Telephone: 716-645-1202; Fax: 716-645-3822
Tissue-Specific Stem Cells
Version of Record online: 27 NOV 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 12, pages 2746–2759, December 2012
How to Cite
Han, J., Mistriotis, P., Lei, P., Wang, D., Liu, S. and Andreadis, S. T. (2012), Nanog Reverses the Effects of Organismal Aging on Mesenchymal Stem Cell Proliferation and Myogenic Differentiation Potential. STEM CELLS, 30: 2746–2759. doi: 10.1002/stem.1223
Author contributions: J.H. and P.M.: collection and/or assembly of data, data analysis and interpretation, and manuscript writing; P.L.: data analysis and interpretation and manuscript writing; D.W. and S.L.: statistical analysis of microarray data; S.T.A.: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS September 4, 2012.
- Issue online: 27 NOV 2012
- Version of Record online: 27 NOV 2012
- Accepted manuscript online: 4 SEP 2012 10:01AM EST
- Manuscript Accepted: 5 AUG 2012
- Manuscript Received: 31 JAN 2012
- National Institutes of Health. Grant Number: R01 HL086582
- New York State Stem Cell Science. Grant Number: NYSTEM Contract #C024316
Additional Supporting Information may be found in the online version of this article.
|sc-12-0114_sm_SupplFigure1.tif||242K||Figure S1: Immunostaining for Nanog in nBM-MSC (green). Cell nuclei were counterstained with Hoechst 33342 (blue).|
|sc-12-0114_sm_SupplFigure2.tif||18K||Figure S2: Nanog alters the transcription level of p16INK4a regulators. Quantitative RT-PCR for (A) MEOX2 and (B) EZH2. Expression level of the indicated transcripts was normalized to GAPDH. The relative expression level was defined as the ratio of the normalized expression level of each gene in BM-MSCs to that in control nBM-MSCs. Real-time RT-PCR experiment was repeated with RNA from three independent experiments. (*) p< 0.05, n=3.|
|sc-12-0114_sm_SupplFigure3.tif||335K||Figure S3: Karyotype analysis based on DAPI banding (nBM-MSC) or Giemsa banding (aBMMSC) of Nanog expressing BM-MSC showed 54 diploid chromosomes as expected for normal ovine cells.|
|sc-12-0114_sm_SupplFigure4.tif||44K||Figure S4: Nanog increased transcription of TGF-β1 in nBM-MSC and aBM-MSC. Realtime qRT-PCR for TGF-β1. GAPDH served as a loading control. Results from 3 independent experiments were plotted as average±SE. (*) p<0.05, n=3.|
|sc-12-0114_sm_SupplFigure5.tif||216K||Figure S5: Nanog did not affect the amount of total Smad2. (A) Western blotting for total Smad2 in nBM-MSC and aBM-MSC; GAPDH served as loading control. (B) The band intensity of total Smad2 was determined using Image J software and normalized to GAPDH. Results from 3 independent experiments were plotted as average ± SE.|
|sc-12-0114_sm_SupplFigure6.tif||199K||Figure S6: TGF-β-neutralizing antibody inhibited nBM.N-CM-induced hydrogel contraction and Smad2 phosphorylation. (A) Exogenous TGF-β1 (2ng/ml) was neutralized by different concentrations of anti-TGF-β neutralizing antibody (R&D systems, Minneapolis, MN) as indicated and the level of p-Smad2 in control nBM-MSCs was measured by WB. (B) Gel compaction profile and (C) p-Smad2 level of control nBM-MSCs in the presence of nBM.C-CM or nBM.N-CM with or without TGF-β neutralizing antibody (5μg/mL) as indicated. (*) p<0.05 between the indicated samples and corresponding control at the same time point; (#) p<0.05 between the indicated sample and nBM.N-CM treated sample at the same time point (n=3).|
|sc-12-0114_sm_SupplFigure7.tif||20K||Figure S7: Exogenous TGF-β1 pathway increased Nanog-induced hydrogel compaction. Gel compaction profiles of (A) control BM-MSCs and (B) Nanog+ BM-MSCs with or without TGF-β1 (2 ng/ml). The TGF-β receptor inhibitor SB431542 (10 μM) was added in some samples as indicated.|
|sc-12-0114_sm_SupplFigure8.tif||27K||Figure S8: Differential effects of Oct4 on proliferation and contractility of nBM-MSC and aBM-MSC. (A) Number of colonies larger than 2mm in diameter from the clonogenic assay. (B) Doubling time. (C) Vascular reactivity (kPa) of nBM-MSC and aBM-MSC-derived TEVs in response to Endothelin-1 (20 nM), U46619 (10-6 M) or KCl (118 mM). All values are the mean±SE of three independent experiments (n=3). The symbol (*) denotes p<0.05 between the indicated samples and their corresponding controls.|
|sc-12-0114_sm_SupplTable1.xls||7847K||Table S1: Genes expressed in at least one of four cells: aBM.C, aBM.N, nBM.C, or nBM.N. Fold changes of up- and down-regulated genes (FC ≥ 2, p<0.05) are highlighted red and green, respectively and p-values of less than 0.05 are highlighted yellow.|
|sc-12-0114_sm_SupplTable2.xls||630K||Table S2: Venn diagram depicting DEG in each of the three group comparisons. Fold changes of up- and down-regulated genes (FC ≥ 2, p<0.05) are highlighted red and green, respectively and p-values of less than 0.05 are highlighted yellow. A: All DEG in at least one of the three group comparisons B: Common DEG between nBM.C vs. aBM.C and aBM.N vs. aBM.C groups C: Common DEG between nBM.C vs. aBM.C and nBM.N vs. nBM.C groups D: Common DEG among all three groups|
|sc-12-0114_sm_SupplTable3.xls||56K||Table S3: Estimated list of DEG promoters in nBM-MSC and aBM-MSC with Nanog binding sites.|
|sc-12-0114_sm_SupplTable4.xls||99K||Table S4: Gene ontology classification of DEG in each of the three group comparisons.|
|sc-12-0114_sm_SupplTable5.pdf||43K||Table S5: Primer sequences used for RT-PCR.|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.