257. Persistence of Luciferase Expressing Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs) in Non-Obese Diabetic (NOD) and NOD/Scid Mice
Authors: Dario Gerace, Rosetta Martiniello-Wilks, Ann M. Simpson
Journal: Molecular Therapy (2015)

Background: A potential alternative therapy for the treatment of Type 1 diabetes (T1D) is the generation of "artificial" β-cells via the transfer of β-cell transcription factors to non-pancreatic tissue. This approach has had varying success as challenges in overcoming immune responses towards engrafted cells requires strategies such as cell encapsulation or the administration of immunosuppressive drugs to permit a sustained therapeutic effect. Due to the reported immune-evasive properties of mesenchymal stem cells (MSCs), their therapeutic potential as a treatment for T1D has been pursued. By modifying bone marrow-derived MSCs (BMSCs) to express the codon optimised luciferase reporter gene Luc2, we suggest that bioluminescence imaging (BLI) can be used to monitor the persistence of engrafted BMSCs in immune-competent and immune-compromised models of T1D. The aim of this study is to determine whether Luc2 is a suitable reporter for monitoring the persistence of BMSCs in an immune-competent model of T1D. Methods: Bone-marrow was harvested from the femurs of 20 NOD mice and cultured for 2 passages prior to FACS sorting. Adherent cells were sorted according to expression of two surface markers (Marker 1: hematopoietic marker) and (Marker 2: stem cell antigen). Marker 1−/Marker 2+ cells were subjected to tri-lineage differentiation and clonogenicity assays to confirm MSC identity. Luc2 was ligated into the mammalian expression plasmid pVITRO2-mcs. Using nucleofection pVITRO-Luc2 (10μg) was introduced into 1×106 BMSCs, and positive clones selected in culture containing hygromycin (200μg/ml). BMSC-Luc2 were transplanted subcutaneously in NOD and NOD/Scid mice at three cell concentrations (1×106, 1×105 and 1×104 cells/injection) in duplicate, and imaged via BLI following administration of D-luciferin (15mg/ml) for 38 days. Results: FACS sorting of adherent cells yielded 15.3% Marker 1−/Marker 2+ with 95% purity. These cells successfully underwent tri-lineage differentiation and formed colonies, confirming their identity as BMSCs. Nucelofected BMSC-Luc2 expressed functional Luc2 in vitro and in vivo in NOD and NOD/Scid mice. BMSC-Luc2 cells were detected in NOD mice up to 10 days post-transplantation, after which no signal was detected. By comparison, transplanted BMSC-Luc2 cells in NOD/Scid mice were detected up to 38 days post-transplantation (experimental endpoint). Conclusions: The markers used to isolate a unique subpopulation of cells from the stromal cell fraction of NOD mouse bone-marrow yielded cells that conform with the typical characteristics of MSCs. The in vivo data shows that in the immune-competent NOD mouse, the BMSC-Luc2 cells are cleared within 2 weeks post-transplantation and suggest immune targeting against the cell graft. Autologous transplantation of NOD BMSCs suggests immune-targeted clearing of the cell graft may be due to expression of the Luc2 reporter gene. Therefore, the use of Luc2 as a reporter gene for long-term in vivo research may be limited to immune-compromised models.