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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 26 OCT 2010
Copyright © 2010 AlphaMed Press
Volume 28, Issue 10, pages 1782–1793, October 2010
How to Cite
Chung, T.-L., Turner, J. P., Thaker, N. Y., Kolle, G., Cooper-White, J. J., Grimmond, S. M., Pera, M. F. and Wolvetang, E. J. (2010), Ascorbate Promotes Epigenetic Activation of CD30 in Human Embryonic Stem Cells. STEM CELLS, 28: 1782–1793. doi: 10.1002/stem.500
Author contributions: T.-L.C.: overall planning and design, collection and/or assembly of data, and data analysis and interpretation, and manuscript writing; J.T.: conception and design, collection and/or assembly of data, and data analysis and interpretation; N.T.: collection and/or assembly of data, data analysis and interpretation; G.K.: conception and design, collection and/or assembly of data, microarray data analysis and interpretation, and manuscript writing; J.C.-W.: conception and design, and financial support; S.M.G.: conception and design; M.F.P.: conception and design, data analysis and interpretation, and manuscript writing; E.W.: overall planning and design, financial support, and provision of study material or patients, 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 CELLS EXPRESS August 16, 2010.
- Issue online: 26 OCT 2010
- Version of Record online: 26 OCT 2010
- Accepted manuscript online: 16 AUG 2010 12:00AM EST
- Manuscript Accepted: 9 AUG 2010
- Manuscript Received: 24 FEB 2010
Additional Supporting Information may be found in the online version of this article.
|STEM_500_sm_SuppFig1.tif||3791K||Supporting Information Figure 1. Karyotype analysis of HES2, HES3 and HES4 passaged for 5, 13 and 6 passages in KOSR medium, respectively, shows normal karyotypes for all hESC lines.|
|STEM_500_sm_SuppFig2.tif||6608K||Supporting Information Figure 2. (A) Immunofluorescence microscopy of CD30 expression (green) in HES3 hESCs cultured with KOSR medium without b-FGF. (B) KOSR-batch independent induction of expression of CD30 on hESCs. Three different batches of KOSR (1342641, 1342683 and 1313351 obtained from Invitrogen) were used to test its ability to induce CD30. hESCs were grown under serum-free conditions for 4 weeks. (1) Flow cytometric analysis and (2) immunofluorescent detection of CD30 (green) and Oct4 (red) indicates that CD30 is induced by all three batches of KOSR.|
|STEM_500_sm_SuppFig3.tif||3997K||Supporting Information Figure 3. (A) CD30 expression is not reversible upon switching KOSR cultured hESCs back to standard FCS conditions. MEL3 hESC, a line that was originally established in KSOR, and consequently are entirely CD30 positive, remained uniformly CD30 positive when cultured for 14 weekly passages under standard FCS conditions. (B) Ascorbate acts directly on hESCs to induce CD30. Standard FCS cultured HES3 hESCs were seeded on Matrigel and cultured for 1 week with 1) mouse embryonic fibroblast feeder (MEF) conditioned KOSR medium (CM) 2) MEF conditioned KOSR minus Ascorbate (CM-Asc) 3) MEF conditioned KOSR minus ascorbate with the addition of 50 μg/ml ascorbate ((CM-Asc)+Asc). hESCs in 1) show weak but positive staining (most likely due to the short half life of ascorbate (12 hrs), hESCs in 2) remain CD30 negative while HES3 hESCs in 3) display clear CD30 staining. These data demonstrate that ascorbate in KOSR induces CD30 expression in hESCs through a direct effect on hESCs and not through a paracrine effect via MEFs. (C) Immunofluorescent analysis of CD30 expression (green) in HES3 hESCs cultured in commercially available VitroHES and mTesR serum-free media that contain 50?μg/ml ascorbate shows positive CD30 staining (nuclei staining with DAPI, blue).|
|STEM_500_sm_SuppFig4.tif||448K||Supporting Information Figure 4. CD30 induction in hESCs by ascorbate is dosage-dependent. Flow cytometric analysis of CD30 induction in HES2 cultured in KOSR medium with different concentrations of ascorbate for 4 passages. (50 ug/ml as standard concentration contained in KOSR medium)|
|STEM_500_sm_SuppFig5.tif||7277K||Supporting Information Figure 5. CD30 is highly expressed in human iPSC line ES4CL1 derived and cultured in KOSR-based medium. (A) Flow cytometric analysis of CD30 expression in human iPSC line ES4CL1 indicated 56℅ CD30+. (B) Immunofluorescence microscopy of CD30 (green) and Oct4 (red) expression and nuclei (blue) in ES4CL1.|
|STEM_500_sm_SuppFig6.tif||1617K||Supporting Information Figure 6. Karyotype analysis of HES2 GFP, HES2 CD30v, HES4 GFP and HES4 CD30v cultured in serum-containing medium, respectively, shows normal karyotypes for all GFP only and CD30v transduced hESC lines.|
|STEM_500_sm_SuppFig7.tif||27660K||Supporting Information Figure 7. CD30v activates P-ERK1/2 in hESCs. (A) Western blot analysis of ERK1/2, and phosph-ERK1/2 (P-ERK1/2) proteins in HES2 control (lane 1) and HES2 CD30v (lane 2) cells grown as pieces culture in FCS. P-ERK1/2 is upregulated in HES2 CD30v. A representative experiment of three independent experiments is shown. (B) Average densitometry quantification of ERK1/2, P-ERK1/2 in HES2 control and HES2 CD30v cells relative to β-tubulin as based on three western blots obtained from three independent experiments.|
|STEM_500_sm_SuppFig8.tif||16323K||Supporting Information Figure 8. (A) Histological analysis shows the presence of all three primary germ-layer derivatives in intramuscular teratomas derived from HES2 GFP, HES2 CD30v, HES4 GFP and HES4 CD30v, respectively (10X). Gl, glandular epithelium (endoderm) ; Sk, skin (ectoderm) ; Mu, muscle (mesoderm) ; Ch, chondrocyte (mesoderm) ; Ne, neural epithelium (ectoderm) (bar : 200 μm). (B) Evaluation of pluripotentcy markers in cultured embyoid bodied (EBs) by real-time quantitative PCR. 0-, 6- and 12-day EBs from the parental GFP or CD30v hESC lines were compared in terms of expression of Oct4 and Nanog.|
|STEM_500_sm_SuppTable1.xls||67K||Supporting Information Table 1. Microarray analysis of changes in expression level of genes upregulated in HES2 CD30v vs HES2 GFP lines with B-statistic greater than zero.|
|STEM_500_sm_SuppTable2.xls||93K||Supporting Information Table 2. Microarray analysis of changes in expression level of genes downregulated in HES2 CD30v vs HES2 GFP lines with B-statistic greater than zero.|
|STEM_500_sm_SuppTable3.doc||102K||Supporting Information Table 3. Genes which were significantly differentially regulated between CD30V and GFP samples were uploaded into Ingenuity Pathways Analysis (Ingenuity).|
|STEM_500_sm_SuppTable4.doc||58K||Supporting Information Table 4. Upregulated zinc finger proteins in CD30v expressing HES2. Majority (27 genes) of these zinc finger proteins are located in chromosome 19.|
|STEM_500_sm_SuppMaterials.doc||65K||Supporting Information Materials.|
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