The Differentiation Mechanism Of Mouse Induced Pluirpotent Stem Cells Deriving Into Neural Stem Cells

Background and Objective：Induced pluripotent stem cells(iPSCs) which were first produced by ShinyaYamanaka’s team at Kyoto University are remarkably similar to embryonic stemcells(ESCs). They have widely application potential in regeneration and implantationtherapy because of their pluripotency while there is neither ethic issue norimmunological rejection.iPSCs are potential seed cells of implantation therapy fortreating stroke, cerebral trauma, Parkinson’s disease, Alzheimer’s disease and othercentral nervous system diseases, because they could differentiate into neural glialcells, neurons and neural stem cells(NSCs). However the molecular mechanismduring iPSCs differentiating into NSCs is not well understood.According to published data, iPSCs and ESCs share similar biological character-istics. The self-regeneration and differentiation of ESCs are affected by variousfactors, such as extracellular environment, miRNAs modulation, Notch and Sirt1signal pathway. Notch signal pathway plays an important role in proliferation anddifferentiation of stem cells. Activating Notch signal pathway will poke theproliferation of stem cells, while inhibiting will accelerating their differentiation. AndSirt1, a target of mir-34a, is critical in protecting the integrity of neurons andguaranteeing neural differentiation smoothly.Our lab proved that serum free medium with retinoic acid（RA）could improvethe NSCs differentiation efficiency of iPSCs. Then we down-regulated the expressionof Sirt1by nicotinamide(NAM) to observe the changing of Notch, Sirt1, mir-34a andother relevant neural markers through qPCR, Western blot and immunofluorensence.All these experiments explored the functions of Sirt1, Notch signal pathway andmiRNAs modulation in iPSCs differentiating to NSC, established the foundation forfiguring out the mechanism of the process. Methods：1. Feeder—mouse embryonic fibroblast cells(MEF) were isolated from E12.5dICR mouse embryos and purified by0.05%trypsin. Passage3MEF were treated in10μg/ml mitomycin C for2.5h before using.2. iPSCs culture and NSCs induction—Mouse iPSCs were induced todifferentiate into NSC in serum free medium with RA. Neural markers Nestin, SOX2,Tublin3and GFAP were detected by immunofluorencense.3. Expression levels of Notch, Sirt1, mir-34a, Nestin etc. were detected throughqPCR during the whole differentiation process.4. Sirt1inhibitor NAM was added to explore its function during mouse iPSCsdifferentiating into NSCs. Data of natural differentiation group, RA group andRA+NAM group were compared. Protein level of Nestin, SOX2, β-tubulinШ, GFAP,Notch1snd Sirt1were detected through immunofluorensence or Western blot.Expression level of Notch1, Sirt1, mir-34a and Nestin were tested through qPCR.Results：1. Serum free medium with RA successfully induced iPSCs to differentiate intoNSC and OCT4level in cells was down-regulated gradually. Neural like cellsmigrated from neuralsphere and constructed neural net work after adherentculturing14d. Nestin, SOX2and other NSCs markers were up-regulated afterinducing. NSCs were capable of growing at least10passages in serum free medium.2. qPCR result—Expression level of Notch1and Sirt1were down-regulatedcompared with iPSCs during differentiation. However they were up-regulated duringNSCs proliferation. Mir-34a and Nestin were sustainedly up-regulated in the wholeprocess.3. After adding NAM, neural sphere attached quicker and neuron-like cellsmigrated quicker. Cells treated by RA and NAM expression more neural markersthan cells treated with RA alone and natural differentiating cells, however Sirt1andNotch were inhibited. These all accelerate iPSCs differentiating into NSCs andfacilitate further differentiation into terminal nerve cells. Conclusions：1. Serum medium with RA could effectively induces mouse iPSCs todifferentiate into NSCs;2. As a target of mir-34a, Sirt1might modulate mouse iPSCs differentiating intoNSCs through Notch signal pathway. Inhibiting Sirt1could accelerate iPSCsdifferentiating into NSCs and facilitate further differentiation into terminal nervecells.