| Objective(1)This study was to establish a induced differentiation method system of hiPSCs into dopaminergic neurons,to systematically analyze the changes of gene expression and functions of lncRNAs in this induced differentiation method system,and to further screen and explore the role of key lncRNA MIAT in hiPSCs and induced differentiation.(2)The changes of gene expression in different brains from 6-OHDA rats was to detect and the key genes and pathways of PD was to select,providing a new way to study the molecular mechanism and prevention of PD.(3)This study was systematically to compare and evaluate the transplantation effect of neural stem cells(NSCs),mesenchymal stem cells(MSCs)and MSCs-derived induced cells,hiPSCs-derived induced dopaminergic cells in the treatment of PD,providing an experimental basis for the selection of graft cells and the determination of transplantation time point.Methods(1)The method of adherent culture was used to establish the induced differentiation system of hiPSCs toward dopaminergic neurons.The phase of the cells were identified by corresponding biomarkers,which were detected by RT-qPCR and immunofluorescence staining.RNAs of cells in different periods(iPSCdO,iPSCd7,iPSCdll and iPSCd25)were extracted for transcriptome sequencing.The data were analyzed using bioinformatical methods including analysis of gene expression,differently expressed genes(DEGs),target gene prediction and enriched functions.LncRNA MIAT was selected for the further study.The expression of MIAT and its predicted target gene MAPK10 were verified by RT-qPCR.The overexpressed MIAT lentivirus cell line(hiPSC-MIAT)was established by Cas9 transcriptional activation.The effects of MIAT on hiPSCs and the induced differentiation were studied at the gene and protein levels by RT-qPCR,alkaline phosphatase staining and immunofluorescence staining.(2)6-OHDA was injected by stereotactic brain surgery to establish PD rat model.The PD rat was identified by apomorphine(APO)-induced rotation test.The damage degree of dopaminergic neurons was detected by immunohistochemistry.The concentration of DA,dihydroxyphenyl acetic acid(DOPAC)and homovanillic acid(HVA)was detected by high performance liquid chromatography(HPLC).Five brain regions(olfactory bulb/OB,subventricular zone/SVZ,striatum/Str,substantia nigra/SN,hippocampus/Hippo)of rats were isolated and their RNAs were extracted for transcriptome sequencing.Gene expression,DEGs and functional enrichment were analyzed by bioinformatics.The genes were validated by RT-qPCR.The ultrastructure change was observed by transmission electron microscope.(3)The transplanted cells are grouped as follows:neural stem cells(NSCs),human placental chorionic-derived mesenchymal stem cells(hpcMSCs),cord blood mononuclear cells(CBC-MNC),adipose-derived stem cells(ADSCs)and their induced cells at 6,12 and 24 days,and hiPSCs induced cells at 11,18 and 25 days.Stem cells were labeled with molday ion rhodamine B(MIRB)and transplanted into the striatum of 6-OHDA PD rats by stereotaxic brain surgery.Behavioral evaluation included APO-induced rotation,open field test(OFT)and grasping force test.MRI was used to observe the graft cells in living brain.Immunofluorescence staining was used to observe the migration,proliferation and differentiation of the graft cells and the changes of astrocytes.The ultrastructural changes were observed by transmission electron microscopy.Results(1)The induced differentiation system of hiPSCs toward DA neurons was established.The cells were in the stage of NSCs at 5-7 days in this induced system,entered the stage of precursor of DA neurons on the 11th day,and the proportion of TH+neurons was about 40%on the 25th day.The changes of gene expression and functional regulation of lncRNAs were widely observed in the induced differentiation system.There were a large number of differentially expressed genes(DEGs)at transcription level and gene level.The functional enrichment results of DEGs and target genes of differentially expressed lncRNAs were consistent on the whole,including axon guidance,signaling pathways(Wnt/TGF-?/Hedgehog/MAPK/p53),neurodegenerative disease(PD/AD/HD),metabolic pathway and cell cycle.The top 20 statistically enriched terms in iPSCdO vs iPSCd7 also included regulation of neuronal differentiation,development of the central nervous system,Wnt signaling and apoptosis,closely related to induced differentiation.The expression of MIAT and its target gene MAPK10 in iPSCd7 group was higher than that in iPSCdO group(p<0.05),which was consistent with the results of sequencing.The hiPSC-MIAT cells proliferated in a clonal form,and the expression levels of pluripotent genes were not statistically different from those of hiPSCs.Alkaline phosphatase staining of hiPSC-MIAT was positive and a large number of OCT4 positive cells were expressed in hiPSC-MIAT cells.At the 5th day of the differentiation,a large number of cells extended and growth outward with the morphology of cell differentiation in hiPSC-MIAT,the expression levels of OCT4,EN1 and NURR1 genes were higher in hiPSC-MIAT than those in the control group(p<0.05),the genes expression levels of SOX1 and PAX6 were lower in hiPSC-MIAT than those in the control group(p<0.05).(2)6-OHDA rat PD models were successfully established,in which TH+neurons of SN were reduced by more than 90%,and in Str the contents of DA,DOPAC and HVA were respectively 4.28%,8.40%and 4.96%of the control group.The DEGs were in all five brain regions of PD models.The DEGs were mainly related to the biological processes of synapses,mitochondria,inflammation,immunity,metabolism,development,damage repair,memory,oxidative stress,apoptosis and neurodegenerative diseases.Venn analysis showed that Ephx2 and Fam111a were the common shared genes of five brain regions.PPI analysis showed the major node genes of Str including Gng2,Npsrl,Bdnf,Penk and Crh.Dopaminergic synapse and retrograde endocannabinoid signaling were two key pathways of PD,among which there are common cascades(Gi/o-GIRK,Gi/o-AC-PKA,Gi/o-MAPK)of dopaminergic,glutamatergic and GABAergic synapses.Ultrastructural abnormalities in mitochondrial morphology were found in five brain regions of the PD models.(3)The APO-induced rotation test showed that ADSCd12,iPSCd18 and iPSCd25 got better effectiveness and reached the end point of observation for 32 weeks.Among them,iPSCd18 group had the highest effective proportion.Compared with the control group,the muscle strength in groups of the cell transplantation increased(p<0.05),the total activity distance and total exercise time in groups of cell transplantation were significantly increased(p<0.05),and there was no statistical difference between the transplantation groups.Compared with the control group,the number of transregion increased in the transplantation group,and the number of transregion in the ADSCd12 group was higher than that in the iPSCd25 group(p<0.05).The MIRB-labeled transplanted cells could grow in the brain.The migration mode included local migration,migration toward the corpus callosum and long distance migration across the brain regions.At 8 weeks after transplantation,part of the transplanted cells were stained positive with PCNA,while there were fewer PCNA+cells at 32 weeks after transplantation.At 32 weeks after transplantion in iPSCd18 group,part of graft cells were differentiated into DA neurons with TH positive expression.At 8 weeks after transplantation,a large number of astrocytes around the needle passage were activated,presenting a fibrous network or an enlarged and thickened cell body,while their expression decreased at 32 weeks after transplantation.8 weeks after cell transplantation,mitochondrial morphological abnormalities were improved in all 5 brain regions of rats.Conclusion(1)In the induced differentiation system of hiPSCs toward DA neurons,cells at about 5-7 days were in the stage of NSCs,cells at about 11 days were differentiated to precursors of dopaminergic neuron,and the proportion of TH+cells at 25 days was about 40%.(2)There were extensive changes of gene expression and functional regulation of IncRNAs during the induction and differentiation of hiPSCs toward DA neurons,among which some functional regulations were closely related to the induction and differentiation.(3)LncRNA MI AT and its target gene,MAPK10,were increased at 7th day of the induction differentiation system.Overexpression of MIAT did not affect the pluripotency of hiPSCs,but promoted the differentiation of hiPSCs into midbrain precursors of dopaminergic neuron in the induction and differentiation system.(4)The 6-OHDA rat PD model can well simulate human PD at the level of pathology,biochemistry and gene.The DEGs were in all five brain regions(OB,SVZ,Str,SN and Hippo)of 6-OHDA rats.The DEGs were mainly related to the biological processes of synapses,mitochondria,inflammation,immunity,metabolism,development,damage repair,memory,oxidative stress,apoptosis and neurodegenerative diseases.(5)Common shared genes(Ephx2 and Fam111a)in 5 brain regions and major node genes(Gng2,Npsrl,Bdnf,Penk and Crh)in Str may be key targets of molecular mechanism and therapy of PD.(6)The dopaminergic synapse and retrograde endocannabinoid signaling were the key pathways of PD,among which there were common cascades(Gi/o-GIRK,Gi/o-AC-PKA,Gi/o-MAPK)of dopaminergic,glutamatergic and GABAergic synapses,which may be the molecular mechanism of synaptic damage in PD.(7)Transplantation of these kinds of stem cells and their differentiated cells in the treatment of PD rats can improve behavior disorders of rats(APO-induced rotations,muscle strength,autonomous activity)to a certain extent.The treatment effect of ADSCd12,iPSCd18 and iPSCd25 was relatively better and can be maintained for a long time,among which iPSCd18 had the highest effective proportion of behavioral evaluation.(8)The·transplanted cells can grow in the brain and migrate in different ways.The graft cells had the ability to proliferate and to promote the expression of a large number of astrocytes at the initial stage,and decreased with time.The graft cells can differentiate to dopaminergic neurons and ameliorate the damage of mitochondrial ultrastructure in brain.The age-dependent decline in stem cell function plays a critical role in aging,although the molecular mechanisms remain unclear.PTRF(polymerase I and transcript release factor)is an essential component in the biogenesis and function of caveolae,which regulates cell proliferation,endocytosis,signal transduction and senescence.This study aimed to analyze the role of PTRF in hematopoietic stem cells(HSCs)senescence using PTRF transgenic mice.Flow cytometry was used to detect the frequency of immune cells and hematopoietic stem/progenitor cells(HSCs and HPCs);and was also used to detect the phenotype of cell cycle,senescence and ROS of HSCs.RT-qPCR and western blotting were performed to analyze the expression levels of senescence-related genes and proteins.The results showed than the HSC compartment was significantly expanded in the bone marrow of PTRF transgenic mice compared to age-matched wild-type(WT)mice,and exhibited the senescent phenotype characterized by G1 cell cycle arrest,increased SA-?-Gal activity and high levels of reactive oxygen species(ROS).The PTRF-overexpressing HSCs also showed significantly lower self-renewal and ability to reconstitute hematopoiesis in vitro and in vivo.PTRF induced HSCs senescence via the ROS-p38-p16 and caveolin-1-p53-p21 pathways. |
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