Telephone: 650-736-1707; Fax: 650-736-1705
Tissue-Specific Stem Cells
Article first published online: 16 NOV 2011
Copyright © 2011 AlphaMed Press
Volume 29, Issue 12, pages 2018–2029, December 2011
How to Cite
Levi, B., Hyun, J. S., Nelson, E. R., Li, S., Montoro, D. T., Wan, D. C., Jia, F. J., Glotzbach, J. C., James, A. W., Lee, M., Huang, M., Quarto, N., Gurtner, G. C., Wu, J. C. and Longaker, M. T. (2011), Nonintegrating Knockdown and Customized Scaffold Design Enhances Human Adipose-Derived Stem Cells in Skeletal Repair. STEM CELLS, 29: 2018–2029. doi: 10.1002/stem.757
Author contributions: B.L. and D.C.W.: conception and design, administrative support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; E.N.: administrative support, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; S.L., J.S.H., F.J., M.H., and J.P.G.: collection and/or assembly of data and data analysis and interpretation; A.W.J.: collection and/or assembly of data, data analysis and interpretation, and manuscript writing; D.T.M.: collection and/or assembly of data; M.L.: provision of study material or patients; N.Q.: conception and design, administrative support, collection and/or assembly of data, and data analysis and interpretation; G.C.G., J.C.W. and M.T.L.: conception and design 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 October 13, 2011.
- Issue published online: 16 NOV 2011
- Article first published online: 16 NOV 2011
- Accepted manuscript online: 13 OCT 2011 09:37AM EST
- Manuscript Accepted: 14 SEP 2011
- Manuscript Received: 27 JUN 2011
- National Institutes of Health
- National Institute of Dental and Craniofacial Research. Grant Numbers: 1 R21 DE019274-01, RC2 DE020771-01
- Oak Foundation and Hagey Laboratory for Pediatric Regenerative Medicine. Grant Numbers: R01EB009689, RC1HL099117, R33HL089027
- National Institute of Arthritis, and Musculoskeletal and Skin Diseases. Grant Number: 1F32AR057302-02
- Nog/Cre transgenic mice were a kind gift of Lisa Brunet and the Richard Harland Laboratory at the University of California at Berkeley
Additional Supporting Information may be found in the online version of this article.
|STEM_757_sm_supplFigure1.pdf||528K||Supplemental Figure 1: Noggin suppression and BMP signaling in Nog/Cre inducible cre mouse adipose derived stromal cells. (a)Western blot image of pSmad1/5, Smad5, noggin and alpha tubulin control for nog/cre knockdown mice versus flox control. (b-d) Quantification of total psmad 1/5, smad5 and noggin of nog/cre knockdown mice versus flox control. (e) Gene expression profile of mASCs cultured in ODM for three days. Markers examined include osteogenic specific genes alp, col1a, and ocn. (mean expression +/− S.D. *p<0.05). (f-g) Alkaline phosphatase staining and quantification of mASCs at three days differentiation. Scale bars represent 200 μm. (h-i) Alizarin red staining and quantification of mASCs from flox control mice and Nog/Cre knockout mice after seven days of differentiation.|
|STEM_757_sm_supplFigure2.pdf||437K||Supplemental Figure 2: Validation of transduction and noggin-directed shRNA construct. (a) FACS plot of non-transfected hASCs on the FITC channel. (b) FACS plot of control lentiviral transfected shRNA GFP+ transfected hASCs demonstrating over 99% of the cell population with stable transduction. (c) Evaluation of mean transcripts using QRT-PCR analysis demonstrated significant suppression with noggin directed shRNA (*p<0.05) by lentiviral transduction methodologies. (d) Western blot images demonstrates significantly decreased noggin protein levels in noggin shRNA treated cells with quantification by lentiviral transduction methodologies. (e) Western blot quantification of noggin protein knockdown after lentiviral noggin knockdown.|
|STEM_757_sm_supplFigure3.pdf||467K||Supplemental Figure 3: Effect of noggin suppression on proliferation. (a) Cell counting assays of shRNA control and shRNA noggin hASCs performed over 7 days in standard growth medium (SGM) (DMEM,10% FBS). Cells were counted by trypsinization and hemocytometer analysis (n=3 per group). (b) BrdU incorporation assays of hASCs performed over 36 hours of control shRNA and noggin shRNA hASCs. Labeling reagent was applied for 8 hours in culture (n=6 per group). Statistical significance was calculated between groups at individual days using a Paired Student's ttest (*p < 0.05). (c) BrdU immunostaining of calvarial defect tissue at postoperative day five, with noggin shRNA or control shRNA. Hemi-calvaria shown on left for orientation. Scale bars represent 800 μm in first column and 100μm in second and third columns.|
|STEM_757_sm_supplFigure4.pdf||820K||Supplemental Figure 4: Persistence of hASCs in a defect. (a) GFP stain in defect site with implanted shRNA control GFP+ hASCs 6 weeks after calvarial injury (bottom row). Middle column shows GFP (green) and right column shows DAPI stain (blue). Left column demonstrates co-localization of staining. (b) Immunohistochemistry for a human specific nuclear antigen in the region of new bone formation at six weeks. (c) Confocal microscopy of region of calvarial defect with new bone formation at six weeks. Immunofluorecent stain indicates cells positive for human nuclear antigen (green) in the nucleus and osteocalcin (red) in the cytosol. All scale bars represent 100μm.|
|STEM_757_sm_supplFigure5.pdf||574K||Supplemental Figure 5: rhNoggin suppresses hASC mediated calvarial healing. (a) Micro CT images from 0-6 weeks of the following groups:  scaffold alone  scaffold plus control GFP shRNA transduced hASCs and  scaffold plus hASCs with rhNoggin (200ug/ml) delivered to hASC-engrafted defects by subcutaneous injection on days 1–5 postoperatively. (b) Healing was quantified weekly from implantation until six weeks using Adobe Photoshop, expressed as average fraction osseous healing of the original defect size. A one-way ANOVA was utilized to compare the different treatment groups, followed by a post-hoc Paired Student's t-tests (with a Bonferroni correction) to assess significance. Asterisks placed directly over bars show significance when compared to control groups (scaffold alone and scaffold with hASCs) (*p < 0.05).|
|STEM_757_sm_supplFigure6.pdf||625K||Supplemental Figure 6: BMP-2 enhances osteogenic differentiation of hASCs with noggin and control shRNA. (a) Alizarin red stain of noggin and control shRNA cells after seven days in ODM with or without rhBMP2. Scale bars represent 200μm. (b) Alizarin red quantification of control shRNA hASCs at seven days of differentiation demonstrating increased extracellular matrix mineralization with rhBMP-2 (200ng/ml) (mean absorbance+/− S.D., *p<0.05). (c) Alizarin red quantification of noggin shRNA hASCs at seven days of differentiation demonstrating increased extracellular matrix mineralization with rhBMP-2 (200ng/ml) treatment (mean absorbance+/− S.D., p<0.05). (d-f) Gene expression profile of control shRNA and noggin shRNA transfected hASCs at three days of differentiation with or without rhBMP-2 (200ng/ml). Markers examined included osteogenic specific human genes, ALP, RUNX2, and COL1A. Up-regulation of all genes was noted in noggin-suppressed cells (mean expression +/− S.D. *p<0.05). All transcript levels were expressed relative to shRNA control.|
|STEM_757_sm_supplFigure7.pdf||354K||Supplemental Figure 7: In vitro release profile of BMP-2 from HA coated PLGA scaffold. Release profile of BMP-2 from scaffolds was measured by incubating the scaffolds in PBS. Approximately 12% of the initially loaded BMP-2 was released from scaffolds during the first day, followed by steady release of up to 30% during the first 10 days and 47% by 30 days.|
|STEM_757_sm_supplFigure8.pdf||932K||Supplemental Figure 8: Slow releasing BMP-2 releasing scaffold enhances hASC treated calvarial defects. (a) Representative microCT images of defect sites at stratified time points postoperatively. All defects were treated with an HA coated PLGA scaffold (Scaff). Scaffolds differed in amount of BMP-2 they released and whether or not they were seeded with hASCs. These scaffolds had different levels of BMP-2 release and either had 150,000 hASCs or no hASCs. Row 1: Scaff w/ no hASCs and no BMP-2, Row 2: Scaff w/ hASCs and no BMP-2, Row 3: Scaff w/ slow releasing BMP-2 (50μg/ml) without hASCs, Row 4: Scaff w/ slow releasing BMP-2 (50μg/ml) w/ 150,000 hASCs, Row 5: Scaff w/ slow releasing BMP-2 (100μg/ml) without hASCs, Row 6: Scaff w/ slow releasing BMP-2 (100μg/ml) w/ 150,000 hASCs, Row 7: Scaff w/ slow releasing BMP-2 (200μg/ml) without hASCs, Row 8: Scaff w/ slow releasing BMP-2 (200μg/ml) w/ 150,000 hASCs. (b-e) At each week between 1-6 weeks, healing was quantified using Adobe Photoshop, expressed as average fraction osseous healing of the original defect size. A Paired Student's t-test was utilized to compare the BMP-2 releasing scaffold alone with the BMP-2 scaffold and hASCs to assess significance. Asterisks placed directly over bars show significance when compared to control group (scaffold alone) (*p < 0.05). At all time points and in all BMP-2 treatment groups, those scaffolds with hASCs healed more than those scaffolds without hASCs.|
|STEM_757_sm_supplTable1.pdf||7K||Supplemental Table 1.|
|STEM_757_sm_supplTable2.pdf||8K||Supplemental Table 2.|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.