Supplemental Information. Direct Reprogramming of Fibroblasts. via a Chemically Induced XEN-like State

Transcription

1 Cell Stem Cell, Volume 21 Supplemental Information 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, and Hongkui Deng

2 SUPPLEMENTAL FIGURES Figure S1

3 Figure S1: Expressional and Functional Analysis for Chemical Reprogramming, Related to Figure 1 (A) Numbers of TauEGFP-positive and -negative colonies induced from XEN-like colonies. (B) Phase contrast and fluorescent images of chemically induced neurons after co-culturing with astrocytes. (C) qrt-pcr analysis of neural genes, fibroblast genes and XEN master genes regulated by components in Stage2 neural induction medium included N2, B27, BDNF, GDNF, bfgf, FSK, CHIR, Compound C (abbreviated as CC, also named as: Dorsomorpin). n = 2. (D) Number of genes that are activated or suppressed during the XEN-to-neuron conversion process. upregulated represents genes whose expression level was upregulated by more than 2-fold compared to fibroblasts, while downregulated represents genes whose expression level was downregulated by more than 2-fold compared to fibroblasts. (E) GO analysis for genes that were most robustly upregulated or downregulated by chemical induction (>10-fold differentially expressed). (F) Electrophysiological functional properties of the induced TauEGFP-positive cells without co-culturing with astrocytes. n = 3. (G) Generation and characterization of XEN-like cells-derived neurons from mouse newborn fibroblasts (NBFs). (H) Generation and characterization of XEN-like cells-derived neurons from mouse adult fibroblasts (MAFs). The data are presented as the mean +/ SEM. The scale bars are 100 μm.

4 Figure S2

5 Figure S2. Lineage Tracing Confirms the Fibroblast-Origin of the Induced Neuronal Cells, Related to Figure 2 (A) Scheme of lineage tracing system. (B) Representation of a TauEGFP and tdtomato-double positive colony (top) and TauEGFP-positive and TdTomato-negative colony (bottom). (C) Quantification of TauEGFP-positive colonies induced from TdTomato-positive and -negative cells. (D) The induced TdTomato-positive cells induced from XEN-like cells expressed pan-neuronal markers TUJ1, NF-H and SYN. (E) Patch-clamp recordings on TdTomato-positive cells induced from XEN-like cells after co-culturing with astrocytes. (F) Whole-cell voltage-clamp recording of TdTomato-positive cells induced from XEN-like cells and inward and outward currents were recorded. n = 4. (G) qrt-pcr analysis for hallmark neural-progenitor genes (Sox1 and Olig2) through the chemical induction process. n = 3. The data are presented as the mean +/ SEM. The scale bars are 100 μm.

6 Figure S3

7 Figure S3. Embryo-derived XEN Cells Resemble the Chemically-induced XEN-like Cells for Neuronal Induction, Related to Figure 4 (A) Diagram of CeXEN cell line establishment and induction of the CeXEN cells into neuronal cells. (B) Representative images of CeXEN cells (at P0 and P11) and the CeXEN cells were TauEGFP-negative. (C) CeXEN cells co-expressed XEN master genes Sall4, Gata4, Gata6 and Sox17. (D) Representative image of TauEGFP-positive cells induced from CeXEN cells. (E) Induced TauEGFP-positive cells induced from CeXEN cells co-expressed pan-neural markers (TUJ1 and NF-H). The scale bars: (D), 50 μm; (B), (C), (E), 100 μm.

8 Figure S4

9 Figure S4. Hepatocyte-like Cells Were Generated from the XEN-like Cells, Related to Figure 3 and Figure 4 (A) Gene expression heat map of fibroblast genes from eight single XEN-like colonies analyzed by RNA-seq. (B) Gene expression heat map of neural-ectoderm genes, mesoderm genes and endoderm genes from eight single XEN-like colonies analyzed by RNA-seq. (C) (D) Diagram of XEN-like state based hepatic lineage reprogramming. Representative phase contrast images of hepatocyte-like cells induced from XEN-like cells (> passage 20). (E) qrt-pcr analysis of hepatic hallmark genes in fibroblasts, XEN-like cells, induced hepatocyte-like cells from XEN-like cells (from two batches of experiments) and primary hepatocytes (isolated from 2-day postnatal mouse). n = 3. (F) Co-immunostaining of typical hepatocyte markers AFP and ALB for hepatocyte-like cells induced from XEN-like cells (> passage 20). (G) Hierarchical clustering analysis for XEN-derived hepatocytes, primary hepatocytes, hepatic tissues, XEN-like cells (P3 and P17) and fibroblasts. (H) Number of genes that are upregulated or downregulated during the XEN-to-hepatocytes conversion process. upregulated represents genes whose expression level was upregulated by more than 2-fold compared to fibroblasts, while downregulated represents genes whose expression level was downregulated by more than 2-fold compared to fibroblasts. (I) Quantitative analysis of ALBUMIN and Urea secretion among XEN-like cells, iheps from XEN-like cells (> passage 20) and primary hepatocytes by ELISA. n = 3. Scale bars: 100 μm. The data are presented as the mean +/ SEM. *P<0.05; **P<0.01; ***P<0.001 (Student s t-test).

10 SUPPLEMENTAL TABLES Table S1. Small molecules used in this study, related to Figure 1 and STAR Methods. FULL NAME ABBREVIATION DOSE SOURCE MOLECULAR WEIGHT VPA V 500 (μm) Sigma, cat. no.p CHIR99021 C Synthesized by WUXI APPTEC Synthesized by WUXI APPTEC Tranylcypromine T 5-10 Enzo, cat. no.bml-ei Forskolin F Enzo, cat. no.bml-cn AM580 A 0.05 Tocris, cat. no EPZ E 5 Selleckchem, cat. no.s TD114-2 TD Synthesized by WUXI APPTEC 529 Dorsomorpin D 1-2 Tocris, cat. no LDN L 0.1 Selleckchem, cat. no.s SB S 2-10 Tocris, cat. no

11 Ch Tocris, cat. no L-Ascorbic acid 2-phosphate Vc 50 (μg/ml) Sigma, cat. no.a Table S2. Electrophysiological properties of chemically-induced neurons, related to Figure 2, Figure S1 and Figure 4 Table S2. (A) Electrophysiological properties of chemically-induced neurons from embryonic fibroblasts (MEFs), related to Figure 2 and Figure S1. Induction Rm(MO) Cm(pF) RMP(mV) Threshold(mV) APamp(mV) Ina-max(pA) N ± ± ± ± ± ± /13 1* / / / / / / / /13

12 / / /13 *without co-culturing with astrocytes. Rm: input resisitance; Cm: membrane capacitance; RMP: resting membrane potential; APthreshold: action potential threshold; APamp: action potential amplitude; Ina-max: maximum amplitude of sodium current. All the data are mean+/ SEM. Table S2. (B) Electrophysiological properties of chemically-induced neurons from postnatal fibroblasts (NBFs), related to Figure S1. Induction Rm(MO) Cm(pF) RMP(mV) Threshold(mV) APamp(mV) Ina-max(pA) N ± ± ± ± ± ± / / / /8

13 / / / / /8 Rm: input resistance RMP: resting membrane potential Cm: membrane capacitance Threshold: action potential threshold APamp: action potential amplitude Ina-max: maximum amplitude of sodium current Table S2. (C) Electrophysiological properties of the chemically-induced neurons from passage 20 XEN-like cells, related to Figure 4. Induction Rm(MO) Cm(pF) RMP(mV) Threshold(mV) APamp(mV) Ina-max(pA) N ± ± ± ± ± ± /13 1* /13 2* / / / /13

14 / / / / / / /13 *without co-culturing with astrocytes. Rm: input resisitance; Cm: membrane capacitance; RMP: resting membrane potential; APthreshold: action potential threshold; APamp: action potential amplitude; Ina-max: maximum amplitude of sodium current. All the data are mean+/ SEM. Table S3. CNVs analyzed by CGH assay, related to Figure 4 (See supplemental excel file).

15 Table S3. (A) Overview of CGH profiling, related to Figure 4. Table S3. (B) Gene mutations found in XEN-like P2, related to Figure 4. Table S3. (C) Gene mutations found in XEN-like P10, related to Figure 4. Table S3. (D) Gene mutations found in XEN-like P13, related to Figure 4. Table S4. Primers used for real-time qpcr, related to Figure 1, Figure 3, Figure S1, Figure S4, and STAR Methods. GENE NAME FORWARD (5' TO 3') REVERSE (5' TO 3') APPLICATION Gapdh CATGTTCCAGTATGACTCCACTC GGCCTCACCCCATTTGATGT real-time qpcr Sox2 CGGGAAGCGTGTACTTATCCTT GCGGAGTGGAAACTTTTGTCC real-time qpcr Oct4 CAGGGCTTTCATGTCCTGG AGTTGGCGTGGAGACTTTGC real-time qpcr Esrrb GTGGCTGAGGGCATCAATG AACCGAATGTCGTCCGAAGAC real-time qpcr Nanog AGTTATGGAGCGGAGCAGCAT AGGCCTGGACCGCTCAGT real-time qpcr Gata6 TGAGGTGGTCGCTTGTGTAG ATGGCGTAGAAATGCTGAGG real-time qpcr Gata4 GAGCTGGCCTGCGATGTCTGAGTG AAACGGAAGCCCAAGAACCTGAAT real-time qpcr

16 Sox17 GTCAACGCCTTCCAAGACTTG GTAAAGGTGAAAGGCGAGGTG real-time qpcr Ascl1 ACTTGAACTCTATGGCGGGTT CCAGTTGGTAAAGTCCAGCAG real-time qpcr Brn2 GACAAGATCGCAGCGCAAGG GGCTTAGGGCATTTGAGGAA real-time qpcr NeuroD1 ACGCAGAAGGCAAGGTGTCC GTTCCTCGTCCTGAGAACTG real-time qpcr Ngn2 TGGCTGGCATCTGCTCTATT TAGGCATTGTGACGAATCTG real-time qpcr Col1a1 CATGTTCAGCTTTGTGGACCT GCAGCTGACTTCAGGGATGT real-time qpcr Fsp1 GTGTCCACCTTCCACAAATACTCA ACTTCATTGTCCCTGTTGCTGTC real-time qpcr Cyp2a5 GCACTTCCTAGATGACAAGGGACA CAGGCTCAACGGGACAAGAA real-time qpcr Hnf4α CATCACCACCATCGTCAA CCTCGTGTCACATCTTCTT real-time qpcr Alb ATGTTACCAAGTGCTGTAGT AATCTGCTTCTCCTTCTCTG real-time qpcr Afp TTCCTGTCTCAGTCATTCTAAG AGTCTCCTAAGGTCTGGTAG real-time qpcr Cyp3a11 TACTGTGATGGAGATGGAATAC GGTGAAGAGCATAAGATGGA real-time qpcr Ttr CTCACCACAGATGAGAAG GGCTGAGTCTCTCAATTC real-time qpcr

17 Hnf6a AAATAAGCGTCCGTCCAAAGAA GACGATGAACTGCCTGAGTTG real-time qpcr Cyp3a13 AGTGCTGGTGAAGGAATG CTGGTGAAGGTTGGAGAC real-time qpcr Cyp2b10 GTCCATCCTCCAGAACTTC TTCCAATACCACTCTCCTTG real-time qpcr Gjb1 CGTGAATCGGCACTCTACAGC TGCACCTTGTGTCTCTTTACCTC real-time qpcr Sult1a1 AACATGGAGCCCTTGCGTAAA ATGAGCACATCATCAGGCCAG real-time qpcr Gstt1 AGGCTCGTGCTCGTGTAGA CAGGGAACATCACCTTATGCC real-time qpcr Actin CGCCACCAGTTCGCCATGGA TACAGCCCGGGGAGCATCGT real-time qpcr