Fig 1.
iNSCs directly reprogrammed from MEF cells by conditional induction of classic iPS transcription factors OSKM or OSK.
(A) OG2/rtTA MEF or GFP/rtTA MEF (4×105) cells were transfected with a tetracycline (tet)-on controlled OSKM lentivirus and induced for 6 days by doxycycline. iNSC colonies are shown onday 13. (B) The morphology of GFP/rtTA MEF-derived OSKM-induced iPS clones, OSKM-induced iNSC clones, and OSK-induced iNSC clones. (C) Comparison of iNSC neurospheres with those derived from primary NSCs from the adherent brain tissues. (D) Expansion of GFP/rtTA iNSCs and the expression of GFP in vitro. Scale bar = 100 μm.
Fig 2.
Immunostaining and mRNA expression profiling of iNSC lines for NSC markers.
Expression of NSC markers Nestin, Pax6, and Sox2 in (A) OSKM-induced iNSC1 cells (P19), (B) OSKM-induced iNSC11 cells (P19), and in (C) OSK-induced iNSC9 cells (P11). Scale bar = 25 μm. (D) qRT-PCR analysis of neural progenitor specific genes expressed by OSKM-induced cell lines iNSC1 and iNSC11, and OSK-induced iNSC9 cells vs. those of NSCs derived from the mouse brain.
Fig 3.
Differentiation of iNSC lines in vitro.
GFP was inherited from GFP/rtTA MEFs. Immunostains were performed on differentiated iNSCs with antibodies against the indicated markers: GFAP (astrocyte marker); TUJ1 (pan-neuronal marker); MAP2 (mature neuronal marker); O4 and CNPase (oligodendrocyte markers). (A) Differentiation of NSCs derived from the mouse brain. (B) Differentiation of iNSC1 cells. (C) Differentiation of iNSC11 cells. (D) Differentiation of iNSC9 cells. GFAP+ astrocytes, MAP2+ neurons, and Oliogodendrocytes-like cells were differentiated. Scale bar = 25μm.
Fig 4.
Tumor risk determination of iNSCs.
(A) 2×105 iNSC1 cells or ESCs were injected into the subcutaneous region of the same immunodeficient BALB/c nude mouse (n = 3) and tumor formation was assessed on day 45. Tumors were found in ESCs injected sites, no tumor were found in iNSC1 injected sites. (B) Tumor formation analysis of 2×105 iNSC11 cells and GFP/rtTA iPSCs in the same immunodeficient BALB/c nude mouse on day 45. Tumors were observed in iPSCs injected sites but not in iNSC11 injected sites.
Fig 5.
Behavior tests, MRI and MRS and neuron protection mechanism analysis of MCAO rats treated with iNSCs.
(A) mNSS test of MCAO rats that received PBS(n = 12) or NSCs (n = 12) or iNSC1 (n = 12) treatment post MCAO surgery. NSCs or iNSCs treatment was significantly less mNSS score than PBS treatment on days 7, 10, 14 after transplantation (p <0.05). (B) Adhesive sticker removal test. NSCs or iNSCs treatment was significantly less adhesive removal time than PBS treatment on days 7, 10, 14 after transplantation (p <0.05). (C) T2 MRI of rats with identical lesion size in the same region of the right brain 2 days before treatment and 2 weeks post-MCAO after PBS or iNSC1 cell treatment. (D) MRS images of the MCAO rat treated with PBS or (E) with iNSCs in the ROI2 days and 2 weeks post-MCAO. (F) Magnetic resonance spectroscopy (MRS) images of normal adult rat. (G) Mean value of Mc represents changes of metabolic status in the right brain ROIs of MCAO rats treated with PBS (n = 12 in 3 rats) or iNSC (n = 8 in 2 rats), *P<0.05. The error bars are SE. NAA, N-Acetylaspartate. Cr, Creatine. Cho, Choline. (H) Elisa tests of nerve growth factors BDNF, GDNF, and NT3 secreted by iNSC1 cells and NSCs derived from the E14 of C57 mouse brain. (I) Comparation of neurons in the penumbra of the rats received PBS or iNSCs treatment 8 weeks post-MCAO. Scale bar = 5μm.
Fig 6.
Migration and differentiation of iNSC-derived cells in MCAO rats.
(A) Diagram showing the sites on the A–P axis at which iNSCs were transplanted and the migration pathway of the transplanted iNSCs (green dots) in the lesion (shaded area). (B) One day post-transplantation, iNSCs with GFP had assembled at the injection site. (C) Migrated graft cells expressing the GFP marker across the lesion of MCAO rats 6 weeks after transplantation. (D) Higher magnification of demarked area in C. Migrated cells survived in the lesion of the right cerebral cortex. Scale bar = 50 μm. (E) Confocal images: Graft cells labeled with GFP are green, nuclei stained with DAPI are blue, and various phenotypic markers are in red. iNSC1 cells differentiated into GFAP-positive astrocytes in 2 weeks, and Tuj1-positive neurons in 6 weeks. Undifferentiated iNSCs were detected by Nestin antibody. Scale bar = 25 μm.