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Translational and Clinical Research
Version of Record online: 19 DEC 2012
Copyright © 2012 AlphaMed Press
Volume 31, Issue 1, pages 203–214, January 2013
How to Cite
Fan, W., Cheng, K., Qin, X., Narsinh, K. H., Wang, S., Hu, S., Wang, Y., Chen, Y., Wu, J. C., Xiong, L. and Cao, F. (2013), mTORC1 and mTORC2 Play Different Roles in the Functional Survival of Transplanted Adipose-Derived Stromal Cells in Hind Limb Ischemic Mice Via Regulating Inflammation In Vivo. STEM CELLS, 31: 203–214. doi: 10.1002/stem.1265
Author contributions: W.F.: collection and assembly of data and manuscript writing; K.C., Y.W., and J.C.W.: data analysis and interpretation; X.Q. and S.H.: provision of study material; K.N.: manuscript writing and editing; S.W.: assembly of data; Y.C.: collection of data; L.X.: administrative support; F.C.: conception and design, financial support, and final approval of manuscript. W.F. and K.C. contributed equally to this article.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS October 18, 2012.
- Issue online: 19 DEC 2012
- Version of Record online: 19 DEC 2012
- Accepted manuscript online: 18 OCT 2012 07:32AM EST
- Manuscript Accepted: 23 SEP 2012
- Manuscript Revised: 19 SEP 2012
- Manuscript Received: 1 MAY 2012
- National Basic Research Program of China. Grant Number: 2012CB518101
- National Natural Science Foundation of China. Grant Numbers: Nos. 30970845, 81090274, 81090270, FCao (BWS12J037)
- Innovation Team Development Grant by China Department of Education. Grant Number: 2010CXTD01
- China's Ministry of Science and Technology 863 Program. Grant Number: 2012AA02A603
Additional Supporting Information may be found in the online version of this article.
|sc-12-0412_sm_SupplFigure1.tif||334K||Supplemental Figure 1. Bioluminescence and fluorescence imaging (BLI/FRI) for Tg(Fluc-egfp) mice. Colored scale bars represent optical radiance intensity in photons/second/cm2/steridian (P·s−1·cm−2·sr−1).|
|sc-12-0412_sm_SupplFigure2.tif||401K||Supplemental Figure 2. Multipotent differentiation of mADSCsFluc+GFP+ in vitro. Osteogenesis was identified using alkaline phosphatase (A) and alizarin red-S staining (B); adipogenesis and chondrogenesis were determined using Oil red-O staining (C) and collagen-II immunohistochemistry staining (D) respectively. Scale bar represents 50 μm (A,B) and 5 μm (C,D).|
|sc-12-0412_sm_SupplFigure3.tif||888K||Supplemental Figure 3. In vivo bioluminescence imaging and ex vivo luciferase assay of the engrafted mADSCsFluc+GFP+ in murine ischemic gastrocnemius muscle. (A) Bioluminescence imaging (BLI) tracked the survival and kinetics of engrafted mADSCFluc+GFP+ (1.0×107) in murine ischemic gastrocnemius muscle (n=10 for each group). Quantitative analysis of (B) in vivo Fluc optical signals and (C) ex vivo Fluc enzymatic activity on POD14 (n=5) demonstrated the progressive death of mADSCsFluc+GFP+ following transplantation. Low-dose rapamycin (RapaLD, 50 nM·kg− 1·day−1) treatment also significantly facilitated the mADSCs' survival in the ischemic gastrocnemius muscle. Colored scale bar represents BLI average radiance intensity in P·s− 1·cm−2·sr−1. Error bars represent mean ± SD. *P<0.01 vs. PBS, #P<0.001 vs. ADSC.|
|sc-12-0412_sm_SupplFigure4.tif||538K||Supplemental Figure 4. Immunofluorescence assay of angiogenesis. Confocal images of adductor muscle frozen sections double stained with endothelial marker CD31 (PECAM−1, red) and 4′,6-diamidino−2-phenylindole (DAPI, blue) on POD14. Scale bar represents 50 μm.|
|sc-12-0412_sm_SupplFigure5.tif||191K||Supplemental Figure 5. Expression of inflammatory cytokines in the adductor and gastrocnemius muscles differ after the induction of ischemia. Levels of (A) interleukin−1β (IL−1β), (B) tumor necrosis factor-alpha (TNF-α), (C) IL-6 and (D) IL−10 in the left adductor and gastrocnemius muscle were measured using ELISA on post-operative day1 (POD1), POD7 and POD14. Results are presented for the left adductor muscle in black columns, and the left gastrocnemius muscle in grey columns. Following the induction of ischemia, IL−1β/TNF-α levels are significantly higher, while IL−10 level is significantly lower in the gastrocnemius muscle than the adductor muscle on POD7 and POD14. Error bars represent mean ± SD. *P<0.01 vs. Adductor.|
|sc-12-0412_sm_SupplFigure6.tif||242K||Supplemental Figure 6. Matrigel plug assay of infiltrating cells. Fluorescence activated cell sorting (FACS) and flow cytometry analysis of cells infiltrating into the cellular graft site (Matrigel plug) in ischemic hindlimb, with quadrant percentage. The Ly-6Chigh cells (black square, left panels) were sorted to be further analyzed using FACscan cytometer for CD115/F4-80 (right panels). Macrophages were defined as Ly-6Chigh/CD115+/F4-80+ cells; neutrophils were defined as Ly-6Ghigh/Ly- 6Cdull/CD115neg cells.|
|sc-12-0412_sm_SupplFigure7.tif||314K||Supplemental Figure 7. Apoptosis assay of mADSCs over hypoxia/reoxygenation in vitro. Fluorescence microscopy showed the TUNEL-positive apoptotic cells (green) following hypoxia/reoxygenation. n=20 random fields. Scale bar represents 10 μm.|
|sc-12-0412_sm_SupplTable1.pdf||30K||Supplementary Table 1.|
|sc-12-0412_sm_SupplTable2.pdf||71K||Supplementary Table 2.|
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