Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice

Abstract

BackgroundCell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics.ResultsIn this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-γ-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation.ConclusionWe here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.

Toward Cell Therapy Using Placenta-Derived Cells: Disease Mechanisms, Cell Biology, Preclinical Studies, and Regulatory Aspects at the Round Table

“…Such immune-mediated rejection, which has also been observed with bone marrow MSC in an allogeneic setting [60][61][62][63], constitutes a major challenge for the development of future clinical applications [ 64 ].…”

Labeling of Luciferase/eGFP-Expressing Bone Marrow-Derived Stromal Cells with Fluorescent Micron-Sized Iron Oxide Particles Improves Quantitative and Qualitative Multimodal Imaging of Cellular Grafts In Vivo

“…While BLI detects light produced following a biochemical reaction between the luciferase reporter protein and its substrate (e.g., D-luciferin) [10,11], FLI uses an external light source to excite fluorescent reporter proteins (e.g., eGFP and mCherry) [9]. Both techniques are currently used for noninvasive and twodimensional imaging of stem cell implantation and tracking [12,13]. However, despite their high sensitivity and novel possibility to perform three-dimensional acquisition, both BLI and FLI are restricted to low tissue penetration of light and have a very low spatial resolution caused by the light scattering properties of tissues as compared to other imaging modalities.…”

Distinct In Vitro Properties of Embryonic and Extraembryonic Fibroblast-Like Cells are Reflected in their in Vivo Behavior following Grafting in the Adult Mouse Brain

“…In our preceding studies regarding mMSC and mEF grafting in the CNS of immune-competent mice, we noted that mMSC and mEF grafts became highly infiltrated by microglia (at week 2 postgrafting, up to 50-80% of cells within the graft site are microglia) and surrounded by microglia and astrocytes (4,5,10,21). From the representative Iba1/eGFP, S100b/eGFP, and GFAP/eGFP images provided in Figure 2A, it is clear that mFMSC grafts, too, become highly infiltrated by microglia and surrounded by both microglia and astrocytes.…”

“…However, while extensive in vitro experiments have shed light on potential working mechanisms for the improved clinical outcome following fibroblastic cell grafting in several disease models, including those of the central nervous system (CNS), currently little is known about the actual in vivo behavior of these cellular grafts (1,7). In our preceding studies, we contributed to a better understanding of the cellular events following syngeneic reporter gene-modified murine bone marrowderived mesenchymal stromal cell (mBMMSC) and murine embryonic fibroblast (mEF) grafting in the healthy and injured CNS of immune-competent mice (4,5,10,21). In the course of these studies, it was noted that both mMSC and mEF grafting in the CNS, independent of a preceding injury, results in the activation of strong microglial and astroglial cell responses.…”