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.
Highlights
Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS)
In order to allow expression of the luciferase protein in bone marrow-derived stromal cells (BMSC) derived from ROSA26-L-S-L-Luciferase transgenic mice, a "floxed" neomycin resistance cassette needs to be excised by the Cre recombinase protein
One clonal line was chosen and used for further characterisation and transplantation experiments described below. This clonal luciferaseexpressing BMSC line was further characterised in vitro based on (1) BMSC morphology (Figure 1B, right picture) and immune phenotype (Figure 1E), for which no difference was observed with parental BMSC derived from ROSA26-L-S-L-luciferase transgenic mice, and based on (2) luciferase activity, which was increased as compared to the described polyclonal luciferase-expressing BMSC cells and remained stable over at least 20 passages as demonstrated by standard in vitro luminescence assays (Figure 1D, n = 10; and see Additional file 1)
Summary
Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). A more realistic aim for stem cell therapy to restore injuries to the CNS might be the implantation of genetically modified stem cell populations in order to produce neurotrophic factors (like BDNF, NT3 or GDNF), with the potential to enhance survival of existing neurons and endogenous neuroregeneration [6,7]. This approach is currently well-described by several research groups including ours [8,9,10,11]. While it has been clearly documented that the enhanced green fluorescent protein (eGFP), which is currently the main reporter gene for histological analysis of cell implants, is a strong immunogenic antigen and requires the need for immune suppressive therapy during cell implantation experiments in non-CNS tissues, it is at the moment rather unclear whether the eGFP or luciferase reporter proteins are tolerated by the immune system following cell implantation in the CNS of immune competent animals [16,17,18,19]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
