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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 5 JUL 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 7, pages 1278–1286, July 2013
How to Cite
Luo, M., Ling, T., Xie, W., Sun, H., Zhou, Y., Zhu, Q., Shen, M., Zong, L., Lyu, G., Zhao, Y., Ye, T., Gu, J., Tao, W., Lu, Z. and Grummt, I. (2013), NuRD Blocks Reprogramming of Mouse Somatic Cells into Pluripotent Stem Cells. STEM CELLS, 31: 1278–1286. doi: 10.1002/stem.1374
Author contributions: Z.L., W.T., and I.G.: conception and design, data analysis and interpretation, writing and approval of manuscript; Z.L., M.L., T.L., H.S., and W.X.: collection and assembly of data, data analysis and interpretation; Y.G.Z.: data analysis and interpretation; Q.Z., M.S., L.Z., and G.L.: collection and assembly of data; Y.Z., T.Y., J.G.: other (support of manuscript); M.L., T.L. W.X., and H.S. contributed equally to this article.
Disclosure of potential conflicts of interest is found at the end of this article.
first published online in STEM CELLS EXPRESS March 26, 2013.
- Issue online: 5 JUL 2013
- Version of Record online: 5 JUL 2013
- Accepted manuscript online: 26 MAR 2013 08:22AM EST
- Manuscript Accepted: 15 FEB 2013
- Manuscript Received: 24 SEP 2012
- National Natural Science Foundation of China. Grant Numbers: 30971453, 31171255, 3107116, 30700411, 91219101
- National Basic Research Program of China. Grant Number: 2013CB530700
- Shenzhen Bureau of Science Technology and Information. Grant Number: JC201005260239A
- Chinese Academy of Sciences. Grant Number: XDA01010405
Additional Supporting Information may be found in the online version of this article.
|sc-12-0890_sm_SupplFigure1.pdf||209K||Figure S1 Knockdown of Mbd3 increases the efficiency of iPSC generation. (A) Western blot showing the expression of Mi-2, HDAC1&2 and Mbd2&3 in MEFs, iPSCs and mESCs. (B-D) MEFs infected with Oct4/Sox2/Klf4/c-Myc (OSKM) in the presence of different Mbd3-specific RNAs were cultured in mESC medium and assayed for reprogramming 16 days after infection. (B) Left: Average numbers of AP+ and Oct4-GFP+ clones from 1.5×104 MEFs. Data are from three independent experiments; error bars represent standard deviation. Right: FACS analyses showing the percentage of Oct4-GFP positive cells. (C) Representative experiments showing differential interference contrast (DIC) and fluorescent images of MEFs, scale bars = 300 μm. (D) RT-PCR analysis of mESC marker genes. (E-F) Characterization of iPSCs derived from MEFs expressing OSKM and shMbd3. (E) AP staining and immunofluorescence of mES marker genes (SSEA-1 and Nanog). (F) Immunofluorescence showing embryoid body (EB)-mediated differentiation of iPSCs into ectoderm, mesoderm and endoderm using antibodies against Nestin, SM-actin and ARF. Scale bars = 100 μm. Abbreviations: MEF, mouse embryonic fibroblast; iPSCs, induced pluripotent stem cells; mESCs, mouse embryonic stem cells; RT-qPCR, reverse transcription quantitative polymerase chain reaction; OSKM, Oct4, Sox2, Klf4, c-Myc; DIC, differential interference contrast; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting; AP, alkaline phosphatase; SM-Actin, smooth muscle actin; AFP, α-fetoprotein.|
|sc-12-0890_sm_SupplFigure2.pdf||74K||Figure S2 Depletion of Mbd3 does not alter cell cycle progression. (A) Cell proliferation assay. MEF cells were infected with retroviruses encoding control (shCtrl) or Mbd3-specific shRNAs (shMbd3-1, 2&3). Cell numbers were counted in 24 h intervals 5-11 days after infection. (B) MEFs were infected with retroviruses encoding control (shCtrl) or Mbd3-specific shRNAs and subjected to FACS analysis 10 days after infection. Abbreviations: MEF, mouse embryonic fibroblast; FACS, fluorescence-activated cell sorting.|
|sc-12-0890_sm_SupplFigure3.pdf||125K||Figure S3 Depletion of Mbd3/NuRD substitutes for Sox2 in iSF1 medium. (A) Strategy for iPSC induction in iSF1 culture medium. (B) Knockdown of Mbd3 replaces Sox2 in iSF1 medium. Top: 2,500 MEFs infected with Oct4/Sox2/Klf4/c-Myc (OSKM) alone or together with Mbd3 or shMbd3 were cultured in iSF1 medium and assayed for reprogramming 12 days after infection. AP- and Oct4-GFP-positive clones were counted (top left) and the percentage of Oct4-GFP positive cells was analyzed FACS (top right). Bottom: 1.5×104 MEFs infected with Oct4/Sox2/Klf4 (OSK) alone or together with Mbd3 or Mbd3 shRNA were cultured in iSF1 medium and assayed for reprogramming 18 days after infection. AP- and Oct4-GFP-positive clones were counted (bottom left) and the percentage of Oct4-GFP-positive cells was monitored by FACS (bottom right). Error bars represent standard deviation. (C) RT-PCR analysis of showing expression of mESC marker genes in Oct4/ Klf4/c-Myc (OKM) plus shMbd3 derived iPS cells (sriPS). (D) 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) based assay monitoring viability and proliferation of sriPS cells. Abbreviations: MEFs, mouse embryonic fibroblasts; iPSC, induced pluripotent stem cell; mESC, mouse embryonic stem cell; RT-PCR, reverse transcription polymerase chain reaction; OSKM, Oct4, Sox2, Klf4 and c-Myc; OSK, Oct4, Sox2 and Klf4; OKM, Oct4, Klf4 and c-Myc; GFP, green fluorescent protein; AP, alkaline phosphatase; FACS, fluorescence-activated cell sorting; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide.|
|sc-12-0890_sm_SupplFigure4.pdf||140K||Figure S4 Microarray analysis of gene expression in control and Mbd3-depleted MEFs. (A) Cluster analysis of Mbd3 regulated genes in mES cells, Mbd3-depleted and VPA treated MEFs. Individual genes are listed in Table S2. (B) Immunoblots showing the expression of iPSC promoting factors upon depletion of Mbd3. MEFs were infected with retroviruses encoding control (shCtrl) or Mbd3-specific shRNAs (shMbd3-1&2) and the level of Nanog, Oct4 and Sox2 was monitored 10 days after infection. (C) Cluster analysis of Mbd3 target genes that are involved in activation of MAPK. Left: Gene expression in mESC, Mbd3-depleted and VPA-treated MEFs. Right: Binding of Mbd3 or Mbd3-containing complexes to gene promoters Individual genes are listed in Table S3. Abbreviations: VPA, valproic acid; MEF, mouse embryonic fibroblast; iPSC, induced pluripotent stem cell; mESC, mouse embryonic stem cell; MAPK, mitogen-activated protein kinase.|
|sc-12-0890_sm_SupplFigure5.pdf||94K||Figure S5 Gene Ontology clustering of Mbd3 regulated genes. Gene Ontology analysis of Mbd3 regulated genes based on the GO categories (A) biological process and (B) molecular function.|
|sc-12-0890_sm_SupplTable1.pdf||15K||Supplemental Table 1|
|sc-12-0890_sm_SupplTable2.pdf||2350K||Supplemental Table 2|
|sc-12-0890_sm_SupplTable3.pdf||53K||Supplemental Table 3|
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