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Tissue-Specific Stem Cells
Article first published online: 5 APR 2011
Copyright © 2011 AlphaMed Press
Volume 29, Issue 4, pages 725–735, April 2011
How to Cite
Jun, D., Garat, C., West, J., Thorn, N., Chow, K., Cleaver, T., Sullivan, T., Torchia, E. C., Childs, C., Shade, T., Tadjali, M., Lara, A., Nozik-Grayck, E., Malkoski, S., Sorrentino, B., Meyrick, B., Klemm, D., Rojas, M., Wagner, D. H. and Majka, S. M. (2011), The Pathology of Bleomycin-Induced Fibrosis Is Associated with Loss of Resident Lung Mesenchymal Stem Cells That Regulate Effector T-cell Proliferation. STEM CELLS, 29: 725–735. doi: 10.1002/stem.604
Disclosure of potential conflicts of interest is found at the end of this article.
Author contributions: D.J.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; C.G., K.C., T.S., C.C., T.S., and M.R.: collection and/or assembly of data, data analysis and interpretation; J.W., N.T., and A.L.: data analysis and interpretation; T.C. E.C.T., E.N.-G., and S.M.: collection and/or assembly of data; M.T., B.S., and B.M.: provision of study material or patients; D.K.: collection and/or assembly of data, data analysis and interpretation, manuscript writing; D.H.W. and S.M.M.: conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript.
First published online in STEM CELLSEXPRESS February 3, 2011.
- Issue published online: 5 APR 2011
- Article first published online: 5 APR 2011
- Accepted manuscript online: 3 FEB 2011 01:14PM EST
- Manuscript Accepted: 10 JAN 2011
- Manuscript Received: 8 NOV 2010
- American Heart Association (AHA). Grant Number: GIA0855953G
- NIH. Grant Number: 1R01 HL091105-01
- AHA. Grant Number: SDG073512N
- American Federation for Aging Research
- University of Colorado Cancer Center (UCCC). Grant Number: 5 P30 CA 46934-15
- NCI. Grant Numbers: P30 CA 46934-14, RO1DK075013
- Diabetes Digestive & Kidney Diseases
- Kleberg Foundation
Additional supporting information available online.
|STEM_604_sm_suppinfofig1.eps||33622K||Supplemental Figure 1. Surface phenotype and multipotent differentiation of luMSC. The lung MSC population can be enriched by flow cytometric sorting of Hoechst 33342 stained lung tissue to identify a CD45neg Hoechstdim side population (SP) of cells. (A) These cells express characteristic cell surface MSC markers (CD73, CD105, CD106, CD 44, CD90, CD133 and Sca1) and lack of hematopoietic and vascular markers, mainly CD45, F480, c-kit and CD146. (B) The luMSC also differentiate into three characteristic mesenchymal lineages, osteoblast, chondrocye and adipocyte, identified by von kossa stain to detect calcium, alizarin red to detect bone matrix, oil red o to distinguish lipid droplets and aggrecan and alcian blue to identify chondrocytes matrix.|
|STEM_604_sm_suppinfofig2.eps||672K||Supplemental Figure 2. CD45 positive cells in BAL peak on day 14 following bleomycin injury. Intratracheal bleomycin or PBS was administered to mice and BAL collected on 1,4,7,14 and 35 days post injury. BAL was stained to detect CD45 expressing cells and the mean percentage quantitted by flow cytometry.|
|STEM_604_sm_suppinfofig3.eps||325K||Supplemental Figure 3. Proliferating T cells were rarely identified on days 7 following bleomycin injury. Immunoflourescent staining was performed to localize Tcells (CD3) and proliferating cells (PCNA) following bleomycin injury and cell treatment. T cells were present in all bleomycin injured groups however double labeled cells were infrequently identified. Arrows indicate double positive cells.|
|STEM_604_sm_suppinfofig4.eps||4669K||Supplemental Figure 4. Validation of gene microarray analysis. Quantitative PCR analysis of select markers of inflammation, angiogenesis, WNT targets and fibrosis in bone marrow (BM 2), bleomycin treated lung (luBleo), luFB and luMSC. Data for each marker were normalized to GAPDH signals.|
|STEM_604_sm_suppinfofig5.eps||617K||Supplemental Figure 5. Gating strategy to detect GFP. BM transplants were performed with whole BM from GFP-expressing donor mice. Gates for positive and negative GFP expressing cells were set by the C57Bl6 WT and GFP mouse lung SP and whole lung. Representative scattergrams are shown for each.|
|STEM_604_sm_suppinfofig6.eps||604K||Supplemental Figure 6. LacZ staining of donor luMSC and luFB. Donor lung MSC and lung FB were isolated from C57Bl6/ROSAlacZ mice and expanded in culture using chamber slides, fixed with 4% paraformaldehyde for 30 minutes and LacZ staining was performed as previously described (17).|
|STEM_604_sm_suppinfofig7.eps||10855K||Supporting Information FIgure 7|
|STEM_604_sm_suppinfotables.doc||275K||Supplemental Table 1. Antibodies use in Flow Cytometry Analyses Supplemental Table 2. qPCR Primer Sets Supplemental Table 3. Changes in Gene Expression for luFB vs. luMSC Corresponding to Heatmaps Supplemental Table 4. A Comparison of Gene Expression between BM-MSC, LuMSC and LuFB|
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