Transplantation Of Human Fetal Neural Stem Cells Into The Neonatal Rat Hippocampus Following Hypoxic-ischemic Injury

ObjectiveTo investigate the migration and differentiation of human neural stem cells (NSC) in neonatal rat hippocampus following hypoxic-ischemic brain injury (HIBI) . This study was designed to explore the effective strategies for neonatal hypoxic-ischemic encephalopathy (HIE). Methods1. Cell prepared : Fetal brain tissues derived form human embryonic 12 weeks brains were made to single cell suspension and primaryly cultured in N₂ media(D/F12+N2+B27) for 10 days containing epidermal growth factor(EGF, 20ng/ml), basic fibroblast growth factor(bFGF 10ng/ml) and leukemia inhibitory factor(10ng/ml); lately differentiated in the serum supplemented with 10%fetal bovine serum (FBS) for another 4-5 days;then the eugenic hNSC spheres were obtained(each sphere includes about 5-30 of cells). The cell density of hNSC cell suspension prepared in N₂ media for transplantation was approximately 5.0×l0⁴cells/ul.2. Transplantation of NSCs: H1B1 models were made as follows: Seven-day-SD rat pups were anesthetized. The left common carotid artery was ligated. After the surgery animals were allowed to recover for 2-4h. The animals were then placed in a heated (34-35℃),humidified box with a mix of 8% oxygen and 92% nitrogen for 2.5h. Following this period, the animals were returned to the mother and rearednormally.HIBI medols were made as described above, 3 days after that, the rats were anesthetized with pentobarbital(30mg/kg, i.p.) and steretaxic surgery was performed. hNSCs were implanted into the left hippocampus (the damaged side) at following coordinates: AP=-3mm, ML=-2.5mm, DV=-2mm. Each rat received the cell suspension about 2?l(5.0xl04cells/wl) via a glass capillary. The animals were not immunosuppressed throughout the experiment. Rats were anesthesized and perfused tanscardially with 4% paraformaldehyde at 1, 2, 4 weeks and 3 months postransplantation. Brains were then removed and emdedded in paraffin. Coronal sections(5um) were made for immunohistochemical analysis by using mouse anti-hNuc, rabbit anti-hNF, rabbit anti-hTublin, rabbit anti-hGFAP antibodies. This study was designed to observe the survival, migration and differentiation of the implanted cells.ResultsThe 80% cells of the NSC spheres were identified by detecting the neural stem cell's marker Nestin by immumocytochemistry. After differentiation-inducing, the spheres could differentiate into astrocytes and neurons by detecting the astrocyte's marker GFAP and the neuron's marker NF. The spheres were identified as the stem cell characteristic.Cells expressing human nuclear protein (hNuc) were apparent in the CA1 pyramidal cell layer (CA1) at 1 week;most differentiated to neuron expressing neurofilament protein (NF) and Tublin, few differentiated to astrocyte expressing glial fibrillary acidic protein (GFAP) in the dentate granule cell layer (DG) and CA1 at 2 weeks;extensively migrated and obviously reduced at 4 weeks. ConclusionHuman fetal neural stem cells can extensively migrate and completely differentiate to neuron in neonatal rat hippocampus following hypoxic-ischemic brain injury (H1B1) .