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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Article first published online: 22 OCT 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 11, pages 2437–2449, November 2012
How to Cite
Hamasaki, M., Hashizume, Y., Yamada, Y., Katayama, T., Hohjoh, H., Fusaki, N., Nakashima, Y., Furuya, H., Haga, N., Takami, Y. and Era, T. (2012), Pathogenic Mutation of ALK2 Inhibits Induced Pluripotent Stem Cell Reprogramming and Maintenance: Mechanisms of Reprogramming and Strategy for Drug Identification. STEM CELLS, 30: 2437–2449. doi: 10.1002/stem.1221
Author contributions: T.E.: designed all experiments and edited the manuscript; M.H., Y.Y., Y.T., and T.K.: performed the experiments, analyzed data, and edited the manuscript; Y.H.: designed and performed part of the drug candidate experiments and edited the manuscript; N.F.: produced and provided Sendai virus vectors and edited the manuscript; H.H.: designed and performed part of the shRNA experiments and edited the manuscript; Y.N., H.F., and N.H.: provided the patient samples and data and edited the manuscript; T.E and Y.T.: wrote and prepared the manuscript and edited the manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS August 7, 2012.
- Issue published online: 22 OCT 2012
- Article first published online: 22 OCT 2012
- Accepted manuscript online: 4 SEP 2012 10:01AM EST
- Manuscript Accepted: 3 AUG 2012
- Manuscript Received: 23 MAR 2012
- Ministry of Health, Labor, and Welfare of Japan and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency
Additional Supporting Information may be found in the online version of this article.
|sc-12-0288_sm_SupplTable1.pdf||95K||Supplementary Table 1|
|sc-12-0288_sm_SupplTable2.pdf||33K||Supplementary Table 2|
|sc-12-0288_sm_SupplTable3.pdf||18K||Supplementary Table 3|
|sc-12-0288_sm_SupplFigure1.tif||2169K||Figure S1. Confirmation of mutations in ALK2 gene and colony morphology of FOPderived iPS cells. (A): Genomic sequencing in ALK2 gene from FOP-derived iPSC lines. The mutations of 617G>A (R206H) and 1067G>A (G356D) were indicated by arrows. (B): Phase contrast picture of FOP-derived iPSCs with the treatment of Dorsomorphin (DM). Left panel: iPSCs derived from the patient, F2; right panel: iPSCs derived from the patient, F4. Scale bars, 200 μm.|
|sc-12-0288_sm_SupplFigure2.tif||173K||Figure S2. Gene expression of teratoma derived from FOP-iPSC line Normal and FOP-derived iPSC lines were transplanted into the subcutaneous tissues of the immunodeficient mice. Eight to twelve weeks after injection, the iPSC lines tested formed teratomas. After RNA was purified from the teratomas, the markers such as MESP1 and MESOGENIN (Mesoderm), FOXA2 and SOX17 (endoderm) and SOX1 and NEUROD1 (ectoderm) were examined by qRT-PCR. Both teratomas expressed the markers of three germ layers.|
|sc-12-0288_sm_SupplFigure3.tif||356K||Figure S3. Effects of LDN-193189 and Dorsomorphin on Smad 1/5/8 phosphorylation in FOP-derived iPSC lines. Immunoblot analyses of phosphorylated Smad1/5/8 of FOPderived iPSC lines (F1-1, F2-1, -2, -3, -4, -5, F4-1 and -2) and Normal iPSC lines (N3-1) treated with or without LDN-193189 (LDN) and Dorsomorphin (DM). The normal iPSC line, N3-1, was treated with BMP-4. The relative levels of Smad1/5/8 phosphorylation in each iPSC lines were suppressed by the treatment with LDN and DM. The density of all bands is measured by Image J software, and relative phosphorylation level of Smad 1/5/8 is normalized to Smad1.|
|sc-12-0288_sm_SupplFigure4.tif||128K||Figure S4. Effects of Dorsomorphin on colony formation and maintenance of iPSCs. The number and ratio of three types of colonies from FOP-derived iPSC lines. The iPSC F2-1 and iPSC F4-2 lines were cultured with or without DM. Undifferentiated (u), partially differentiated (p) and completely differentiated (d) colonies are defined as described in Fig. 3A. Scale bars, 200 μm.|
|sc-12-0288_sm_SupplFigure5.tif||194K||Figure S5. The effect of BMP-4 on the expressions of the pluripotency- and differentiation-related genes in normal iPSC lines. DiPS and N3-1 iPSC lines were cultured with or without 10 ng/ml of BMP-4 in the absence of bFGF for 5 days. The data are normalized to GAPDH and represent as relative expression levels to respective control of DiPS.|
|sc-12-0288_sm_SupplFigure6.tif||732K||Figure S6. Inhibitory mechanism of iPSC generation from FOP-derived fibroblasts. (A): The effect of shR206H on the iPSC colony formation. shR206H-infected fibroblast N1 and F2 were induced to iPSC by SeV vectors in the presence or absence of LDN from day 8 to day 30. At day 30 of induction, AP+ colony with undifferentiated colony morphology was counted as a typical colony. The atypical colony is described in Fig. 1D and 1E. (B): On day 7 of iPSC induction, Tra-1-60+ and Tra-1-60- cells were purified by FACS and then pluripotent marker genes (SOX2, GDF3, REX1, DNMT3b and KLF5) were measured by qRT-PCR. The data are means ± SD of three independent experiments. NS, not significant; ND, not detected. (C and D): Immunoblot analyses of the phosphorylation of Smad2 and 3. TGF-β3 stimulation can enhance the phosphorylation of Smad2 and 3 in HeLa cells. This result indicates that the system works well. The relative phosphorylation levels of Smad2 and 3 are summarized in (D). The density of all bands is measured by Image J software, and relative phosphorylation levels of Smad2 and 3 are normalized to total Smad2 and 3, respectively. (E and F): TGF-β/Activin signals during iPS cell generation from FOP-derived fibroblasts. Immunoblot analyses of phosphorylated Smad2 and 3 of normal and FOP-derived fibroblasts on day 7 of iPS cell induction. On day 7 of iPS cell induction, Tra-1-60+ and Tra-1-60- cells were purified by FACS and the phosphorylation of Smad2 and 3 was then measured by immunoblot analyses. The graph (F) shows relative phosphorylation levels of Smad2 and 3. Smad2 and 3 phosphorylations were not enhanced during iPS cell inductions. The density of all bands is measured by Image J software, and relative phosphorylation levels of Smad2 and 3 are normalized to total Smad2 and 3, respectively. PC: Positive control, TGFβ3-treated HeLa cell line|
|sc-12-0288_sm_SupplFigure7.tif||158K||Figure S7. Schematic representation of measurement ALK2 kinase activity for chemical screening. MDA-D-BREFluc/Rluc cell line which were cultured with ALK2 inhibitor candidates in the presence of BMP-4 or BMP-6. After 24 hr cultivation, luciferase activity (both Fluc and Rluc) were measured.|
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