| Cell replacement therapy has become a promising intervention for spinal cord injury (SCI) recently. Embryonic stem cells (ESCs) proliferate indefinitely without signs of senescence and have potential to differentiate into neural cells, such as neurons, oligodendrocytes and astrocytes and so on. These properties make ESCs an attractive source for cell transplantation. However, transplantation of undifferentiated ESCs leads to the formation of teratoma in the recipients. Therefore, ESCs should be induced into specific neural cells or neural progenitors in vitro before beging transplanted into the injured spinal cord. On the whole picture, the application of ESCs treatment to spinal cord injury should be processed as two steps:Firstly, in vitro, ESCs are differentiated into neural cell or neural progenitors with high efficiency, large quantity, and good functioning. Subenquently, in vivo, differentiated cells are injected into the spinal cord which provides a microenvironment for the transplanted cells to migrate, proliferate and integrate with the host tissue.Although the induced differentiation of ESCs in vitro is a well-developed approach, there are still some problems, including low efficiency, concentration and quantity. Thus, in our study, we tried to promote the differentiation rate of Olig2+-GFP+-mES. The procedure of differentiation of neural cell is controlled by multiple signaling pathways, in which the signaling pathway of SHH makes an important effect. Purmorphamine, a low-mass chemical which is the derivate of purine, was previously found to activate the SHH signaling pathway by targeting smoothened, a crucial element of SHH signaling pathway. Purmorphamine plays an important role in development of organism. Here, we raised our hypothesis. First of all, Whether Purmorphamine could replace SHH in inducing the differentiation of ESCs into functional spinal motor neurons and oligodendrocytes with a high concentration and quantity? And even if this possibility holds true, would Purmorphamine cause a subsequent change in the expression associated genes linked with the signaling pathways of SHH, dorsal-ventral axis, as well as the development of motor neuron and oligodendrocyte? Secondly, whether the induced differentiated oligodendrocyte progenitor cells are functioning well, and can migrate in the injured spinal cord, integrate with the host for the next migration and differentiation to perform its function to accelerate the recovery? The details were as follows:In the first part of the observation, in vitro, we investigated the function and possible mechanism of Purmorphamine during the neural differentiation of Olig2+-GFP+-mES by immunofluorescence, fluorescence-activated cell sorter and RT-PCR. The results showed that Purmorphamine can substitute SHH completely, and efficiently induced Olig2+-GFP+-mES to differentiate into spinal motor neurons and oligodendrocytes by up-regulating the expression of Olig2 gene on EB8D and EB12D respectively. The induced rate was over 50% and 80% in the differentiation process of EB8D and EB12D into spinal motor neurons and oligdendrocytes, respectively. Meanwhile, Purmorphamine also caused the change in the expression of relevant genes in the process of motor neuron and oligodendrocyte development, dorsal-ventral axis and SHH signaling pathway. The data above indicated that Purmorphamine may efficiently induce Olig2+-GFP+-mES to differentiate into functional spinal motor neurons and oligodendrocytes.In the second part, in vivo, we further explored whether the transplantation of Olig2+-GFP+-OPC could enhance remyelination and functional recovery after SCI. In this part, the spinal cord injury model was firstly established through the application of NYU-Impactorâ…¡, and then we observed if the transplanted cells could improve the recovery of spinal function via immunofluorescence, transmission electron microscopy, western blot, neural behavior analysis and electrophysiological assessment. Olig2+-GFP+-OPC was transplanted into the contused adult thoracic spinal cord 7d after injury. Four weeks after transplantation, the grafted survived and integrated into the injured spinal cord. The grafted Olig2+-GFP+-OPC retained the proliferative ability and continued to differentiate and mature in vivo and expressed MBP as well. Western blot analyses showed that the expression of NF and MBP protein in the spinal cord were elevated after transplantation. The transmission electron microscopy analyses further demonstrated the improvement axonal myelination in the lesioned spinal cord. The recovery of hindlimb locomotor function of the injured rats was significantly enhanced in animals receiving grafts of Olig2+-GFP+-OPC. The outcome of electrophysiological experiments indicated that the recovery of the conductive ability of injured spinal cord was significantly better than before in Olig2+-GFP+-OPC transplanted group, indicating that conduction was partially restored.In conclusion, Purmorphamine is a good substitution of SHH to induce Olig2+-GFP+-mES to differentiate into a highly concentrated and functional motor neurons and oligodendrocytes. The transplantation of induced Olig2+-GFP+-OPC can enhance remyelination and facilitate functional recovery after traumatic SCI. Our results suggest that transplantation of ESC is one of effective approach for more optimal functional recovery after SCI. |
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