1. Direct Conversion of Mouse Fibroblasts into Neural Stem Cells by Chemical Cocktail Requires Stepwise Activation of Growth Factors and Nup210 
Yuewen Tang, Shumin Xiong, Pei Yu, Feng Liu, Lin Cheng 
Cell Reports 
Volume 24, Issue 5, Pages e3 (July 2018)
DOI: /j.celrep
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2. Cell Reports 2018 24, 1355-1362.e3DOI: (10.1016/j.celrep.2018.06.116)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1 Direct Conversion of Fibroblasts into Neural Stem Cells
(A) NES-GFP− MEFs and TTFs showed NES-GFP+ signal 7 days after the chemical cocktail treatment in M5300. Scale bar, 25 μM.
(B) NES-GFP+ cell numbers gradually increased from day 4 to day 8 to day 12 after NES-GFP− MEFs and TTFs were treated with the chemical cocktail. Left panel shows representative figures at each time point. Right panel shows the quantified data of the left panel. Scale bar, 25 μM.
(C) ciNSCs with high purity were generated. Scale bar, 25 μM.
(D) ciNSCs acquired the proliferation property.
(E) ciNSCs could be passaged, and both cells at passage 24 could still form neurospheres and maintained the neural stem cell markers. Scale bar, 100 μM.
Data presented as mean ± SEM. See also Figure S1.
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4. Figure 2 RNA-Seq and ATAC-Seq Analyses of Cell Conversion Process
(A) Heatmap of differentially expressed genes from RNA-seq data.
(B) Principle component analysis of RNA-seq data.
(C) Heatmap of subset of selected NSC-, fibroblast-, and pluripotency-specific genes.
(D) Left panel shows heatmap of three clusters (I, II, and III) of ATAC-seq peaks activated by the chemical cocktail. Two subgroups of ATAC-seq peaks within cluster III are activated at different time points after the chemical cocktail treatment. Right panel shows distribution of ATAC-seq peaks shown at left with respect to the transcription start site of genes using GREAT analysis.
(E) Genome browser view of the Sox2, Sox3, and Sox1 locus. ATAC-seq peaks activated in NES-GFP+ cells are highlighted in pink.
See also Figure S2.
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5. Figure 3 Growth Factors Contribute to Cell Conversion
(A) Real-time qPCR analysis of the expression of growth factors Il-6, Fgf5, and Lif in cells at different stages during the cell conversion, including initial MEFs, bulk MEFs treated with the chemical cocktail for day 1, NES-GFP− cells and NES-GFP+ cells at day 4 and day 8 after the chemical treatment, and purified ciNSCs derived from MEFs.
(B) Representative figures and the quantitative analysis of the chemical cocktail-induced NES-GFP+ cell generation from MEFs treated with antibodies against IL-6 (from day 0 to day 2), FGF5 (from day 2 to day 6), and LIF (from day 2 to day 6).
(C) Representative figures and the quantitative analysis of the chemical cocktail-induced NES-GFP+, Sox2+, and double-positive cell generation from MEFs treated with Il-6 for early 2 days.
(D) Real-time qPCR analysis of the expression of Sox1, Sox2, and Sox3 in cells from (C).
(E) Schematic diagram of sequential addition of growth factors to convert mouse fibroblasts into neural progenitor-like cells (NPLCs) in the absence of the chemical cocktail.
(F) Representative images for complete growth factor-induced bipolar NES-GFP+ cells from NES-GFP− cells on day 12, expressing Sox1, Sox2, and Sox3.
∗p < 0.05, ∗∗p < 0.01, compared with control (ctrl). Scale bar, 25 μM. Data presented as mean ± SEM. See also Figure S2 and S3.
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6. Figure 4 Nup210 Is Essential for Cell Conversion
(A) Representative figures and quantified data of the nuclear size of the chemical cocktail-treated NES-GFP− MEFs for 12 days. ∗p < 0.05, compared with NES-GFP−.
(B) Expression levels of nucleoporins.
(C) Nup210 knockdown in MEFs by small interfering RNA (siRNA)-1 (siNup210-1) and siRNA-2 (siNup210-2). Upper panel of representative figures shows the NES-GFP+ and Sox2+ cell generation from NES-GFP− MEFs by the chemical cocktail treatment for 12 days. Nuclear size and cell number were quantified and shown in lower panels. ∗p < 0.05, ∗∗p < 0.01, compared with ctrl.
(D) Nuclear size quantification of growth factor (GF)-induced cells from Nup210 knockdown MEFs on day 12. ∗p < 0.05, compared with GFs.
(E) Quantified data for NES-GFP+ cell generation via GF induction from Nup210 knockdown NES-GFP− MEFs on day 12. ∗p < 0.05, ∗∗p < 0.01, compared with GFs.
(F) Real-time qPCR analysis of the expression of Sox1, Sox2, and Sox3 in GF-induced cells from Nup210 knockdown MEFs on day 6. ∗p < 0.05, ∗∗p < 0.01, compared with GFs.
Scale bar, 25 μM. Data presented as mean ± SEM. See also Figure S4.
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