ABSTRACT
Objective To study the possibility of inducing human umbilical cord blood mesenchymal stem cells(MSCs) to differentiate into neuron-like cells by lithium chloride(LiCl) in vitro.Methods Human umbilical cord blood was collected from mature neonates.All samples were obtained sterilely with 20 U/ml heparin.The cord mononuclear cells were isolated with lymphocyte separation medium(density 1.077 g/ml), then purified by wall sticking screening and expanded with slight sugar DIEM containing 15% FBS.The third passage of the expanded MSCs were pre-inducted with DIEM containing 15% FBS and 20 ng/ml bFGF for 24 hours, then induced with DIEM without serum but 3 mol/L LiCl for 6 days in group A.The MSCs were induced with DIEM containing 3 mol/L Licl for 7 days in group B.The MSCs were normally cultured with DIEM containing 15% FBS in group C.The morphological changes of the cells were observed under phase contrast microscope.The neuron specific markers containing neuron specific enolase(NSE), microtubule associated protein2(MAP2) and glial fibrillary acid protein(GFAP) were evaluated by indirect immunocyto-chemistry staining.Results After inducted for 3 days, morphological changes were observed obviously in group A and B.6 days later, the differentiated cells showed typical neuronal morphology.The expression of NSE and MAP2 were positive for the majority cells in group A and B, and that of group A[(73.6 ± 7.8)%, 75.5 ± 8.5)% respectively]were obviously higher than group B[(31.0 ± 4.3)%,(33.5 ± 5.0)% respectively], few expressed GFAP in both groups.Conclusion The combination of LiCl and growth factor may induce the human umbilical cord blood MSCs into neuron-like cells in vitro.
ABSTRACT
BACKGROUND: Basso, beattie and bresnahan (BBB) score and tilt table test are associated with the height of spinal cord injury degree in study of spinal cord models. OBJECTIVE: To establish modified complete spinal cord transected rat models, and to observe effects of BBB score and tilt table test on function following human umbilical cord blood mesenchymal stem cell transplantation. DESIGN, TIME AND SETTING: The observation control experiments were performed at the Key Laboratory of Tissue Construction and Detection of Guangdong Province from April 2007 to July 2008. MATERIALS: Cord blood was collected from healthy puerperants after full-term delivery. Fifty SPF healthy adult female Sprague Dawley rats were randomly divided into group A (sham operation) (n=10), group B (spinal cord transection and phosphate buffered saline, PBS injection) (n=20), group C (spinal cord transection and hMSCs transplantation) (n=20). METHODS: Human umbilical cord blood stem cells were harvested and cultured in vitro. After incising rat dural sac, arachnoid membrane, spinal cord, bilateral wall and ventral dura mater of spinal cord were completely incised in the dural mate of spinal cord to establish complete spinal cord transected rat models. Vertebral plates were only opened in the group A. Rats in the group C were injected with human umbilical cord blood mesenchymal stem cells (6?109 -7?109L-1) at two broken ends of fractured spinal cord. Phosphate buffer saline was injected into rats in the group B. MAIN OUTCOME MEASURES: At 12 weeks after surgery, a behavioral testing was performed every 2 weeks upon each hindlimb for all animals by using the BBB scoring system and tilt table test. RESULTS: No significant difference in hindlimb motor function was detected in the group A. From the second weeks after surgery, motor function of hindlimbs was gradually recovered in the groups B and C. BBB scoring system and tilt table test showed a consistent increasing tendency in a positive correlation. From the fourth week after surgery, significant difference in tilt table test was detected between rats with BBB score