Repair Effect Of IPSCs-derived GABA Progenitor Cells Stereotactically Transplantation In Parkinson’s Disease Rats

Objective:To explore the method of gradually inducing miniature-swine iPSCs to differentiate into GABA progenitors;To establish a stable rat model of unilateral substantia nigra lesion induced by 6-hydroxydopamine(6-OHDA),and to improve the effective evaluation system of dyskinesia in Parkinson’s disease model rats;To study the repair mechanism of transplanted miniature-swine iPSCs-GABA progenitor cells to improve the motor function of Parkinson’s ratsMethods:Miniature-swine iPSCs were induced to GABA progenitor cells in two stages.In the first stage,miniature-swine iPSCs were cultured in suspension.On the third day,the embryoids were induced by NIM(SB431542,DMH1,FGF2),and differentiated into primitive neuroepithelial cells(NECs)on the 12 day.In the second stage,the NIM containing Pur and B27 was used to form neurospheres(NS),At the 21 day,the cells form GABA progenitors and at the same time,the phenotypic and functional effects of NECs and GABA progenitors were detected The rat model of Parkinson’s disease was made by injecting 6-OHDA into the right substantia nigra(SN)at two coordinates.Two weeks after operation,APO induced rotation test was used to choose successfully the Parkinson’s disease rat model.Miniature-swine iPSCs-GABA progenitors were transplanted into the substantia nigra-striatum area of Parkinson’s rat brain.The APO-induced rotation test,open field assay,water maze test and rotarod test were used to compare the motor coordination ability of rats in control group,model group and transplantation group.HE staining was performed in the control group,model group and transplantation group to observe the number and morphological changes of cells in the injured site.Immunohistochemical method was used to detect the expression of tyrosine hydroxylase(TH)in each group.At 8 weeks and 32 weeks after cell transplantation,the survival,migration and differentiation of GABA progenitors in the host brain microenvironment were observed by immunofluorescence.Western Blot assay was used to detect the protein expression of TH,GFAP and SynaptophysinResults:Miniature-swine iPSCs was stably passaged on feeder layer cells,expressing pluripotent markers OCT4,Nanog,SSEA1 and TRA-160,and karyotype analysis showed that there was no cell contamination from other species.At day 12,About 90%of the differentiated rosette structures could express the surface markers PAX6,SOX2 and Nestin as well as the neural tubulin marker TUJ1.By day 21,more than 90%of the cells were induced to GABA progenitors that express NKX2.1+ and the forebrain marker FOXG1+.Compared with the model group,there was significant difference in behavior between the cell transplantation group and the model group in APO-induced rotation test,open field test,water maze test and the rotarod test(p<0.01).The above behavioral tests showed that the motor function of Parkinson’s rats was significantly improved after transplantation.After 8 weeks of transplantation,the results of HE staining showed that the number of cells in the injured area of the model group was significantly less and arranged disorderly.The number of cells in the transplantation group was more than that in the model group,and the arrangement of cells was more regular.The results of TH immunohistochemistry showed that the expression of TH in the injured area of the model group was significantly lower than that of the control group,while the expression of TH in the transplantation group was significantly higher than that of the model group.At 8 and 32 weeks after transplantation,the immunofluorescence results showed that CM-Dil positive cells were observed,and the neuron markers Nestin and tubulin-Ⅲ,dopamine neuron marker TH,GABA neuron marker GABA,synaptic marker synaptophysin and astrocyte marker GFAP were also expressed.The results of Western Blot showed that the contents of TH and Synaptophysin in the model group were significantly lower than those in the control group,while the expressions of TH and Synaptophysin in the transplantation group were significantly higher than those in the model group,GFAP is a specific marker of astrocytes.The content of GFAP in the model group was not significantly lower than that in the control group,but the content of GFAP in the model group was significantly higher than that in the model group at 8 weeks after transplantation.Conclusion:1.Through two stages,miniature-swine iPSCs was gradually induced to forebrain GABA progenitor cells,which laid a foundation for the induction of GABA progenitors transplantation for the treatment of nerve injury diseases.2.Two-point injection of 6-OHDA into the right medial forebrain bundle to establish the model of laterally damaged Parkinson’s rats is an efficient method,and the evaluation system of motor behavior of Parkinson’s rats is improved by using APO-induced rotation test,open-field test,water maze test and rotarod test.3.The transplanted cells can survive for 32 weeks in the brain microenvironment of Parkinson’s rats,migrate widely,and differentiate into different functional neurons,which make synaptic connections with autologous neurons.