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SC_11-0848_sm_supplFigure1.pdf342KFigure S1 The expression of neural markers in diNSC-derived cells. (A) After a 14 day differentiation period, cells differentiated from diNSC-derived neurospheres were subjected to immunostaining. The proportion of cells with or without the expression of each neural marker is shown. (B)(C) GFP fluorescence in neurospheres derived from diNSCs established from adult Nestin-EGFP mouse fibroblasts. Phase-contrast (B) and fluorescence (C) micrographs are shown. Scale bar=100 μm
SC_11-0848_sm_supplFigure2.tif316KFigure S2 Detection of Nanog-GFP positive pluripotent cells on day 4 following the introduction of KSOM reprogramming factors. On day 4, corresponding to the start of the suspension culture for the fabrication of neurospheres, no Nanog-GFP positive cells were detected by FACS calibur.
SC_11-0848_sm_supplFigure3.pdf251KFigure S3 The effect of cAMP analogues on diNSC-derived neurosphere forming efficiency. (A) Dose-dependent effect of pCPT-cAMP on the formation frequency of diNSC-derived neurospheres from fibroblasts. pCPT-cAMP was most effective at a concentration of 100 μM. (B) 8-pCPT-2-O-Me-cAMP did not increase neurosphere-forming efficiency at a concentration of 100 μM (n=4; no significant difference), while pCPT-cAMP did (n=8; *P<0.05).
SC_11-0848_sm_supplFigure4.tif1674KFigure S4 Neurons and Astrocytes derived from diNSCs grown in EGF diNSC-derived neurospheres grown with EGF were allowed to differentiate without growth factors for 7 or 14 days, followed by immunostaining. The βIII-tubulin-positive neurons and GFAP-positive astrocytes were similar to those obtained from cultures without EGF.
SC_11-0848_sm_supplFigure5.pdf520KFigure S5 Microarray analysis of diNSC-derived neurospheres (A) Table of twelve samples including four diNSC-derived neurospheres prepared for microarray analysis. (B) The total RNA from the twelve kinds of samples was extracted and subjected to microarray analysis. The heat map of the hierarchically clustered microarray data is shown. (C) Expressions of fibroblast marker genes are shown. Values presented are the signal intensity detected by microarray analysis. (D) Expression levels of mesoderm and endoderm marker genes are shown. Values are the signal intensity detected by microarray analysis.
SC_11-0848_sm_supplFigure6.tif2071KFigure S6 Retroviral transgenes are silenced in diNSC-derived cells FACS analysis of GFP-positive cells on days 0 and 14 of the suspension culture used to make diNSC-neurospheres. On day 0 of suspension culture, 61.2% of the fibroblasts were GFP-positive. The GFP-positive cells were separated by FACS and subjected to suspension culture. On day 14, only 14.4% of the total cells in these neurospheres were GFP-positive. 4F = KSOM. (B) Microscopic observation of the GFP-positive cells in a neurosphere. (C) Transgene expression following 14 days of suspension culture was measured by quantitative PCR and the delta Ct method.
SC_11-0848_sm_supplFigure7.tif608KFigure S7 Retroviral transgenes are silenced in the diNSC-derived cells produced with EGF diNSC-derived neurospheres were established from Nanog-GFP fibroblasts transduced with the four KSOM reprogramming factors (4F) and DsRed in the presence of EGF. Although no Nanog-GFP-positive cells were observed in the EGF-dependent diNSC-neurospheres, a few DsRed-positive cells were observed in these neurospheres on day 14 of suspension culture.
SC_11-0848_sm_supplFigure8.pdf142KFigure S8 Teratomas generated from diNSCs transplanted into mouse striatum Hematoxylin-eosin staining of teratomas formed in the striatum of C57b6j mice showing the differentiation of transplanted cells into cell types of all three germ layers: ectoderm, mesoderm, and endoderm.
SC_11-0848_sm_supplFigure9.tif598KFigure S9 Oct4-positive cells in differentiated human diNSC-derived neurospheres. Human diNSC-derived neurospheres were allowed to differentiate without growth factors for 10 days and were then subjected to immunostaining. A small number of Oct4 positive cells was observed.
SC_11-0848_sm_supplFigure10.tif2622KFigure S10 Efficiency of diNSC-derived neurosphere formation from embryos and adult animals. The number of neurospheres derived from 5x105 partially reprogrammed cells is shown. The frequency of diNSC-derived neurospheres was 3 times higher in embryonic cells (n=3; *P<0.001).
SC_11-0848_sm_supplTable1.pdf84KSupplementary Table 1
SC_11-0848_sm_supplTable2.pdf123KSupplementary Table 2
SC_11-0848_sm_supplTable3.pdf32KSupplementary Table 3
SC_11-0848_sm_supplTable4.pdf7KSupplementary Table 4

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