| Background:Parkinson’s disease(PD)is a neurodegenerative disease whose main pathological feature is dysfunction of dopaminergic neurons(DAns)in the substantia nigra(SN).The treatment of PD is mainly based on drugs.Early patients can control symptoms through drug treatment.Although the drug still has a certain role in the mid-disease,it often leads to a decline in quality of life due to the occurrence of motor complications such as switch effects.Cell therapy of PD using pluripotent stem cells(PSCs)has become the most promising method for fundamentally curing patients,inducing pluripotent stem cells(iPSCs)and embryonic stem cells(ESCs).The research and application of)is most noticeable.iPSCs can be reprogrammed from the patient’s own somatic cells,and ethical controversy and immune rejection are relatively minor.However,due to individual differences,one-to-one construction must be used when using them,and there are large differences among cells.The ESCs are of a single source,and a single system can be used by many people,but at the same time it also brings about immune rejection and ethical problems.In recent years,countries such as the United States,Japan,and China have successively reported cases of using pluripotent stem cells to treat PD.However,in clinical applications,it is not possible to perform in-vivo tracing,section staining,etc.of the grafts to study the details of their treatment,rather than human spirits.Long animal species are most similar to humans in comparison with other animal models.Using this as an animal model can solve this problem successfully.In this study,the iPSCs cell line derived from the PINK1 knockout monkey and the ESCs cell line derived from normal embryos were established.The differentiation and differentiation system of neural stem cells and dopaminergic neurons was established to prepare cells for transplantation.Objectives: 1.Detecting the knock-out efficiency and mutation type of PINK1-targeted cynomolgus monkey model;obtaining the pluripotent stem cells and neural stem cells induced by PINK1 knockout cynomolgus monkey model;2.Establish rhesus monkey/cynomolgus monkey ES cell lines with different systems to find an optimal method to obtain a more versatile Rhesus monkey/cynomolgus monkey ES cell line.Methods:1.Reprogramming of PINK1 knockout cynomolgus monkey fibroblasts into i PS cells using Sendai virus carrying SOX2,OCT4,Klf4,c-Myc four factors;2.Embryonic stem cells were isolated from blastocyst inner cell mass using three different ES culture systems.The pluripotency of the embryonic stem cells obtained by the three methods was compared to find the most versatile system for the establishment of embryonic stem cells.Results: 1.Three strains of PINK1 knockout i PSCs were obtained,and 10 strains of ESCs derived from normal embryos were successfully obtained using three different embryonic stem cell lines.2.PINK1 knockout iPSCs successfully induce into neural stem cells and dopaminergic neurons.Conclusions: We successfully obtained neural stem cells and dopaminergic neurons to prepare for cell transplantation in the next step;three systems can successfully establish ESCs cell lines,and the first and second systems have higher efficiency,which is 31.25%(5/16),22.22%(4/18),the third has the lowest efficiency,about 20.8%(5/24),but its morphology is closer to mouse Na?ve stem cells,and the other two systems are typical Primed state stem cells.. |
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