| Background:Intracerebral hemorrhage (ICH) refers to bleeding caused in vascular ruprure, namely primary parenchymal hemorrhage, with high morbidity,mortality and morbidity. There are still no effective treatment methods to improve the neural function of survivors. Experiments showed neural stem cell (NSC) transplantation could promote the neural function recovery after ICH. Currently, neural stem cells mainly derived from induction of the early embryonic stem cells or directly separation and culture from central nervous system of the developing and adult mammals. However, in a certain extent, transplantation of neural stem cells have been limited because of ethics, safety problems and the limited cell numbers as well as sources. Therefore, it is necessary to find other ways to obtain neural stem cells to overcome these limitations. Research demonstrated that bone marrow stromal cells could be directly induced into neural stem cell using basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Umbilical cord (UC) were recently found as a good source of mesenchymal stem cells, so here we want to study whether Umbilical cord mesenchymal stem cells (UC-MSC) could be induced into the neural stem cells, which could promote the neural function recovery after incerebral hemorrhage.Objective:We plan to explore the feasibility of generation of neural stem cells induced from UC-MSC, and then transplant these cells into the rat brain of intracerebral hemorrhage induced by collagenase and investigate the survival, distribution and differentiation of the grafted cells in order to provide the theory basis and the experimental data in neuroscience clinical application of UC-MSC.Methods:We chose the umbilical cord from healthy neonatal with full-term pregnancy cesarean section using the method of enzymatic digestion and cell adherent to gain the primitive cells. Passage 4-6 were used in this study. UC-MSCs were pre-induced in DMEM/DF-12 media containing basic fibroblast growth factor (bFGF),fibroblast growth factor 8 (FGF8),sonic hedgehog (SHH) and leukemia inhibitory factor (LIF)within 6-8 days, and then replated in culture plastic dishes after trypsinization. After about 20 days of culture in 2%N2/B27containing neurobasal media with bFGF,FGF8 and SHH, we gained the floating nestin-positive aggregates with morphologies identical to typical neural stem cells(NSC). The characteristics of neural stem cells derived from UC-MSC were detected the expression of NeuroDl, Tubulin, Nestin, GFAP, Galc and Fibronectin in mRNA and protein level respectively by Real-time RT-PCR and immunofluorescence stain. The differentiation potentials to neurons and glia were also detected to further identify the the characteristics of neural stem cells derived from UC-MSC. In the other hand, the UC-MSC phenotypes of UC-NSC were confirmed by Flow cytometry analysis and the potentials to differentiate into osteoblasts and adipocytes by staining. In addition, to further determine the function of neurospheres derived from UC-MSCs (UC-NSC), we transplanted these cells into the collagenase-induced ICH rats. The CM-Dil labeled cells were transplanted in the boundary of hemotama 24 hours later. The neural function was estimated by two methods (mNSS and MLPT) every 7 days within 7 weeks. We also detected the brain water contents on day 3 after transplantation and the expression of IL-1βaround hemotama in the brain on day 3 and 7 after ICH. Otherwise, the glial thickness, hemorrhage volume and differentiation of grafted cells were investigated on day 35 after execution.Results:The neural stem cells induced from UC-MSC grew in neurospheres-like structures, expressed high level of neural progenitor cell markers, but lost the characteristics of mesenchymal stem cells. In addition, they also could be differentiated into the neurons and astrocytes in vitro. Aftrer transplantation into ICH rat brains, we found they could decrease brain water content and glia thickness as well as the expression of IL-10 around hemotama in the brain, differentiate into neurons and astrocytes, and improve significant behavioral recovery. These results suggest that UC-NSC may be a new and useful therapeutic armament for ICH.Conclusion:1. Nenral stem cells could be efficiently gained from human UC-MSC; 2. Nenral stem cells derived from human UC-MSC ameliorated neurological deficits in a rat model of incerebral hemorrhage. Spinal cord injury (SCI) that is one of human diseases with highest morbidity mainly causes paraplegina. There is no effective treatment for patients with SCI at present. The methods all over the world are confined to fix and install the vertebral fracture and dislocation as well as drug treatment in order to relieve compression of spinal cord, reduce edema and secondary injury, and improve microcirculation. Cell transplantations for SCI have become a hot point of research in recent years. Studies showed that grafted cells could survive migration and differentiate into nerve cells and then promote neural function recovery. The transplanted cells were used for the treatment of spinal cord injury, including embryonic stem cells, neural stem cells, mesenchymal stem cells, umbilical cord blood stem cells, schwann cells and olfactory ensheathing cells. Among them, mesenchymal stem cells especially seem to be a more promising one regarding their advantages over other types of transplanted cells. It has been proven that mesenchymal stem cells derived from bone marrow (BMSC) could be beneficial to promote neural function recovery of SCI in rats. However, BMSC is restricted to clinical application because of difficult materials, limited donors and complication with virus infection as well as the declining proliferate capacity and multi-differentiation caused with age. Umbilical cord tissue, as a "waste", has been recently regarded as a rich and ideal source of mesenchymal stem cells. In comparison with BMSC, UC-MSCs derived from rich sources without virus infection risk are more primitive and powerful in proliferation ability, thus UC-MSCs are deserved to be.investigated in the treatment of SCI as seed cells.Objective:This study was designed to preliminarily study if transplantation of UC-MSC could promote the recovery of neurological functions after canine spinal cord injury, in order to provide theoretical and experimental basis for seeking a promising seek cell to use in cell transplantation in SCI.Methods:Human UC-MSCs are derived from the full-term pregnancy cesarean section umbilical cord from healthy children using the method of enzymatic digestion and cell adherent to gain the primitive cells. After digestion and passage, passage 4-6 were used to transplantation. The characteristics of UC-MSC were confirmed by Flow cytometry analysis to detect cellular markers and by the potential to differentiate into osteoblasts and adipocytes to detect the muti-differetiation ability. The canine spinal cord injury animal model was induced by closing hydraulic impact. The dogs were used and randomly divided into two groups:namely UC-MSC group and PBS group. 1) UC-MSC group:transplantation 1×106 UC-MSC into animals by lumbar puncture 1 weeks after spinal cord injury; 2) injection equal volume of PBS into animals by lumbar puncture 1 weeks after spinal cord injury. Functional outcome measurements were preformed using the modified Tarlov score at lday, 1,2,4,6,8,16,24 weeks after cells transplantation. In addition, Dynamic observations by MRI (SIEMENS MagnetomVision) were investigated to observe the injury spinal cord at 1 week after SCI and at 1,6 weeks after transplantation. The animals were sacrificed 24 weeks later transplantation and then removed injury spinal cord tissue to prepare paraffin sections. Luxol fast blue/cresyl violet staining was used to observe the pathological changes of spinal cord tissue.Results:FCAS revealed that human UC-MSCs highly expressed cellular phenotypes of CD90, CD29, CD73 and CD105, but not hematopoietic markers CD34 and CD45, and endothelial cell markers CD31. In addition, they also expressed moderate level of HLA-ABC but not express HLA-DR, suggesting that UC-MSCs could be used to allograft. UC-MSCs were also able to differentiate into osteoblasts and adipocytes under special culture condition. After.canine SCI, significant improvements in neurological functional outcome were noted using Tarlov score in animals of human UC-MSCs transplantation group in comparison with PBS group. MRI showed that edema zone was observed around the damage in animals of human UC-MSCs transplantation group, however, hematoma around the damage in PBS group. Pathological changes were found by Luxol fast blue/cresyl violet staining. In contrast with PBS group, the fibrous tissue filling necrosis is significantly reduced in UC-MSC group. In addition, many neurons were found around the fibrous tissue without pyknotic and the Nissl bodies were dyeing deep.Conclusion:Transplantation of human UC-MSCs following spinal cord injury may improve neurological outcome in dogs.
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