Studies Of Human Umbilical Cord Mesenchymal Stem Cells Transplantation In Rats With Ischemic

Objective: Investigate biological characteristics of human umbilical cord mesenchymal stem cells (hUMSCs). Exploring the effect and mechanism of hUMSCs transplantation on treating ischemic stroke in rats, providing a theoretical and experimental foundation for the clinical application of hUMSCs in the neuroscience field.Methods: The full-term umbilical cords from healthy neonates were washed with D-Hank's, and umbilical cord arteries and vein were stripped off.Then remaining parts of umbilical cord were cut into small fragments of 1 mm3, digested by 0.2% collagenase II and plated in growth medium consisting of DMEM/F12, 20% fetal bovine serum (FBS), 2 ng/mL epidermal growth factor (EGF), 25 mM L-glutamine (L-Glu), 100 units/mL penicillin, and 100μg/mL streptomycin. Obsevre the primary culture cells'morphological changes. Once adherent cells reached approximately 80~90% confluence,they were detached with 0.25% trypsin -0.01%mM EDTA solution and were passaged.After trypsization, collect the adherent cells. Then flow cytometry analysis was performed by FACScalibur flow cytometry with mouse monoclonal phycoerythrin (PE) or fluorescein isothiocyanate (FITC) labeled antibodies, which includes PE-CD13 , PE-CD29 , PE-CD31 , PE-CD44 ,PE-CD73,PE-CD105,PE-CD133,PE-CD166,FITC-CD34,FITC-CD45 , FITC-CD90 , FITC-HLA-ABC (HLA-I) and FITC-HLA-DR (HLA-II),. FITC or PE conjugated Mouse IgG1 were used as controls.A number of adult male Sprague-Dawley (SD) rats weighing 230~250 g, were employed in all our study. A monofilament nylon suture, 0.235 mm in diameter, with its tip covered by paraffin, was advanced from the right common carotid artery into the lumen of the internal carotid artery until it blocked the origin of the middle cerebral artery, to induce the middle cerebral artery occlusion (MCAO). Two hours after MCAO, animals were reanesthetized, and reperfusion was performed by withdrawal of the suture. The neurological function of experimental animals was evaluated using modified neurological severity score (mNSS) 24 hours after MCAO. And the rats awarding 7~12 points were randomly divided into groups as follows: (1) hUMSCs transplant group, n = 12; (2) Phosphate Buffered Saline (PBS) control group, n = 12; (3) MCAO alone group, n = 8. And another (4) sham operation group, n = 4.The rats were anesthetized again, and the head was fixed on stereotaxic frame. For hUMSCs transplant group and sham operation group rats, 1, 1' - dioctadecyl - 3, 3, 3', 3' - tetramethylindocarbocyanine perchlorate (DiI) labeled hUMCSs were then transplanted into the right striatum and cortex, using a 10-μL Hamilton syringe, at the following coordinates, calculated from bregma: (AP=-1 mm; ML=2.0 mm; DV=4.5 mm); (AP=-1 mm; ML=2.0 mm; DV=2 mm). Each rat received a 10-μL injection, approximately 5×105 hUMSCs; 7μL was initially injected into the striatum and 3μL into the cortex slowly. After retraction of the syringe, fibrin glue was applied to enclose the pinhole and burr hole. Equal quantity of PBS was injected in PBS control group in the same way, served as the vehicle control.In all animals, a set of mNSS tests was performed before MCAO and at 1, 7, 14, 21, 28 days after MCAO.Spatial learning was assessed using a 5-day Morris water maze test from 24 to 28 days after MCAO. Finally, after all the behavioral tests, brain tissue of rats was removed promptly and successive sections were made using a cryomicrotome. Immunofluorescence staining was performed to examine the survival, and expression of neuron specific enolase (NSE), Microtubule-associated protein 2 (MAP2), glial fibrillary acid Protein (GFAP), platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) and von Willebrand factor (vWF) of DiI-labeled hUMSCs.The SPSS (Statistical Package for the Social Science) 15.0 for Windows was used in this sutdy to analyse the data.Results: Adherent cells with different morphology could be found 24~48 hours after cultivation. Then expanding after 6~10 days, they formed colonies with fibroblastic morphology. These colonies present a radiating appearance. Two weeks after plating, when the cells reached 80%~90% confluence, they were detached and passaged at a ratio of 1:2 to 1:3. Several hours after plating, cells became adherent rapidly. After approximately 5~6 days,cells cultures reached 90%~95% confluence again and formed a compact, whirling-like appearance in low power field.FACS shown that hUMSCs express CD13, CD29, CD44, CD73, CD90, CD105, CD106, CD166, HLA-ABC (HLA-I), and not express CD31, CD34, CD38, CD45, CD133, HLA-DR (HLA-II).Before and 24 hours after MCAO, there was no significant difference in mNSS between hUMSCs transplant group, PBS control group and MCAO alone group (p>0.05). From 14 days, rats of hUMSCs transplant group had significant improvement on mNSS compared to control animals (p<0.05). In the Morris water maze test, hUMSCs transplant group showed a shorter mean latency time, which was significantly different from control groups. Immunofluorescence staining demonstrated that, after 28 days from hUMSCs transplantation, not only could a number of transplanted hUMSCs survive in the host brain, but also some of them expressed NSE, MAP2, GFAP, CD31 and vWF.Conclusion: In this study, we found that umbilical cord was a rich source of MSCs, which could be easily isolated, cultured and passaged in vitro. Transplanted hUMSCs could survive, differentiate in host brain and improve functional recovery after MCAO in rats.