The Recovery Effect Of Ischemia Brain Injury By The Neural Stem Cells Derived From Human Induced Pluripotent Stem Cells

Background and Objective:Induced pluripotent stem cells(iPSCs)，a kind of pluripotent stem cells which aresimilar to the Embryonic Stem Cells(ESC), can differentiate into cells of all germlayers in theory. Presently, iPSCs differentiated into nerve cells including neural stemcells (NSC) and neurons in vitro, it can be used for the cell replacement therapy ofcentral nervous system diseases, has foreground of wide clinical application.However, the mechanism of iPSCs differentiate into NSC is still unknown, it’sdifficult to achieve amount of neural cells derived from iPS cells, so it’s veryimportant and meaningful for investing the mechanism of iPSCs differentiation.Studies had shown that self-renewal and directed differentiation of ESC regulated byexternal signals such as Notch, Wnt pathway, transcription factors and epigeneticmodification. The Notch pathway is one of the important signal transductionpathways in the organisms, plays an important role in stem cells renewal anddifferentiation. Stem cells performance proliferation through the combination ofactivated Notch receptor and ligand, while the Notch signaling pathway is inhibited,the stem cells to differentiate into functional cells. MiRNAs are short, non-codingsmall RNAs that regulate gene expression by controlling translation or stability ofmRNAs; it plays an extremely important role in regulating the organism development,proliferation and differentiation of stem cells and tumor development. In human,bioinformatic predictions indicate that approximately30%of all protein-coding genesincluding the Notch signaling molecule could be regulated, suggesting their importantroles in almost all biological process investigated so far. Many research shown thatmiRNAs can down-regulated the level of specific gene expression to maintain theundifferentiated state of ESC, while activation the lineage-specific gene for theirdirected differentiation of those cells during the NSC differentiation.However，there were no research report about the mechanism of iPSCs inducedinto NSC. To investigate the mechanism and differentiation of human iPSCs intoNSC, human iPSCs were differentiated into NSC at the beginning of the research, in vitro, the real-time quantitative PCR and Western blot were used to detect theexpression of Notch Signaling(Notch1, Hes1) and Notch-related miRNAs (mir-9,mir-34a, mir-200b, etc), and then theγ-secretase inhibitor-DAPT were used forfurther defined the function of notch signaling pathway during the iPSCs induced intoNSC; in vivo, the NSC derived from iPSCs were transplanted into the models ofMCAO, to provide a new theoretical foundation and technical preparation for theuse of stem cells and regenerative medical technique in treating diseases of the centralnervous system.Methods:1In vitro experiment1.1Human iPSCs were induced into the NSC through RA and serum-freemedium methods. Immunostaining was used to detect the expression of the neuralmakers in protein level after the induction.1.2The qRT-PCR was used to detect the expression changed of mir-9，mir-34a、mir-200b.1.3The qRT-PCR was used to detect the mRNA expression changed of Notch1and Hes1.1.4The immunostaining and Western blot were used to detect the proteinexpression changed of Notch1and Hes1.1.5Confirm the function of Notch signaling pathway during the iPSCs inducedinto NSC used the DAPT. The experiments were divided into four groups: RA controlgroups, RA and DAPT induction groups, RA and DMSO control groups, DAPTinduction groups. The changes of iPSCs appearance in4groups were observed underinvert microscope. The RT-qPCR and immunostaining were used to detect theexpression of genes and protein, include neural markers, Notch1and Hes1on the7thdays after induction process.2In vivo experiment2.1Middle cerebral artery occlusion (MCAO) models were prepared byobstructing MCA using a single strand nylon thread, CM-DiI labeled the NSCderived from iPSCs transplanted into rat’s striaturm through the stereotactic.Neurological function was evaluated with beam-walking, grab rope experiment and water maze. Immunofluorescence assay (Nestin, β-tublinIII, GFAP)-associatedantigen expression of rat nerve cells.Results:1In vitro experiment1.1IPSCS were induced into NSC through the RA and serum-free mediuminduction way. On the third days of induction, rosette constructs formed in RA andserum-free medium, most cells expressed Nestin and Sox2markers, after cultured forone month, those cells formed the neural net construct, and also the immunostainingshows the cells expressed the NSC markers.1.2Compared with the iPSCs groups, the expression levels of mir-9,-34a,-200bwere significantly up-regulated during the induction process.1.3Compared with the iPSCs groups, the mRNA expression levels of Notch1and Hes1were up-regulated during the iPSCs form the EB process, whereasdown-regulated after the RA and serum-free medium were for the induction.1.4Relative to the natural differentiation group, the immunostaining showsNotch1and Hes1positive cell rates in RA and serum-free medium induction groupwas increased respectively; Western blot also shows the protein levels of Notch1and Hes1decreased after induction, lowest on the14thdays after adherent culture,and then up-regulated after the adherent culture for14days.1.5Most of the cell clones adherent easily after DAPT added, and someantennas were observed around the clones; on the third day, mounts of rosetteconstruct were observed in the adherent clones, and then those clones differentiatedinto neural net construct until the second weeks. RT-qPCR and immunostaining wereused to detect the mRNA and protein expression on the7thday; the results shown thatDAPT can inhibit the Notch signaling pathway effectively, and it can affect theprocess of iPSCs differentiated into NSC.2In vivo experiment2.1The transplanted of NSC derived from iPSCs can survive in the modelsbrain,β-tubullinIII and GFAP positive cells were observed in the striatum andischemia region of model’s brain after cells transplanted for one and two weeksthrough the Immunofluorescence assay, it showed that the NSC derived from iPSCs could migrated into the ischemia region and differentiate into neural cells forischemia brain injury recovery. The behavioral deficits in experimental groups andcontrols were assessed according to the grab rope experiment, beam walking andMorris water maze scale at0d，1W，2W and3W after MCAO models prepared. Therewere no obvious differences among the groups before the MCAO or transplantation.It was found that there were no obvious sensorimotor and function difference amongthe three groups at0d, the first and the second weeks after models prepared; but at thethird weeks, the transplantation group showed greater function recovery than those ofthe other groups (PBS and sham groups) as time went on. These results demonstratedthat the NSC derived from iPS cells could improve the behavioral and sensorimotorfunction of the MCAO of rats models through transplanted those cells in the striatumof rats brain.Conclusions:1. RA and serum-free medium can promote the iPSCs differentiated into NSC;2. Notch signaling pathway can regulate the process of iPSCs differentiated intoNSC;3. mir-9、-34a and mir-200b may regulate the process of iPSCs differentiated intoNSC through the Notch signaling pathway;4. NSC derived from iPSCs can survive in the brain of MCAO rats and furthermigrated into the ischemia region and differentiate into neural cells for ischemia braininjury recovery. Also it could improve the behavioral and sensorimotor function ofthe models.