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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 22 MAY 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 6, pages 1121–1135, June 2013
How to Cite
Son, M. J., Son, M.-Y., Seol, B., Kim, M.-J., Yoo, C. H., Han, M.-K. and Cho, Y. S. (2013), Nicotinamide Overcomes Pluripotency Deficits and Reprogramming Barriers. STEM CELLS, 31: 1121–1135. doi: 10.1002/stem.1368
Author contributions: M.J.S., M.S.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript. B.S., M.K., C.H.Y.: Collection and/or assembly of data. M.H.: Conception and design. Y.S.C.: Conception and design, Financial support, Data analysis and interpretation, Manuscript writing, Final approval of manuscript. MJS and MS 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 February 4, 2013.
- Issue online: 22 MAY 2013
- Version of Record online: 22 MAY 2013
- Accepted manuscript online: 25 MAR 2013 01:56AM EST
- Manuscript Accepted: 5 FEB 2013
- Manuscript Received: 26 SEP 2012
- Ontario Genomics Institute, the Stem Cell Network
- Canadian Institutes of Health Research
Additional Supporting Information may be found in the online version of this article.
|sc-12-0905_sm_SupplFigure1.tif||537K||Supporting Information Fig. S1. The effect of FK866 alone and in combination with NAM on intracellular NAD+ levels (A) and hESC self-renewal (B). H9 hESCs were cultured under feeder-free conditions with chemically defined mTeSR1 for 6 days.|
|sc-12-0905_sm_SupplFigure2.tif||2356K||Supporting Information Fig. S2. NAM blocks differentiation and supports the maintenance of hPSC self-renewal. (A): AP staining of hESCs (H9 and H1) cultured in the feedercontaining culture system for 6 days. (B): AP staining of hESCs and hiPSCs cultured under feeder-free conditions with chemically defined mTeSR1 for 6 days. (Aa, Ba): Representative scanned images of AP-stained culture dishes. (Ab, Bb): Relative AP expression measured by scanning densitometry. The data are presented as the mean±SE (n= 3). * p < 0.05 and ** p < 0.01 compared to control.|
|sc-12-0905_sm_SupplFigure3.tif||2522K||Supporting Information Fig. S3. NAM increases the cell proliferation and MMP (Ψm) of hESCs under chemically defined culture conditions. H9 hESCs were cultured in mTeSR1 with the indicated NAM concentrations under feeder-free conditions for 6 days. (A): Representative images of BrdU staining (top). Quantification of the number of BrdU+ cells (bottom). (B): Representative images of JC-1 staining (top) and their fluorescence intensity profiles (middle). Active mitochondria (red) and the less active form (green). The image intensity profile was obtained with Axiovision 4.5 software. ;Ψ m is represented as the relative red/green ratio (bottom). The data represent the mean±SE (n= 3). ** p < 0.01 compared to control.|
|sc-12-0905_sm_SupplFigure4.tif||576K||Supporting Information Fig. S4. The positive effects of NAM on the long-term maintenance and self-renewal of hESCs cultured under non-self-renewing condition. Under feeder free condition, H9 hESCs were cultured with UM in the absence (−) or presence of NAM (0.1 mM) over 10 passages. Cells were live-stained with TRA-1-60 primary antibody and visualized by DAB solution (left). The total number of TRA-1-60+ colonies was counted (right). The data are presented as the mean±SE (n= 3). ** p < 0.01 compared to the control.|
|sc-12-0905_sm_SupplFigure5.tif||652K||Supporting Information Fig. S5. NAM promotes Oct4 induction. (A): OSKM-transduced cells at the indicated time points of reprogramming in the absence (−) or presence of NAM were immunofluorescence stained with Oct4-specific antibody (green). (B): The relative counts of fluorescent clusters. The data are presented as the mean±SE (n= 3). * p < 0.05 and ** p < 0.01 compared to the control.|
|sc-12-0905_sm_SupplFigure6.pdf||372K||Supporting Information Fig. S6. Characterization of NAM-hiPSCs. (A): The morphology of a representative NAM-hiPSC colony and immunocytochemical analysis with common hESC markers (OCT4, NANOG, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81). hESC-like morphology of NAM-hiPSC (top left), AP-stained NAM-hiPSC (top right). Scale bar, 200 μm. (B): PCR verification of the genomic integration of retroviral genes OCT4, SOX2, KLF4, and MYC in NAM-hiPSCs was performed with genomic DNA with a viral-specific primer and a gene-specific primer. GAPDH was used as a positive amplification and loading control. (C): Transgene-specific PCR primers permit the determination of the relative expression levels of total, endogenous (Endo), and retrovirally expressed (Trans) genes (OCT4, SOX2, KLF4, and cMYC) via semi-quantitative RT-PCR. GAPDH was used as a positive amplification and loading control. (D): Bisulfite sequencing measuring the methylation status within the promoter regions of the OCT4 and NANOG genes. Genomic DNA was processed for bisulfite modification. The promoter regions of OCT4 and NANOG were amplified by PCR, and their methylation status was analyzed. Each horizontal row of circles represents an individual sequencing result from one amplicon. Open or black circles indicate demethylated or methylated CpGs, respectively. The proportion of methylated CpGs is indicated.|
|sc-12-0905_sm_SupplFigure7.pdf||469K||Supporting Information Fig. S7. NAM-hiPSCs differentiate into lineages of the 3 major germ layers in in vitro and in vivo differentiation assays. (A): RT-PCR analyses of various differentiation markers for the 3 germ layers: endoderm (HGF, Amylase, HAND1, and PECAM1), mesoderm (cTnT, IGF2, Tbx6, and Runx2), and ectoderm (NCAM, PAX6, VIM, and OTX1). (B): Immunocytochemistry of 3 clones of NAM-hiPSCs with FoxA2 and Sox17 (endoderm), Desmin and α-smooth muscle actin (α-SMA) (mesoderm), and Tuj1 and Nestin (ectoderm). Nuclei were stained with DAPI (blue). Scale bar, 200 μm. (C): Histological analysis of teratomas derived from NAM-hiPSCs by hematoxylin and eosin staining. Differentiation into multiple derivatives of the 3 germ layers is shown.|
|sc-12-0905_sm_SupplFigure8.tif||322K||Supporting Information Fig. S8. Karyotype analysis of long-term cultured hPSCs with NAM. H9 hESC (passage 50) and hiPSCs (passage 20) were cultured in the presence of NAM for 10 passages. At passages 60 and 30, the karyotype is normally maintained.|
|sc-12-0905_sm_SupplFigure9.tif||672K||Supporting Information Fig. S9. NAM inhibits p53 activation in hESCs under differentiation conditions. (A): H9 hESCs cultured with CM or UM containing 0.1 mM NAM under feeder-free conditions. Scanned images of AP stained cells at day 6 (left) and relative AP expression normalized to the CM control (right). (B): Western blot analysis of p-p53 (Ser15 and 315) and p53 at D6 in hESC cultures in the absence (−) or presence of 0.1 mM NAM. β-actin was used as an internal control. The data are presented as the mean±SE (n= 3). *p < 0.05 compared to control.|
|sc-12-0905_sm_SupplFigure10.tif||628K||Supporting Information Fig. S10. Effects of 3-ABA and iso-NAM on FK866-induced apoptosis. (A): Reprogramming cultures in combination with FK866 (0.1 nM), 3-ABA (1 mM), or iso-NAM (1 mM) were stained with caspase-3 substrate (green) and annexin V (red) on day 19 of reprogramming. (B): Apoptotic cells were quantified by the percentage of caspase-3-activated cells. The data are presented as the mean±SE (n= 3). ** p < 0.01 compared to the control.|
|sc-12-0905_sm_SupplTable1.pdf||78K||Supporting Information Table S1. NAM (Niacinamide) concentration in basal medium for hESC culture.|
|sc-12-0905_sm_SupplTable2.pdf||130K||Supporting Information Table S2. List of antibodies used in this study.|
|sc-12-0905_sm_SupplTable3.pdf||82K||Supporting Information Table S3. List of primers used in this study.|
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