highly undifferentiated totipotent cell. It can proliferate and maintain its totipotency in the system cultured in vitro. It is one of most promising stem cells in thetreatment of central nerve injury.OBJECTIVE: To observe the survival and migration of induced transplanted ESC in mice with spinal injury and hypoxic-ischemic encephalopathy.DESIGN: A completely randomized grouping design, controlled animal experiment.SETTING: Laboratory of Developmental Biology Research Center of Shanghai Second Medical University.MATERIALS: Sixty C57/BL6J mice, of clean grade and either gender, aged 6 to 8 weeks (n =30) and 7 days (n =30)were provided by the Shanghai Experimental Animal Center, Chinese Academy of Sciences [Permission No, SCXK (hu)2003-0003]. This animal experiment was approved by Animal Ethics Committee. Mouse ESC strain S8, labeled LacZ marker gene (Provided by Shanghai Developmental Biology Research Center). X-gal dyeing reagent (Sigma Company).METHODS: This experiment was carried out in the laboratory of Shanghai Developmental Biology Research Center (Shanghai Key Laboratory) from October 2002 to December 2003. ① Experimental grouping of spinal injury: Sixteen C57/BL6J successful mice models, aged 6-8 weeks, were randomized into 2 groups: experimental group (n =8), in which, following right spinal semi-sectioning, derivated cell suspension for inducing the in vitro differentiation of ESC was injected at 1 cm away from injury through vertebral canal, and control group (n =8), in which, following right spinal semi-sectioning, phosphate buffer solution (PBS) was injected at the peripheral region of injury. ② Hypoxic-ischemic encephalopathy experimental grouping: Sixteen successful C57/BL6J mice models, aged 7 days, were randomized into 2 groups: experimental group (n =8), following ligation of right common carotid artery, mice were placed in the closed container containing 0.08 volume fraction of oxygen and 0.92 volume fraction of Nitrogen gas, and taken out 1.5 hours later; 3 μL ESCs were injected into the right cerebral ventricle at about 1 week, and control group (n =8), in which, the same amount of PBS was injected into the right cerebral ventricle. ③ At 12 weeks after transplantation, the survival and migration of induced ESCs labeled by Lac-Z in the spinal cord and brain were observed by zymologic method.MATN OUTCOME MEASURES: Survival and migration of ESCs in the central nervous system.RESULTS: ①After being induced in vitro and transplanted to spinal injured region, ESCs differentiated into neural precursor cells. Neural precursor cells could survive in the injured region and migrate to 5 mm away from injured region.Immunohistochemistry proved that the neural precursor cells of transplanted ESCs could differentiate into neurons.Morphologically, it was proved that neural precursor cells-derived from ESCs could well integrate peripheral tissue. ② The induced ESCs were injected into the lateral cerebral ventricle of mice. Derived ESCs widely distributed in the injured hippocampal region, cerebral cortex ventricle choroid plexus, vascular endothelium and other regions, and integrated peripheral tissue, which were similar to adjacent cells in morphology, suggesting that induced ESCs also could survive for long time and far migrate.CONCLUSION:The induced ESC can survive and migrate in the host injured brain and spinal cord, and the migration of ESCs is more obvious in the brain than in the spinal cord.
ABSTRACT
BACKGROUND: Embryonic stem cells can be induced to differentiate into neural precursors under certain conditions, and they can effectively integrate with host cells after transplanted into healthy or injured central nervous system, and then repair and rebuild the injured nerve tissue.OBJECTIVE: To observe the effects of transplantation of neural precursors induced by embryonic stem cells on the recovery of neurological function in mice with spinal cord injury.DESIGN: A randomized controlled animal trial.SETTING: Research Center of Developmental Biology, Shanghai Second Medical University.MATERIALS: Twenty-eight C57/BL6J mice of 6-8 weeks old, both sexes, were used. Mice embryonic stem cell strain S8 and carrier LacZ labeling genes were provided by Shanghai Research Center of Developmental Biology. High-glucose Dulbecco's modified Eagle media (DMEM), β-mercaptoethanol (BME), mice leukemia inhibitory factor (LIF) and mitocin-C were all from GIBCO attachment induction medium, which were provided by Shanghai Research Center of Developmental BiologyMETHODS: The experiment was carried out in the central laboratory of developmental biology of Shanghai Second Medical University from April 2003 to April 2004. The embryonic stem cells were cultured and induced to differentiate into neural precursors by means of attachment induction. The mice were anesthetized and made into models of spinal cord hemisection on the T9-T10 plan. The mice were randomly divided into three groups: sham operated group (n =9): Only T9-T10 spinous process and corresponding lamina of vertebra were removed, then the skin was sutured layer by layer;ransplantation group (n =10): After spinal cord hemisection, embryonic stem cells were injected into the vertebral canal about 1 cm away from the injured site; model group (n =9): DMEM was injected into the region around the injured site.The mice were evaluated with Basso, Beattie, Bresnahan (BBB) locomotor rating scale to observe the recovery of neurological function at 1, 2, 4, 6 and 8 weeks (the score ranged from 1 to 21 points, the higher the scores, the better the recovery of neurological function). At 8 weeks, the survival and differentiation of embryonic stem cells at the injured site of spinal cord were observed using X-Gal staining in each group. The positively stained sections with X-Gal at the injured site of spinal cord were detected with fluorescent immunohistochemical staining.MAIN OUTCOME MEASURES: ① Recovery of neurological function; ② Survival and differentiation of embryonic stem cells at the injured site of spinal cord; ③ Results of fluorescent immunohistochemical staining.RESULTS: ① The BBB scores in the transplantation group at 1, 2 and 4 weeks were higher than those in the model group (P ＜ 0.01). ② Survival and differentiation of embryonic stem cells at the injured site of spinal cord: In the transplantation group, there were X-Gal positively stained cells in the tissue sections of the injured spinal cord of mice,the cytoplasm was blue with nucleoli in it, i.e. the cells derived from embryonic stem cells, which were not observed in the sham-operated group and model group. In the transplantation group, the cells derived from embryonic stem cells, which were implanted to the injured spinal cord, distributed around the injured sites, and integrated with the surrounding tissue and had similar form with the surrounding cells. ③ At the injured site, X-Gal positively stained cells in the transplantation group aiso expressed neurofilaments (the specific marker of neurons), but did not express GFAP.CONCLUSION:The embryonic stem cells were cultured and induced to differentiate into neural progenitors, and they could survive, migrate and differentiate into neurons after transplantation, but there was no obvious improvement of neurological function.
ABSTRACT
AIM: Some researches show that stem cell transplantation for damaged spinal cord can improve the function of damaged spinal cord. But the studies about bone marrow mononuclear cell transplantation for injuried spinal cord are seldom. We transplanted fresh bone marrow mononuclear cells isolated from rats into rat models of injured spinal cord to explore the effect of bone marrow mononuclear cells for injured spinal cord functions, nerve regeneration, neovascuarization and long-term effect. METHODS: Experiments were performed in the Experiment Center of Developmental Biology of Shanghai Second Medical University from October 2005 to April 2006. The laboratory is Specific-pathogen free grade. ①Female clean SD rats aged 8 weeks weighting 200-220 g were offered by Animal Experimental Centre of Chinese Academy of Sciences. Animal intervention met the animal ethical standard. ②Rat bone marrow mononuclear cells were isolated from the tibia and the femur by Ficoll-Paque density gradient centrifugation. Rat models of spinal injury were established. The 22 successfully established rat models were divided into 2 groups. Rat models in a model plus cell group (n =11) received the complete T9-10 transection of spinal cord, and then bone marrow mononuclear cells were transplanted into the vertebral canal. Rat models in a model plus DMEM group (n =11) received the complete T9-10 transection of spinal cord, and then DMEM was injected into adjacent region. Rat models in a sham operation group (n =9) received T9-10 spinous process and lamina of vertebra incision and then the incision was sutured. ③Hybridization in situ and immunohistochemistry technique were used to determine the survival of implanted cells in host spinal cord. BBB scale system was applied to assess the functional recovery of spinal cord nerves. RESULTS: ①There was no significant difference in postoperative score at each time point in the sham operation group. The score was 0 point in the model plus DMEM group. The function of spinal cord did not recover. The function of spinal cord became better at weeks 2, 4, 6 and 8 in the model plus cell group. There were significant differences as compared with the model plus DMEM group and the sham operation group (P
