Experimental Study Of The Construction And Neural Differentiation Of Ips ( Induced Pluripotent Cells) Neural Precursor Cells Derived From Mscs

[ Background and objection]:Bone mesenchymal stem cells (MSCs) are one of non-hematopoietic stem cells in bone morrow, which have shown to possess the ability of self-renewal and multiplex differentiation potential. A number of different approaches were reported to trigger this apparent trans-differentiation in vitro,which including cytokines, co cultured with neural stem cells, gene transfection, chemical materials et al. However, these methods were proved to be ineffective, low ratio, and less of neuro-electrophysiological function, which limited the clinical use of these methods. Reprogrammed pluripotent cells was a new excited technology, demonstrating for the first time that the reprogramming process can be achieved without going through a nuclear transfer into the eggs, but with the introduction of some transcription factors: Oct4/Sox2/cMyc/Klf4, was able to convert somatic cells from a terminally state to an embryonic state .The anterior consideration guided our choice for genetic engineering of neural precursor cells. In this study, we choose the transcription factor Ngn2 as an inducer for converting the MSCs into neuronal precursor cells without causing cellular stresses. Following proper induction with some neuro-differentiation factors such as EGF, bFGF, BDNF, the MSCs-derived ips neuronal precursor cells differentiated into functional neurons turly. [Methods]1. Rat MSCs isolation and cell culture, Construction of recombinant lentiviral vector with Ngn2 and transductionRat bone marrow mesenchymal stem cells (BMSCs) was cultured and proliferated from bone marrow of SD rat in vitro sterile. Flow cytometry was used to determine the surface markers of passage-3 MSCs, including: CD34,CD45,CD44,CD29.The full-length mice Neurogenin2 cDNA was isolated and constructed the recombinant lentivirus DNA pHIV-EGFP-Neurogenin2, the recombinant lentivirus was packaged and grew in 293T cells DNA. The recombinant lentivirus were used to transfect rat MSCs.2. Neuronal induction of lenti-Ngn2-MSCs with cykotinesAfter gene transduction, lenti-Ngn2-MSCs were incubated in the neuronal induction medium: L-DMEM/2% FBS containing 10ng/ml epidermal growth factor(EGF, Peprotech ), 20 ng/ml basic fibroblast growth factor(bFGF, Peprotech) for 5 days,and then in L-DMEM/2%FBS medium containing 20 ng/ml Brain-Derived Neurotrophic Factor (BDNF, Peprotech) for 2 weeks. The morphology changes of MSCs were studied after MSCs had been transduced to express Ngn2 during the neuronal induction with EGF,bFGF , BDNF. In the next set of experiments, we examined whether MSCs transduced with neurogenic fate determinants, besides adopting morphological and immunocytochemical neuronal characteristics, also acquire the electrophysiological hallmarks of neurons, such as the voltage-gated Na + and K+ channels.3. To explore the molecular mechanisms of transdifferentiation of MSC into neuronal cellsAs we have known that during the development of CNS, the pro-neural bHLH factors (for example,Ngn1 and Ngn2) and DNA methylation, not only inhibit STAT1/3 from activating glial genes, but also suppress the phosphorylation and activation of the JAK-STAT pathway to inhibiting gliogenesis during the neurogenic period. In order to investigate the intrinsic mechanism of Neurogenin2 in the deriving of neuronal differentiation of MSCs, we detected the phosphorylation expression level of the neuronal development related transcription factors such as STAT3,ERK, JNK by western blot.[ Results]1. The rat MSCs, cultured with anchorage velocity-dependent separation method, proliferated quickly and had a favorable biological nature . MSCs were uniformly positive for CD105 and CD44 but negative for CD34 and CD45 which according with the characteristics of mesenchymal cells by Flow Cytometry technology. The titer of prepared lenti- virus which contained the gene of Neurogenin2 and EGFP were about 1*108TU/ml. And the transfection efficiency of rat MSCs with lenti- virus was about 93%. After gene transfection, MSCs -derived ips neural precursor cells continuously proliferated in growth medium without apparent morphological changes. Western blot analysis of neural precursor markers further revealed that a significant increased expression in nestin, pax6,vimentin and musashi1 after transduction of neurogenin2 of MSCs.2. Following 2 weeks of neuronal differentiation with EGF,bFGF and BDNF,aconsiderable portion of Ngn2-MSCs expressed NeuN and some Ngn2-MSCs also respond forβ-tubulinⅢand MAP2. High level expression of NeuN,β-tubulinⅢand MAP2 but only very low level expression of those protein in the parental and lenti-GFP MSCs by wetern blot analyses. After 2 weeks incubating in neuronal induction medium, we found that Ngn2-MSCs acquire the voltage-gated Na + and K+ channels that were absent in parental MSCs. 3. The phosphorylation (Tyr705) level of STAT3 was decreased after enforced expression of neurogenin2 in MSCs(P<0.05). however, the phosphorylation ERK and JNK had no significant changed after gene transfection of Neurogenin2 in MSCs(P>0.05). The phosphorylation (Tyr705) level of STAT3 still at a relatively low level versus MSCs of gene transfection after neural induction with growth factors (P>0.05), but the phosphorylation expression level of ERK, JNK had significantly up-regulated (P<0.05)[Conclusion]1. Ips neural precursor cells derived from MSCs could be prepared and differentiated into functional neurons by gene transduction of MSCs with Ngn2.2. This internal mechanism underlying this differentiation may be associated with the activation of STAT3 after neurogenin2 enforced expression earlier, but inhibition of STAT3 and activation of ERK,JNK following neuronal induction later.