1. Volume 21, Issue 2, Pages 264-273.e7 (August 2017)
Direct Reprogramming of Fibroblasts via a Chemically Induced XEN-like State 
Xiang Li, Defang Liu, Yantao Ma, Xiaomin Du, Junzhan Jing, Lipeng Wang, Bingqing Xie, Da Sun, Shaoqiang Sun, Xueqin Jin, Xu Zhang, Ting Zhao, Jingyang Guan, Zexuan Yi, Weifeng Lai, Ping Zheng, Zhuo Huang, Yanzhong Chang, Zhen Chai, Jun Xu, Hongkui Deng 
Cell Stem Cell 
Volume 21, Issue 2, Pages e7 (August 2017)
DOI: /j.stem
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2. Cell Stem Cell 2017 21, 264-273.e7DOI: (10.1016/j.stem.2017.05.019)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
Developing a Strategy to Chemically Induce Neuronal-like Cells
(A) Phase-contrast images and induced efficiency of primary colonies by using chemical compounds (TD114-2 cocktail or CHIR cocktail).
(B) qRT-PCR analysis of XEN master genes (Gata4, Sall4, Sox17, and Gata6) by chemical induction with TD114-2 cocktail or CHIR cocktail (n = 2).
(C) Co-immunostaining (Gata4, Sall4, Sox17, and Gata6) for the primary colonies induced by the TD114-2 cocktail.
(D) Diagram of the XEN-like-state-based chemical reprogramming approach.
(E) Efficiency of TauEGFP-positive cells induced by the TD114-2 or CHIR cocktail with different starting cell densities (determined by fluorescence activated cell sorting [FACS]) (n = 2).
(F) Representative images of TauEGFP-positive colonies induced by TD114-2 cocktails before and after further specification.
(G) Hierarchical clustering analysis for chemically induced TauEGFP+ colonies, primary neurons, and fibroblasts.
(H) Gene expression heatmap of neuron-specific genes and fibroblast-specific genes after chemical induction (analyzed by RNA-seq).
Scale bars, 100 μm. Data are presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < (Student’s t test). See also Figure S1 and Tables S1 and S4.
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4. Figure 2 Characterization of Chemically Induced Neurons
(A–D) TauEGFP-positive induced cells co-expressed pan-neuronal markers [TUJ1 (A), NF-H (B), MAP2 (C), and SYN (D)].
(E–H) TauEGFP-positive induced cells expressed genes typical of functional subtypes and mature neurons [vGLUT1 (E), GABA (F), and NEUN (G)] after co-culture with astrocytes. (H) The mature neuronal transcriptional factor NEUN was detected in 80% of the induced TauEGFP-positive cells after further maturation; DAPI also stained the co-cultured astrocytes.
(I) Patch-clamp recordings on TauEGFP-positive induced cells after co-culturing with astrocytes.
(J) Action potentials were elicited on TauEGFP-positive induced cells. One exemplary trace of action potential was highlighted (n = 11).
(K) Whole-cell voltage-clamp recording of TauEGFP-positive cells and inward currents were recorded, and the sodium currents were blocked by tetrodotoxin (TTX) (n = 11).
(L) Spontaneous excitatory postsynaptic currents (EPSCs) were recorded after co-culturing with primary astrocytes, which were blocked with 20 μM CNQX plus 50 μM AP5 (n = 6).
(M) Focal application of 100 μM glutamate induced inward membrane currents (n = 5).
(N) Scheme for transplantation of induced TauEGFP-positive cells into adult mouse brain.
(O) TauEGFP-positive cells survived and matured after transplantation. TauEGFP-positive cells being transplanted into the striatum co-expressed neuronal-specific (TUJ1 and MAP2) and mature neuronal genes (NEUN).
Scale bars represent 100 μm (TauEGFP), 75 μm (NEUN), 25 μm (TUJ1), and 50 μm (MAP2).
(P) Quantification of TauEGFP-positive cells at different distances from the injected site (top) (n = 2). Percentage of mono-nucleated and bi-nucleated cells (bottom left) (total examined cell number = 551, from three independent transplantation experiments). Quantification of the grafted NEUN/TauEGFP-double positive cells (bottom right) (n = 66/234).
(Q) Action potentials were elicited on TauEGFP-postive cells after being transplanted into brain striatum (28 days and 35 days). One exemplary trace of action potential was highlighted (n = 7).
Scale bars represent 100 μm (A, C, E, G, and I), 200 μm (B, D, and H), and 50 μm (F). See also Figure S2 and Table S2.
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5. Figure 3
XEN-like State Based Chemical Reprogramming Bypasses Acquisition of Pluripotency and Undergoes Dynamic Transcriptional Changes
(A) qRT-PCR analysis for hallmark pluripotent genes (Nanog, Oct4, and Esrrb) through the chemical induction process (n = 2).
(B) Diagram of tracing endogenous Oct4 activation during chemical reprogramming.
(C) Representative images of the four Yamanaka factor-induced iPSC colonies (OSKM-iPSCs, with or without 4-OHT induction) using the lineage tracing system. OSKM: Oct4; SOX2; Klf-4; c-Myc. The induced TdTomato-positive OSKM-iPSC colonies co-expressed the hallmark pluripotent genes Oct4 and Nanog.
(D) Representative images of chemically induced Tau-positive colonies via an XEN-like state (with or without 4-OHT induction) using the lineage tracing system.
(E) Percentage of Tau-positive colonies induced from the TdTomato-labeled fibroblasts at the end of the XEN-based chemical reprogramming period (n = 40).
(F) Induced expression of neural master genes (NeuroD1, Ngn2, and Sox2) during stage 1 chemical induction and XEN master genes (Gata4, Gata6, and Sox17) at stage 2 (n = 2).
(G) Expression of Sox2 or Gata6 after small hairpin RNA (shRNA) knockdown. shControl, non-targeting vector shRNA (n = 2).
(H) Inducing efficiency of primary XEN-like colonies and TauEGFP-positive colonies by knocking down a neural master gene (Sox2) or a XEN master gene (Gata6) (n = 2).
(I) Potential principles for using the plastic XEN-like state as a universal platform to obtain other functional cell types.
Scale bars, 100 μm. Data are presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < (Student’s t test). See also Figure S4 and Table S4.
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6. Figure 4
Long-Term Expanding Chemically Induced XEN-like Cells Retain Genome Stability and Neuronal Specifying Potential
(A) Doubling time analysis of induced XEN-like cells (different passages), ESCs, and fibroblasts (n = 3).
(B) Phase-contrast images of XEN-like colonies at passages 2 and 20.
(C) XEN-like cells at passage 20 co-expressed XEN master genes (Sall4, Gata4, Gata6, and Sox17).
(D) Karyotype analysis of chemically induced XEN-like cells.
(E) Distribution of chromosome numbers in passaged XEN-like cells. n indicates the number of cells analyzed.
(F) Comparative genomic hybridization (CGH) analysis of chemically induced XEN-like cells. Average log2 ratio values are plotted using fibroblasts as a reference.
(G) Global gene expression patterns of fibroblasts and induced XEN-like cells.
(H) Induced TauEGFP-positive neurons from long-term expanded XEN-like cells (after 20 passages) express typical neuronal markers (NF-H, TUJ1, and SYN).
(I) Induced TauEGFP-positive neurons from long-term expanded XEN-like cells (after 20 passages) show electrophysiological functional properties (n = 12).
(J) Quantification of inducing efficiency of TauEGFP-positive cells from XEN-like cells at different passages (the inducing efficiency from XEN-like cells at passage 1 was set as 100%) (n = 2).
(K) Hierarchical clustering analysis for XEN-like-cell-derived neurons at different passages (2 and 24), primary neurons, brain, fibroblasts, and direct conversion-derived chemically induced neurons (CiNs). Direct conversion-derived CiNs were induced as previously reported (Li et al., 2015).
(L) Diagram of scalable XEN-like-state-based chemical reprogramming.
Scale bars, 100 μm. Data are presented as mean ± SEM. See also Figures S3 and S4 and Tables S2, S3, and S4.
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