Comparing The Induction System Of UC-iPSC Derived NSC And Preliminary Study Of NSC In HIE Model

ObjectiveSelect an efficient inducing system of urine cells derived induced pluripotent stem cells(UC-iPSC) being differentiated into neural stem cells(NSC) from the two kinds of inducing system. Detect the survival, differentiation and migration of UC-iPSC derived NSC in hypoxic- ischemic encephalopathy(HIE) animal model. MethodsCompared the efficiency of two different inducing system. A inducing system: SB431542(5 ug/ml), Drosomophorin(5 ug/ml) and 100% initial cell density. B inducing system: SB431542(5 ug/ml), Drosomophorin(1ug/ml) and 30-50% initial cell density. We compared the efficiency of two different inducing system by observing morphological changes of the cells, detecting the expression of related genes such as Pax6, Nestin, Sox1, Sox2 by q-PCR and comparing the positive rate of Pax6 at the 16 th day via flow cytometry analysis.Established the HIE animal model: HIE was induced in postnatal day 7(P7) of SD rats by isolating, ligating and cutting the left common carotid artery and then expos ing to hypoxic environment(8% O2,92% N2, 38℃) for 120 mins. Observed the brain damage area b y TTC staining at the 7th days after surgery. NSCs(300,000 in 3ul) were transplanted into the left lateral ventricles of the HIE animal models at the 3th day after surgery. O n the 7th and 28 th day, the HIE animal models subjected to heart perfusion flow and then fixing the brain, sectioning, immunofluorescence staining, analyzing the survival, migration and differentiation of exogenous neural stem cells of the HIE animal model brain by laser scanning confocal microscope. ResultsThe first generation(P1) of NSCs from A system presented bright sphere shapes, got together closely, while the NSCs from B were dark and irregular and aggregated loosely.Q-PCR showed that NSCs from A expressed NSC related genes more highly than that of B system. The positive rate of Pax6 was also higher than that of B system at 16 th day by flow cytometry. Q-PCR and immunofluorescence showed that the P3 NSCs expressed Pax6, Nestin, Sox1, Sox2 highly. The NSCs could form evident nerve fiber bundle at 20 th day of Spontaneous differentiation and immunofluorescence demonstrated the cells expressing TUJ-1, MAP2 and GFAP. TTC staining showed lesions significantly in the cortex and striatum of HIE model after 7 days. Immunofluorescence showed that exogenous NSCs could survive in the left ventricle, nestin negative exogenous cells could migrate from lateral ventricle to lesions at 7 days after transplantation and exogenous NSCs could differentiate into neurons and astrocytes around lesions at 28 days. ConclusionThe inducing system with 5ug/ml of SB431542 and Drosomophorin and 100% initial cell density is better.The UC-iPSC derived NSCs can survive, differentiate and migrate in the brain of HIE model.