title: Real-time in vivo monitoring of viable stem cells implanted on biocompatible scaffolds.
abstract: OBJECTIVE
  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 (99m)Tc-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 (99m)Tc-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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Programs in Neuroscience, Seoul National University, Seoul, South Korea.
authors.forename: Do Won
authors.initials: DW
authors.lastname: Hwang
authors.forename: Sung June
authors.initials: SJ
authors.lastname: Jang
authors.forename: Yun Hui
authors.initials: YH
authors.lastname: Kim
authors.forename: Hyun Joo
authors.initials: HJ
authors.lastname: Kim
authors.forename: In Kyong
authors.initials: IK
authors.lastname: Shim
authors.forename: Jae Min
authors.initials: JM
authors.lastname: Jeong
authors.forename: June-Key
authors.initials: JK
authors.lastname: Chung
authors.forename: Myung Chul
authors.initials: MC
authors.lastname: Lee
authors.forename: Seung Jin
authors.initials: SJ
authors.lastname: Lee
authors.forename: Seung U
authors.initials: SU
authors.lastname: Kim
authors.forename: Soonhag
authors.initials: S
authors.lastname: Kim
authors.forename: Dong Soo
authors.initials: DS
authors.lastname: Lee
mesh_terms: D000818:Animals; D001345:Autoradiography / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D003199:Computer Systems; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D015336:Molecular Probe Techniques; D011877:Radionuclide Imaging; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000166:cytology* / Q000000981:diagnostic imaging*; D023822:Tissue Engineering / Q000379:methods*; D051598:Whole Body Imaging / Q000379:methods*