Study On The Differentiation Of Human Embryonic Stem Cells Into Dopamine Precursor Cells

The aging of the population face a high incidence of various degenerative diseases.Among them,the degenerative diseases of the nervous system account for a large proportion.This subject is mainly focused on the cell therapy of pakinson's disease,which will lay a solid foundation for later study of midbrain The developmental pathway of DA neurons,the pathogenesis of PD and the cell therapy for PD.The main pathogenesis of PD is the degenerative death of dopamine neurons in the substantia nigra pars compacta region,resulting in a decrease in the secretion of dopamine.In order to carry out cell therapy for PD,the most important thing is to obtain a steady stream of donor cells,namely DA neural progentitor cells.The main research objective of this subject is to establish a differentiation system of DA neurons.The strategy is that differentiate human embryonic stem cells into DA progentitor cells to obtain donor cells.First,hESCs were induced to differentiate into neural floor plate cells by adding specific combinations of factors,which were able to express the markers of neural floor cells,LMX1 A,FOXA2,OTX2.Then,the neural floor cells can be differentiated into dopaminergic pregentior cells by induction of factors such as FGF8.These cells are capable of expressing the midbrain markers LMX1 A,FOXA2,EN1.After we matured in vitro,it was able to express mature DA neurons markers TH,TUJ1.To investigate whether there is a difference in the differentiation potential of hESCs,we used 12 hESCs for DA differentiation.The experimental results show that the efficiency of dopamine neural progenitor cells differentiated by different hESCs is different,and screened a higher potential hESCs to provide seed cells for differentiation of DA.The current DA neuron system still has some shortcomings.The first deficiency is that the expression percentage of the DA progentitors marker LMX1 A is low.By investigating the literature,we found that LMX1 A is regulated by the WNT pathway in development,and CHIR99021 is an agonist of the WNT pathway,so we used the reporter cell line of H9-LMX1 A to screen the optimal concentration of CHIR99021,and then up regulated the expression level of LMX1 A.The second drawback is that the level of reagents does not meet clinical requirements.Since the base grade B27 contains the BSA ingredients,we replaced it with clinical grade B27.The experimental results show that clinical grade B27 can better replace the basic grade B27.The third shortcoming is the low differentiation efficiency of DA neurons.One way to improve efficiency is to provide DA neurons with the nutrients needed for differentiation by prolonging the exposing time of the two trophic factors GDNF and TGF?3,thereby increasing the expression percentage of LMX1 A,FOXA2,EN1,TH,TUJ1.Another method is to increase the differentiation efficiency of DA neurons by reducing the concentration of high SHH and increasing the degree of ventralization by adding SHH agonist PP in the process of ES cell differentiation into neural cells.From the perspective of differentiation cost,SHH is a kind of protein,which is more expensive,which can also reduce the cost of differentiation of DA neurons,which provides great help for the subsequent large supply of DA cells to treat PD.To sum up,this project mainly includes three parts: establishment of DA neuron differentiation system,differential research on the potential of DA differentiation from different hESCs,and optimization of DA neuron differentiation system.This not only provides sufficient donor cells for the cell therapy of PD,but also provides valuable experience for the optimization of DA differentiation system.