Abstract
Purpose
Three-dimensional fibrous scaffolds provide an environment that enhances transplanted stem cell survival in vivo and facilitates imaging their localization, viability, and growth in vivo. To assess transplanted stem cell viability on biocompatible polymer scaffolds in vivo, we developed in vivo imaging systems for evaluation of implanted viable neural stem cells (NSC) and mesenchymal stem cells (MSC) on scaffolds using luciferase or sodium/iodide symporter (NIS) genes.
Methods
Firefly luciferase stably expressing-C6 cell was established (C6-Fluc). The human neural stem cell, F3, was infected with adenoviral vector carrying luciferase gene (F3-Fluc) and MSC expressing NIS controlled by ubiquitin C promoter using lentiviral vector was established by treating blasticidine for 2 weeks (MSC-NIS). Chitosan and poly l-lactic acid (PLLA) scaffolds were used for in vivo image. In vivo expression of luciferase and human NIS was examined by bioluminescence image or 99mTc-pertechnetate gamma camera image, respectively. The cell/scaffold complex was implanted into subcutaneous or abdominal area of BALB/C nude mouse. For quantitative evaluation of cell viability, regions of interest were drawn on 99mTc-pertechnetate scintigraphy by manual.
Results
The gradual increase of luciferase activity was observed in C6-Fluc seeded with chitosan according to the increase in the number of cells. C6-Fluc/chitosan complex subcutaneously implanted into nude mice showed longitudinal bioluminescence image until 34 days. Luciferase image of abdominal-injected C6-Fluc/PLLA complex was saturated in only 14 days, showing great cell growth due to abundant nutrients. F3 cells showed well-incorporated pattern with fibrous chitosan scaffold using scanning electron microscopy. F3 infected with Ad-Fluc showed >100-fold higher luciferase activity than luciferase activity in F3. Cell-number-dependent increase of luciferase activity was shown in F3-Fluc seeded on chitosan. F3-Fluc incorporation into chitosan after abdominal injection was clearly visible on bioluminescence image up to 11 days. Radionuclide imaging showed higher uptake by MSC-NIS on PLLA scaffolds than by MSC-NIS not seeded on a scaffold. Quantitative data showed significantly better survival of MSC-NIS on PLLA scaffolds than without scaffold at 72 h post-implantation, which concurred with histologic findings.
Conclusion
These results suggest that NSC-Fluc and MSC-NIS cells incorporated within polymer scaffolds can be monitored on a long-term basis by serial in vivo imaging. We believe that a biocompatible scaffold-based imaging system could be used to assess stem cell viabilities in a non-invasive way to aid the development of regenerative therapeutics.
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Acknowledgments
We thank Dr. Sang Hee Kim of the Asan Medical Center, Seoul, South Korea for generously donating the rat mesenchymal stem cells. This work was supported by Nano Bio Regenomics Project of Korean Science and Engineering Foundation and by Innovation Cluster for Advanced Medical Imaging Technology. This study was made easier using KREONET, Korean Research Network, a nation-wide Giga-bps network system.
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Soonhag Kim and Dong Soo Lee contributed equally to this investigation as corresponding authors and Do Won Hwang and Sung June Jang equally contributed as co-first author.
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Supplementary Table 1
CMCs of 99mTc-pertechnetate uptake at the implantation sites of MSC-NIS with scaffold and MSC-NIS without scaffold. Repeated measurement ANOVA revealed a significant difference between CMCs of MSC-NIS with scaffold and MSC-NIS without scaffold (p = 0.034) (DOC 50.0 KB)
Supplementary Table 2
Ratio of corrected maximum counts on MSC injection site and control area. The paired t test showed no significant difference between groups (DOC 28.5 KB)
Supplementary Table 3
Ratio of corrected maximum counts on scaffold alone and control area. The paired t test showed no significant difference between groups (p > 0.05) (DOC 28.0 KB)
Supplementary Fig. 1
C6 glioma cells stably expressing luciferase showed much higher luciferase activity than parental C6 cells. The values are mean ± SD of tetraplicate wells (GIF 67.2 KB)
Supplementary Fig. 2
The 2 × 106 C6-Fluc cells seeded on a PLLA scaffold were implanted intraperitoneally. Summed activities in C6-Fluc/PLLA complex of peritoneal cavities (region B of Fig. 1c) were quantified, and their time courses were shown (GIF 76.4 KB)
Supplementary Fig. 3
F3-Fluc cells showed markedly higher luciferase activity than in parental F3 cells at 48 h after infecting F3 with Ad-Fluc vector (GIF 70.8 KB)
Supplementary Fig. 4
On these microplate images, luciferase activities gradually increased in F3-Fluc/chitosan scaffolds as cell numbers were increased. Initially administered number of cells was comparable to the final activities in cell–scaffold complexes in vitro. Linearity was preserved up to the cell density of 1 × 106 cells/well (GIF 105 KB)
Supplementary Fig. 5
NIS activity in MSC infected with NIS using a lentiviral system was higher than in parental MSC cells (black bar), and KClO4 completely inhibited NIS expression (white bar) (GIF 70.1 KB)
Supplementary Fig. 6
MSC-NIS was implanted to three or four subcutaneous areas of these mice. These are co-registered images showing ROIs used for quantitative analyses. A: MSC-NIS–PLLA scaffold complex was implanted surgically in right flanks. B: MSC-NIS cells without scaffold were injected subcutaneously using a 22 G needle. C: PLLA scaffold alone without cells was implanted subcutaneously. In the mice 1, 3, and 5, on the left scapular area opposite to region C, MSC without NIS was injected subcutaneously as a negative control (GIF 119 KB)
Supplementary Fig. 7a
To the seven mice, MSC-NIS–scaffold complex and MSC-NIS cells without scaffold were implanted. The number of implanted cells was varied for each mouse (1a, 1b: 3 × 106; 2a, 2b: 6 × 106; 3a, 3b: 9 × 106; 4: two scaffolds with 3 × 106cells. In addition to normal thyroid, stomach, and occasional bladder uptakes, the 99mTc-pertechnetate uptake on the implanted sites by MSC-NIS were shown repeatedly imaged from 3 to 72 h post-implantation. MSCs without NIS were implanted in the left scapular area of subjects 1a, 2a, and 3a (GIF 309 KB)
Supplementary Fig. 7b
(GIF 256 KB)
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Hwang, D.W., Jang, S.J., Kim, Y.H. et al. Real-time in vivo monitoring of viable stem cells implanted on biocompatible scaffolds. Eur J Nucl Med Mol Imaging 35, 1887–1898 (2008). https://doi.org/10.1007/s00259-008-0751-z
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DOI: https://doi.org/10.1007/s00259-008-0751-z