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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 12 FEB 2013
Copyright © 2012 AlphaMed Press
Volume 31, Issue 2, pages 259–268, February 2013
How to Cite
Hu, S., Wilson, K. D., Ghosh, Z., Han, L., Wang, Y., Lan, F., Ransohoff, K. J., Burridge, P. and Wu, J. C. (2013), MicroRNA-302 Increases Reprogramming Efficiency via Repression of NR2F2. STEM CELLS, 31: 259–268. doi: 10.1002/stem.1278
Author contribution: S.H.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; K.D.W. and Z.G.: conception and design, collection and/or assembly of data, data analysis and interpretation; L.H. and F.L.: collection and/or assembly of data, data analysis and interpretation; Y.W.: data analysis and interpretation; K.J.R.: collection and/or assembly of data; P. B.: design; and J.C.W.: conception and design, data analysis and interpretation, financial support.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS November 8, 2012.
- Issue online: 12 FEB 2013
- Version of Record online: 12 FEB 2013
- Accepted manuscript online: 8 NOV 2012 06:54AM EST
- Manuscript Accepted: 9 OCT 2012
- Manuscript Received: 30 JUN 2012
- Burroughs Wellcome Foundation. Grant Numbers: NIH DP2OD004437, R01 HL113006, RC1 AG036142
- Foudation Leducq 11CVD02
- AHA Postdoctoral fellowship. Grant Number: 10POST3730079
Additional Supporting Information may be found in the online version of this article.
|sc-12-0607_sm_SupplFigure1.tif||1027K||Figure S1. Algorithm for correlating miRNAs with their target mRNAs in pluripotent cells.|
|sc-12-0607_sm_SupplFigure2.tif||2545K||Figure S2. (A) The miR-302 inhibits NR2F2 3′UTR reporter activity. The lentiviral miR- 302a/b/c/d (Lv-302) suppresses the activity of T-WT reporter. However, mutations in either binding site reduce this suppression. The reporter with double 3′UTR mutations showed luciferase activity similar to control (pGL3-Con). (B) The miR-367 can not inhibit NR2F2 3′UTR reporter activity.|
|sc-12-0607_sm_SupplFigure3.pdf||106K||Figure S3. Electroporation of GFP reporter into H7 hESCs. The hESCs were electroporated with GFP reporter using the AmaxaR Human Stem Cell NucleofectorR Kit II. With electroporation, typical transfection efficiency is estimated at 80%. Scale bar=50 μm.|
|sc-12-0607_sm_SupplFigure4.tif||829K||Figure S4. Knockdown of NR2F2. The shRNA against NR2F2 led to approximately 50% knockdown of endogenous NR2F2 in HeLa cells.|
|sc-12-0607_sm_SupplFigure5.tif||1294K||Figure S5. The expression level of both miR-302b and NR2F2 upon the reprogramming initiation. (A) The reprogramming with the addition of Lv-shNR2F2 or Lv-NR2F2. (B) The reprogramming with Lv-302 and/or Lv-NR2F2.|
|sc-12-0607_sm_SupplFigure6.pdf||274K||Figure S6. Typical colonies used for the evaluation of reprogramming efficiency. These colonies were AKP+, TRA-1-60+, and had hESC-like morphology. Note that colonies that lacked TRA-1-60 and/or did not have hESC-like morphology were discarded from our analysis. Scale bar=50μm.|
|sc-12-0607_sm_SupplFigure7.pdf||381K||Figure S7. Representative hematoxylin and eosin staining of a solid encapsulated teratoma derived from typical iPSC lines implanted into the sub-renal capsule of SCID mice. Histological analysis of tissue demonstrated presence of glandular structures (endoderm), cartilage (mesoderm), and neural rosettes (ectoderm). Scale bar=250 μm.|
|sc-12-0607_sm_SupplFigure8.pdf||280K||Figure S8. Characterization of KMOS and KMOS3-iPSC-derived endothelial cells (ECs). The differentiated and purified endothelial cells expressed typical EC markers such as CD31 and CD144. Furthermore, these endothelial cells showed tube formation on Matrigel and low-density lipoprotein uptake, indicating that they are functional endothelial cells. Scale bar=50 μm.|
|sc-12-0607_sm_SupplFigure9.pdf||183K||Figure S9. Characterization of KMOS- and KMOS3-iPSC-derived cardiomyocytes. The differentiated cardiomyocytes expressed cardiac-specific TNNT2 and transcription factor MEF2C. Scale bar=50 μm.|
|sc-12-0607_sm_SupplFigure10.pdf||156K||Figure S10. Characterization of iPSC-derived neural stem cells (NSCs). The differentiated NSCs expressed NESTIN and transcription factor SOX2. Scale bar=50 μm.|
|sc-12-0607_sm_SupplFigure11.tif||1440K||Figure S11. Schematic diagram showing predicted miR-302b induced regulatory circuit in pluripotent cells.|
|sc-12-0607_sm_SupplFigure12.tif||1535K||Figure S12. OCT4, miR-302, and NR2F2 regulatory models in pluripotent cells and non- pluripotent cells based on experimental results from our study. Blue line represents clustering in pluripotent cells; black line represents clustering in differentiated cells.|
|sc-12-0607_sm_SupplMovie1.avi||3536K||Movie S1. After 14 days of cardiac differentiation of KMOS iPSCs, spontaneously contracting cell layers are observed under light microscopy.|
|sc-12-0607_sm_SupplMovie2.avi||2033K||Movie S2. After 14 days of cardiac differentiation of KMOS3 iPSCs, spontaneously contracting cell layers are observed under light microscopy.|
|sc-12-0607_sm_SupplTable1.pdf||22K||Supplementary Table 1|
|sc-12-0607_sm_SupplTable2.pdf||26K||Supplementary Table 2|
|sc-12-0607_sm_SupplTable3.pdf||21K||Supplementary Table 3|
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