Tissue-Specific Stem Cells
Article first published online: 24 JUL 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 8, pages 1756–1770, August 2012
How to Cite
Liu, L., Wang, Y., Fan, H., Zhao, X., Liu, D., Hu, Y., Kidd, A. R., Bao, J. and Hou, Y. (2012), MicroRNA-181a Regulates Local Immune Balance by Inhibiting Proliferation and Immunosuppressive Properties of Mesenchymal Stem Cells. STEM CELLS, 30: 1756–1770. doi: 10.1002/stem.1156
Author contributions: L.L.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; Y.W.: collection and/or assembly of data and provision of study material or patients; H.F.: conception and design, and data analysis and interpretation; X.Z.: provision of study material or patients; D.L.: administrative support; Y. Hu: provision of study material or patients; A.R.K. and J.B.: manuscript writing; Y. Hou: conception and design, financial support, 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 June 19, 2012.
- Issue published online: 24 JUL 2012
- Article first published online: 24 JUL 2012
- Accepted manuscript online: 19 JUN 2012 09:10AM EST
- Manuscript Accepted: 21 MAY 2012
- Manuscript Received: 29 FEB 2012
- National Natural Science Foundation. Grant Number: project number: 81072410
- Special Research Grant of Jiangsu Province Department of Health. Grant Numbers: project number: XK200709, JHB2011-1
- Special grant for maternal-fetal medicine from Jiangsu province Health Department of China. Grant Number: project number: 81070508
Additional Supporting Information may be found in the online version of this article.
|SC_12-0204_sm_supplFigure1.tif||606K||Supplementary Figure 1. miR-181a does not affect phenotypic surface antigens of MSCs. Flow cytometry characterization of human MSCs 48 h after transfection with control (pre-nc and anti-nc), pre-miR-181a (A) and anti-miR-181a (B) individually. Expression of MSCs phenotypic surface antigens (CD105, CD73, CD90, HLA-ABC, CD29, CD44, CD45, CD106, HLA-DR, CD19, CD11b, CD14, CD34, CD31) was detected using FACS (n =3).|
|SC_12-0204_sm_supplFigure2.tif||117K||Supplementary Figure 2. PE patient derived MSCs amplified more slowly than normal patient derived MSCs. Cell growth curve of MSCs derived from PE (n=20) and normal (n=20) patients. MSCs of passage 2 were plated in 24-well plates in DF12 supplemented with 10% FBS at a density of 5×103 cells/well. The cells were detached with 0.25% trypsin-EDTA and counted every 2 days and until day 14. Bars show the mean. P values were determined by Mann-Whitney test with Bonferroni correction. P<0.05.|
|SC_12-0204_sm_supplFigure3.tif||239K||Supplementary Figure 3. miR-181a does not affect MSCs apoptosis. MSCs were transfected with control (pre-nc or anti-nc), pre-miR-181a and anti-miR-181a individually. 30 pmol of pre-miR-181a or anti-miR-181a were used for transfection in 12-wells plates at a density of 8×104 cells/well. 48 h after transfection, MSCs were collected for the following experiment. Effects of miR-181a on MSCs discriminated by Annexin- V/PI doublestain 48 h after transfection (n =3). Data are representative of three independent experiments.|
|SC_12-0204_sm_supplFigure4.tif||53K||Supplementary Figure 4. (A) Phosphorylation of Smad2 after SB431542 treatment. Whole-cell lysates of untreated MSCs or MSCs treated with TGF-β1 and the type I TGF-β receptor inhibitor SB431542 for 30 min. Phosphorylation of Smad2 was detected by western blot with a polyclonal antiphospho-Smad2 antibody. (B) MSCs proliferation after 48 h of SB431542 treatment was determined by Cell Counting Kit (n =4).|
|SC_12-0204_sm_supplFigure5.tif||33K||Supplementary Figure 5. qRT-PCR analysis of PAI-1 expression in pre-181a transfected MSCs after 1 h of treatment with TGF-β1 (n =3). Data are representative of three independent experiments.|
|SC_12-0204_sm_supplFigure6.tif||55K||Supplementary Figure 6. qRT-PCR (A) and western blot (B) analysis of the expression of the TGFBR1 and TGFBRAP1 48 h after anti-181a transfection (n =3).|
|SC_12-0204_sm_supplFigure7.tif||45K||Supplementary Figure 7. (A) qRT-PCR analysis of the expression of IL-6 and IDO 48 h after anti-181a transfection (n =3). (B) IL-6 protein expression, as detected by ELISA 48 h after anti-181a transfection (n =3).|
|SC_12-0204_sm_supplFigure8.tif||50K||Supplementary Figure 8. Pre-181a transfected MSCs and pre-nc transfected MSCs were treated with indicated concentrations (0 U, 200 U, 500 U, 1000 U per milliliter) of IFN-γ for 72 h and then cells were harvested and IDO protein was detected by western blot.|
|SC_12-0204_sm_supplFigure9.tif||41K||Supplementary Figure 9. p38 inhibitor blocked pre-miR-181a induced IL-6 protein expression. MSCs were transfected with pre-miR-181a, and, 24 h later, with the Erk1/2 inhibitor PD98059, the p38 inhibitor SB203580, the JNK inhibitor SP600125, the STAT1 inhibitor fludarabine, or the NF-κB inhibitor PDTC for another 24 h. The protein level of IL-6 expressions were detected by ELISA 48h after pre-181a transfection. (n =3)|
|SC_12-0204_sm_supplFigure10.tif||421K||Supplementary Figure 10. T cells activated with or without PHA/IL-2 were cultured with pre-nc transfected MSCs or pre-181a transfected MSCs at MSCs: T cell ratio of 1:10. After 24 hours, CD8+ cells were analyzed for expression of activation molecules using anti-CD25 or anti-CD69 monoclonal antibodies. Cells were permeabilized and the proportion of CD8+/CD69+ and CD8+/CD25+ T cells were quantified (n =4).|
|SC_12-0204_sm_supplFigure11.tif||1149K||Supplementary Figure 11. miR181a attenuates benefits MSCs have on healing in a mouse model of preeclampsia induced by adoptive transfering of activated Th1 cells. (A) Scheme for the experimental course of preeclampsia model induced by activated Th1 cells and MSCs-based therapy. Blood pressure (B) and proteinuria (D) were detected on day on DG14. (C) The fetal rejection rate in pregnant mice after PBS treatment (n=11) or transfer of activated Th1-like cells (n=8) or transfer of activated Th1-like cells and MSCs (n=5) or transfer of activated Th1-like cells and miR-181a transfected MSCs (n=5), as calculated from the ratio of rejected fetuses to total implantation sites. (E,F) Representative picture of placenta and the fetuses from a pregnant mouse. After transfer of activated Th1-like cells or transfer of activated Th1- like cells and miR-181a transfected MSCs, some placenta and fetuses showed placental bleeding (red arrow). (G) Histopathological analysis of kidney after sacrificed. Representative examples of abnormal cell distribution as well as cell clusters in the glomerulum of pregnant cell recipients; normal pregnant animals show no pathological signs in the kidneys. Moreover, fibrosis and glomerular disorganization could also be observed in animals with PE-like signs. MSCs infusion reversed the condition of PE. However, infusion with pre-181a transfected MSCs can not reverse the condition of PE. (H) Histopathological analysis of placenta after sacrificed, animals that received Th1 activated cells and developed PE show large areas of massive hemorrhage (blood), animals that received Th1 activated cells and infusion with pre-181a transfected MSCs also show some areas of massive hemorrhage (blood).|
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