1. Volume 18, Issue 11, Pages 2018-2027 (November 2010)
siRNA Nanoparticle Functionalization of Nanostructured Scaffolds Enables Controlled Multilineage Differentiation of Stem Cells 
Morten Ø Andersen, Jens V Nygaard, Jorge S Burns, Merete K Raarup, Jens R Nyengaard, Cody Bünger, Flemming Besenbacher, Kenneth A Howard, Moustapha Kassem, Jørgen Kjems 
Molecular Therapy 
Volume 18, Issue 11, Pages (November 2010)
DOI: /mt
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Monolayer transfection of human mesenchymal stem cells (hMSCs) grown on tissue culture polystyrene plates, either noncoated (NC) or coated with freeze-dried TransIT-TKO nanoparticles containing EGFP-targeted siRNA (siEGFP), EGFP mismatch siRNA (siMM), TRIB2-targeted siRNA (siTRIB2 #1), or BCL2L2-targeted siRNA (siBCL2L2 #1). (a) The relative fluorescence level of EGFP in hMSCs (EGFP+) after 2 and 7 days of reverse transfection was determined by flow cytometry. Displayed is the average geometric mean fluorescence normalized to nontransfected cells which were set to 100%, the standard deviation is indicated by error bar (N = 3). (b) The relative expression level of BCL2L2 mRNA normalized to GAPDH mRNA after 48 hours of reverse transfection of hMSCs (EGFP−). The graph shows average normalized to untransfected cells which were set to 100%, the standard deviation is indicated by error bars (N = 3). (c) The relative expression level of TRIB2 mRNA normalized to GAPDH mRNA after 48 hours of reverse transfection of hMSCs (EGFP−). The graph shows average normalized to untransfected cells which were set to 100%, the standard deviation is indicated by error bars (N = 3). (d,e) The relative expression level of BCL2L2 mRNA and TRIB2 mRNA normalized to GAPDH mRNA after 48 hours of reverse transfection of hMSCs (EGFP−). The graph shows average normalized to untransfected cells which were set to 100%, the standard deviation is indicated by error bars (N = 5). siRNA #1 corresponds to the same sequence as in graph b and c, although a different siRNA supplier was used. siRNAs #2 targeted different regions of the genes representing a completely different sequence. SScr denotes siRNA #1 where the sequence of four nucleic acids in the seed sequence (position 3–6) has been scrambled. (f) Displays viability after hMSCs (EGFP−) were cultured for 2 days in maintenance medium followed by 12 days in the indicated mediums (M, maintenance medium; O, osteogenic medium without vitamin D3; A, adipogenic medium; C, complex medium; D, osteogenic medium with vitamin D3). The graph shows average normalized to nontransfected cells in maintenance media which were set to 100%, the standard deviation is indicated by error bars (N = 4). EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Scanning electron microscopy of scaffolds. (a) Scaffold before coating with nanoparticles. (b) High magnification of scaffold structure indicated in a. (c) Scaffold after coating with TransIT-TKO/BCL2L2 siRNA nanoparticles. (d) High magnification of scaffold structure indicated in c.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Fluorescence confocal laser scanning microscopy of scaffolds. (a) Scaffold coated with Cy5-labeled small-interfering RNA (siRNA). (b) Scaffold coated with Cy5-labeled siRNA after 24 hours in maintenance medium at 37 °C. (c,d) Scaffolds coated with Cy5-labeled siRNA and seeded with enhanced green fluorescent protein (EGFP)-expressing human mesenchymal stem cells (EGFP+) after (c) 24 hours and (d) 72 hours incubation in maintenance medium. Blue is scaffold autofluorescence, red is Cy5 fluorescence, and green is EGFP fluorescence. All images were acquired with the same settings for the red channel and green channel at ×40 magnification. Image sizes are 100 µm × 100 µm.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
In vitro knockdown on scaffolds. (a) Human mesenchymal stem cells (hMSCs; EGFP+) were seeded onto noncoated (NC) scaffolds and TransIT-TKO particle–coated scaffolds made with EGFP mismatch siRNA (siMM) or EGFP-specific siRNA (siEGFP). After 48 hours, the ratio of EGFP mRNA to GAPDH mRNA was characterized by qPCR. (b,c) hMSCs (EGFP−) were seeded onto scaffolds coated with EGFP-targeted siRNA (siEGFP), BCL2L2-targeted siRNA (siBCL2L2 #1), or TRIB2-targeted siRNA (siTRIB2 #1). After 72 hours, the ratio of (b) BCL2L2 or (c) TRIB2 mRNA to GAPDH mRNA was found. Averages are shown with standard deviation on error bars (n = 5), the values were normalized to (a) noncoated or (b,c) siEGFP control scaffolds which were set to 100%. EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
In vitro adipogenic (a–d) and osteogenic (e–h) differentiation of human mesenchymal stem cells (EGFP−) seeded on noncoated scaffolds (white) and scaffolds coated with TransIT-TKO nanoparticles containing EGFP-targeted siRNA (black) or (a–d) TRIB2 #1- or (e–h) BCL2L2 #1-targeted siRNA (grey). After 2 days of culturing in maintenance medium, differentiation was induced with (a–d) adipogenic or (e–h) osteogenic medium for the indicated number of days. The ratio of (a) AP2, (b) PPARγ2, (c) ADN, (d) LPL, (e) ALP, (f) COL1, (g) OC, and (h) RUNX2 mRNA to B2M mRNA was characterized. Averages are shown with standard deviation indicated by error bars (n = 3 or 4). The values were normalized to the noncoated control scaffold at day group which was set to 100%. Days denote days of culturing in maintenance medium plus days in differentiation medium. ADN, adiponectin; ALP, alkaline phosphatase; AP2, apolipoprotein 2; B2M, β-2 microglobulin; COL1, collagen type I; EGFP, enhanced green fluorescent protein; LPL, lipoprotein lipase; OC, osteocalcin; PPARγ2, peroxisome proliferator–activated receptor-γ isoform 2.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
In vitro osteogenic differentiation of hMSCs (EGFP−) seeded on noncoated scaffolds (NC) and scaffolds coated with TransIT-TKO nanoparticles containing EGFP-targeted siRNA (siEGFP) or BCL2L2-targeted siRNA (siBCL2L2 #1). Scaffolds were cultured 2 days in maintenance medium and 21 days in osteogenic medium. Shown are two representative micro computed tomography slices from the edge and center of each scaffold as well as paraffin sections from the edge of each scaffold (with the edge in the upper right corner) stained with von Kossa (mineralization appears black) or by immunohistochemistry for osteocalcin (osteocalcin appears brown). Cells were counterstained with H&E. The average bone volume/total volume (BV/TV) of two consecutive experiments is also shown.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
In vivo differentiation of human mesenchymal stem cells (EGFP−) seeded on noncoated scaffolds (left column), scaffolds coated with TransIT-TKO nanoparticles prepared with BCL2L2 #1- (middle column) or TRIB2 #1 (right column)-targeted siRNA. After 16 hours of in vitro culturing in maintenance medium scaffolds were implanted subcutaneously in nonobese diabetic/severe combined immunodeficient mice for 8 weeks after which they were surgically removed and studied by histology using the indicated stain or antibody. Representative pictures are shown. In all cases, hematoxylin and eosin staining was used as a counterstain. Positive immunohistochemical staining appears brown. On the Sirius red–stained sections collagen deposition appears red and birefringence appears orange. Pictures are 318-µm wide and 238-µm high.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

9. Figure 8
In vitro and in vivo double differentiation of human mesenchymal stem cells (hMSCs; EGFP−) seeded on scaffolds where one side was coated with TransIT-TKO nanoparticles containing TRIB2-targeted siRNA (#1) and the other side with TransIT-TKO nanoparticles containing BCL2L2-targeted siRNA (#1). (a) In vitro double differentiation. The dual-coated scaffolds were seeded with hMSCs and cultured 2 days in maintenance medium and 8 days in complex medium. The sides were then separated and for each side the ratios of the mRNA levels of PPARγ2, RUNX2, AP2, ADN, OC, and ALP relative to the mRNA level of B2M were determined. The ratios of the marker expression between the side with the TRIB2-targeted siRNA (#1) and the side with the BCL2L2-targeted siRNA (#1) were characterized and plotted. Averages are graphed with error bars representing standard deviation (n = 3 or 4). The y-axis is logarithmic and that markers that were upregulated in the side coated with TRIB2 targeted siRNA compared to side coated with the BCL2L2-targeted siRNA will have values above 1 and vice versa. (b–d) In vivo differentiation on a dual-coated scaffold. The dual-coated scaffolds were seeded with hMSCs for 16 hours in maintenance medium. They were then implanted subcutaneously in nonobese diabetic/severe combined immunodeficient mice for 2 weeks after which they were surgically removed and studied by histology. Whole scaffold sections were stained with (b) hematoxylin and eosin, (c) Sirius red, (d) and von Kossa. Mosaic pictures of whole scaffold sections at ×10 magnification are displayed with the BCL2L2 siRNA–coated side in the top of the pictures, host tissue can be seen surrounding the implant. On the Sirius red–stained section collagen deposition appears red and birefringence appears orange. Images in b and c are 7.6-mm wide and 8.4-mm high and d is 7.6-mm wide and 6.5-mm high. ADN, adiponectin; ALP, alkaline phosphatase; AP2, apolipoprotein 2; B2M, β-2 microglobulin; COL1, collagen type I; EGFP, enhanced green fluorescent protein; LPL, lipoprotein lipase; OC, osteocalcin; PPARγ2, peroxisome proliferator–activated receptor-γ isoform 2; siRNA, small-interfering RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions