Optimizing The Method Of IPSC Derived MDA Neuron To Study The Pathogenesis Of PD And The Therapeutic Effects Of PB-Ⅱ On PD

ObjectiveInduced pluripotent stem cells（iPSCs）can differentiate into midbrain dopaminergic neurons（mDAn）,providing an ideal cell model for in vitro studies of PD.However,the existing methods of inducing iPSC are very inefficient and there are cases of gene integration.There are many methods for iPSC to differentiate mDAn.The differentiation efficiency and purity are generally low,which brings some uncertain factors to the in vitro research of PD.Based on previous studies,this paper intends to establish a gene non-integrated iPSC cell line derived from somatic cells of PD and normal healthy people by optimizing the method system of iPSC induction and its mDAn differentiation.We used FACS to improve the purity of the mDAn obtained from differentiation and obtain higher purity human mDA neurons.We comparatively analyzed the phenotypic differences of cells from PD-mDA and healthy-mDAn,and looked for key data on abnormal PD-mDAn cell performance under in vitro culture conditions to provide data references for exploring the pathogenesis of PD.Using the iPSC directed differentiation mDAn system,we studied the molecular mechanism of traditional Chinese medicine compound PaBing-Ⅱ（PB-Ⅱ）that is commonly used in the treatment of PD to protect neuronal cells in Guangdong Provincial Hospital of Traditional Chinese Medicine.Methods1.Construction of gene non-integrated iPSC.We improve on the induction method of Yamanaka laboratory.We first obtained human skin tissues using a skin sampler with a diameter of 2 mm through the theoretical evidence of the hospital and the patient’s informed consent,and the primary culture was a skin fibroblast cell line.We used Lonza nuclear transfection to transfect Y4 factor（pCXLEhOCT3/4-shp53,pCXLE-hSK,pCXLE-hUL）into skin cells and induce cell reprogramming.Next,we selected the successfully reprogrammed cell clones,performed expansion culture,and performed cell pluripotency and gene non-integrity verification.We chose a gene non-integrated iPSC with better pluripotency for subsequent experiments.2.Optimized iPSC derived mDAn method.This laboratory has been committed to studying the method of iPSC directed differentiation of mDAn.We have improved and optimized the neuro-differentiation method published by Professor Tongxiang Lin in PANS in 2011,and refer to mDAn flow sorting and purification reported by previous researchers（Woodard CM,Cell Report,2014;Pruszak J,Stem Cells,2007）.We use a two-step differentiation method.In the early stage of iPSC-induced differentiation,we induced differentiation of neural stem cells with LDN193189,SB431542,CHIR99021,and FGF8b.After 15 days of culture,we then sorted CD15/CD133 double-positive cells by FACS.Population,these cells are considered NSC cells,and continue to culture and induce differentiation.Then we replaced the medium with a medium containing BDNF,GDNF,dbcAMP,and N2B27,and continued to culture the cells for 30 days.Then we used FACS to sort the CD15/CD184 double negative and CD56/CD24 double positive cell population.After one week of sorting,the cells were analyzed for mDAn cell phenotype and functional verification.We tested the expression of genes and proteins such as TH,TUJ1,FOXA2,and MAP2,which are specific to mDAn cells.We also tested the electrophysiological tests of mDAn cells to ensure that the cells obtained from differentiation and purification are mDAn cells with higher purity.Provide guarantee for the scientificity and reliability of subsequent experimental results.3.Study the pathogenesis of PD.We obtained somatic cells from PD and normal humans,constructed a gene non-integrated iPSC,and directed differentiation into mDAn cells.We compared the phenotypic differences between PD-mDAn and Heal thy-mDAn.We used immunofluorescence to analyze the expression of TH/TUJ1 double-positive cells in PD-mDAn and Healthy-mDAn cells,and counted the difference in mDAn-specific markers between the PD and Healthy-mDAn.Using IF and Western Blot technology,we analyzed the expression of α-Synuclein in PD-mDAn and Healthy-mDAn cells.qRT-PCR was used to check the expression of mDAn-specific genes TH,TUJ1,Foxa2 and Enl in the both cells.We analyze data on these phenotypic differences in an attempt to find the molecular mechanism of PD-mDAn cell phenotype abnormality.4.The molecular mechanism of PB-Ⅱ in the treatment of PD.Since PBⅡ has a significant effect on PD in clinical,we planed to analyze the neuroprotective mechanism of PB-Ⅱ drug-containing serum on iPSC derived mDAn.We prepared rat PB-Ⅱ drug-containing serum.The mDAn cells were divided into a blank control group（Ctrl）,an oxidative damage model（ODM）group,a blank serum group（Blank Serum,BS）,and a drug-containing serum group（Medicated Serum,MS）.The cells of ODM group were treated with 100 μm H₂O₂ for 12 hours.Before H₂O₂ treatment,the cells of BS group and MS group were pretreated with 10%blank serum and 10%PB-Ⅱ containing serum for 24 hours.We used immunofluorescence to analyze the expression of TH and TUJ1 in each experimental group,and FACS to analyze the apoptosis and ROS level in each experimental group.In our previous study,we found that PB-Ⅱ can enhance the antioxidant capacity of cells by activating Nrf2 pathway of rat mDAn.We analyzed the expression of Nrf2 and its downstream genes HO-1 and NQO1 in each experimental group by Western blot and qRT-PCR.Based on the above data,we try to find the molecular mechanism of PB-Ⅱ serum to protect mDAn from H₂O₂ oxidation.Results1.Establishment of genetically non-integrated iPSC cell lines.We used immunofluorescence to detect the expression of pluripotent factors.The results showed that the cells highly expressed SSEA4,Oct4,TRA-1-60 and TRA-1-81.qRT-PCR results also confirmed that these cells highly expressed the mRNA of Oct4,Nanog and Sox2.The results of teratoma experiments showed that the induced cells could differentiate into three germ layers of endoderm,mesoderm and ectoderm in NOD-SCID mice.These experimental results show that the cells have pluripotency.qRT-PCR was used to detect the integration of foreign genes in the cells.The results confirmed that most of these cells did not integrate foreign genes,that is,non-integrated iPSC genes.We removed the gene-integrated cell line and took non-gene-integrated iPSC cells for subsequent experiments.2.Improving the efficiency and purity of iPSC derived mDAn.The experimental results show that in cells cultured for 10 days after two cell sorting and purification,the protein and mRNA expression of the mDAn cell-specific genes TH,TUJ1,FOXA2,and MAP2 are highly expressed.The proportion of cells was reach to 70%,suggesting a higher proportion of mDAn cells.The patch-clamp technique was used to detect the electrophysiological phenomena of these mDAn cells,and it was confirmed that these cells had the electrophysiological characteristics peculiar to human midbrain dopaminergic neurons,including action potential,Na+ current and K+ current.The experimental results showed that these cells have the functionality of mDAn cells.It can provide an ideal cell model for subsequent experiments to study the neuroprotective effect of PB-Ⅱ and the pathogenesis of PD.3.Explore the pathogenesis of PD.We comparatively analyzed the cell phenotypic differences of mDAn from PD and normal people.Immunofluorescence data showed that in the early stage of iPSC-mDAn cell differentiation,that is,the NSC stage and mDAn precursor cell stage,cells from PD and normal healthy human cells did not show cell phenotypic differences,suggesting that iPSC-NSC-mDA Progenitor cell stage,PD cells may not be functionally abnormal.But in the mature mDAn stage,that is,40 days after differentiation,we found that PD-mDAn has more α-synuclein（α-Syn）accumulation than Healthy-mDAn.With the increase of time,the accumulation of α-Syn in PD mDAn cells is more serious.After 60 days of differentiation,the ratio of TH/TUJ1 double positive cells in PD mDAn was lower than that in healthy mDAn.These data suggest that the abnormal accumulation of α-Syn in PD-mDAn cells may be serious over time,resulting in PD-mDAn cells with functional impairment in the late stage of differentiation.These data provide research direction and data support for the study of PD pathogenesis.In the next step,we will study the molecular mechanism of abnormal accumulation of α-Syn in PD-mDAn cells,and try to find the molecular mechanism of PD pathogenesis.4.The protective effect of PD-Ⅱ on mDAn cells injured by H₂O₂.The percentage of TH/TUJ1 double positive cells in MS group was significantly higher than that in BS group and ODM group.The results of flow cytometry showed that the apoptosis rate of MS group was significantly lower than that of BS group and ODM group,which indicated that PD-Ⅱ had the anti-oxidation effect on mDAn.We further found that the expression of Nrf2 and its downstream genes in MS group was higher than that in BS group and ODM group,and the ROS in MS group was lower than that in BS group and ODM group.Combining our previous animal experimental data showing that PD-Ⅱ can activate the Nrf2-ARE pathway of neuronal cells,We conclude that the protective mechanism of PD-Ⅱ on oxidative damaged neurons may be through activating Nrf2-ARE pathway of endogenous cells,which can improve the antioxidant capacity of cells,reduce the level of ROS and reduce the apoptosis rate of neurons,and play a role in neuron protection effect.Conclusion1.On the basis of the traditional method of inducing IPSC by plasmid transfection,by adding small molecular compounds to promote cell reprogramming and IPSC maturation in different stages of skin fibroblast induced IPSC,the induction efficiency is about 10 times higher than that of the traditional method,and the integration of foreign genes is detected as gene non integration,and gene non integration from PD and normal healthy human skin cells is constructed IPSC cell line.2.The traditional iPSC method for directional differentiation of mDAn was optimized,the mDAn differentiation efficiency and purity of the mDAn were improved,and mDAn cells expressing more than 70%of TH/TUJ1 double positive cells were obtained.At the same time,the cells highly expressed human mDAn-specific genes,The mDAn cell line with specific electrophysiological phenomena of human mDAn cells is considered to be more in line with the actual situation of the human body.It is suitable for studying dopaminergic neuron degenerative disease mDAn cell lines,providing in vitro research on the mechanism of drug treatment of PD and the pathogenesis of PD ideal cell model.3.Through systematic comparison and analysis of the cell phenotype of mDAn from PD and its healthy siblings,we found that during the iPSC-NSC-DA Progenitor stage,there were no cell phenotypic differences between these cells from PD and healthy people,Suggesting that early PD dopaminergic neuron precursor cells may not have dysfunction.However,the accumulation of α-Syn appeared in PD-mDAn at maturity stage,and the accumulation of α-Syn increased with time.After 60 days of differentiation,the proportion of TH/TUJ1 double positive cells in PD-mDAn cells decreased,suggesting that PD-mDAn may have abnormal cell function in the middle and late stages.We confirmed that the accumulation of α-Syn in PD-mDAn cells was abnormally accumulated,resulting in a loss of function in PD-mDAn cells in the late stage of differentiation.These data provide data references for the study of PD-mDAn cell phenotype abnormalities,and provide entry points and theoretical basis for further research on the pathogenesis of PD.4.We illustrated that PB-Ⅱ containing serum could enhance the antioxidant capacity of H₂O₂-mDAn cells,reduce the ROS level and apoptosis of H₂O₂-mDAn cells,and increased the expression of TH/TUJ1,which can protect H₂O₂-mDAn cells and provided data support and theoretical basis for the clinical treatment of PD with PB-Ⅱ.