Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice
- Irene Bergwerf1, 2,
- Nathalie De Vocht1, 2, 3,
- Bart Tambuyzer1, 2,
- Jacob Verschueren3,
- Kristien Reekmans1, 2, 9,
- Jasmijn Daans1, 2,
- Abdelilah Ibrahimi4, 10,
- Viggo Van Tendeloo1, 2, 9,
- Shyama Chatterjee5,
- Herman Goossens2,
- Philippe G Jorens6, 9,
- Veerle Baekelandt7, 10,
- Dirk Ysebaert8, 9,
- Eric Van Marck5,
- Zwi N Berneman1, 2, 9,
- Annemie Van Der Linden3 and
- Peter Ponsaerts1, 2, 9Email author
© Bergwerf et al; licensee BioMed Central Ltd. 2009
Received: 18 June 2008
Accepted: 07 January 2009
Published: 07 January 2009
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). 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.
In 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.
We 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.
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). While injuries to the CNS have been shown to trigger neurogenesis from resident neural stem cells, these endogenous self-repair mechanisms are insufficient to induce full functional recovery [1, 2]. Therefore, it is clear that additional therapies, like cell transplantation, might be needed to further enhance restoration of brain function following primary (e.g. impact, stroke) and secondary (e.g. inflammation) injury to the CNS. Although many studies aim to replace necrotic or dysfunctional neural tissue directly by implantation of stem cells, only modest functional recovery following injury has been observed until now [3–5]. 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 [8–11]. For these studies, most ideally one should be able to non-invasively visualise and localise stem cell implants in the brain of living animals at different time-points. For this purpose, both bioluminescence imaging (BLI) and magnetic resonance imaging (MRI) have been proposed as suitable non-invasive methodologies for the follow-up of cell implants in the CNS of rodents [12–15]. While images created by MRI have a high spatial resolution, cells need to be loaded with contrast agents, like super paramagnetic iron oxides (SPIO), which might display some toxicity towards the implanted cells and surrounding tissue. Another disadvantage of these contrast agents is leakage out of necrotic cells and uptake by endogenous cells, which might result in false identification of cell implant survival and localisation. In contrast, for generating images by BLI, cell implants need to express the luciferase reporter protein, which, following administration of the substrate luciferin, can produce light through an ATP-dependent enzymatic oxidation of luciferin. Therefore, despite the lower special resolution than MRI, BLI visualises only viable cell implants, which makes BLI one of the most valuable research techniques in order to monitor survival of cell implants non-invasively. One potential drawback of BLI is the need for genetic modification of cell populations with the Luciferase reporter gene. 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 [16–19].
Homozygous ROSA26-L-S-L-Luciferase transgenic mice (FVB background) were obtained via Jackson Laboratories (strain 005125) and further bred in the specific pathogen free animal facility of the University of Antwerp . Male offspring (n = 90) were used for bone marrow-derived stromal cell (BMSC) culture, cell implantation experiments and/or ELISPOT analysis. For all experiments, mice were kept in normal day-night cycle (12/12) with free access to food and water. All experimental procedures were approved by the Ethics Committee for Animal Experiments of the University of Antwerp (approval no. 2006/36).
Establishment and maintenance of primary BMSC cultures
BMSC were cultured from male ROSA26-L-S-L-Luciferase transgenic mice following a protocol previously described by Peister et al. . Briefly, bone marrow was flushed from tibia and femurs of 3-week old ROSA26-L-S-L-Luciferase mice. Next, harvested bone marrow was washed twice with phosphate-buffered saline (PBS) and the total cell population obtained was plated in a T75 culture flask (one flask per mouse) in 20 ml 'complete isolation medium' (CIM), consisting of RPMI-1640 medium (Invitrogen) supplemented with 8% horse serum (HS, Invitrogen), 8% fetal calf serum (FCS, Hyclone), 100 U/ml penicillin (Invitrogen), 100 mg/ml streptomycin (Invitrogen), and 1.25 mg/ml amphotericin B (Invitrogen). Following 24 hours of culture, non-adherent cells were removed and 20 ml fresh CIM was added to the cultures. For a period of two weeks, CIM was replaced every 3 to 4 days. Next, cultured cells were harvested using trypsin-EDTA (Invitrogen) treatment and replated in a new T75 culture flask in 20 ml CIM. Stromal cell outgrowth in this culture was termed passage 1 and further expanded in 'complete expansion medium' (CEM), consisting of Iscove modified Dulbecco's medium (IMDM, Cambrex) supplemented with 8% FCS, 8% HS, 100 U/ml penicillin, 100 mg/ml streptomycin and 1.25 mg/ml amphotericin B. For routine cell culture, BMSC cultures were split 1:3 every 5 to 7 days. In addition, clonal cultures of luciferase-expressing stromal cells were obtained by limiting dilution.
Immunophenotyping of BMSC cultures derived from ROSA26-L-S-L-Luciferase transgenic mice was performed using the following monoclonal antibodies: fluorescein-isothiocyanate (FITC)-labelled anti-mouse CD31 (eBioscience, 11/0311-82), FITC-labelled anti-mouse CD106 (eBioscience, 11/1081-82), FITC-labelled anti-mouse CD117 (eBioscience, 11/1171-82), FITC-labelled anti-mouse Sca-1 (eBioscience, 11/5981-82), FITC-labelled anti-mouse MHC-I (Becton Dickinson, 5553570), phycoerythrin (PE)-labeled anti-mouse CD45 (Becton Dickinson, 553081), and PE-labelled anti-mouse MHC-II (eBioscience, 12/5321/82). Immunostaining for A2B5 was performed using an unconjugated mouse-anti-mouse A2B5 monoclonal antibody (Chemicon, MAB312R) followed by staining with PE-labelled rat-anti-mouse secondary antibody (Jackson Immunoresearch, 115-116-075). Before staining, harvested cells were washed twice with PBS supplemented with 1% FCS (designated as PBS*) and resuspended in PBS* at a concentration of 5 × 105 cells/ml. For antibody staining, 1 μg of antibody was added to 100 μl of cell suspension for 30 min at 4°C. Following incubation, cells were washed once with PBS*, resuspended in 1 mL PBS*, and analysed using an Epics XL-MCL analytical flow cytometer (Beckman Coulter). For determination of eGFP transgene expression, harvested eGFP mRNA-electroporated or lentivirus-transduced BMSC cultures were washed once with PBS, resuspended in PBS and directly analysed using an Epics XL-MCL analytical flow cytometer. Cell viability was assessed through addition of GelRed (1× final concentration, Biotum) to the cell suspension immediately before flow cytometric analysis. At least 10,000 cells per sample were analysed per sample and flow cytometry data were analysed using FlowJo software.
Messenger RNA electroporation
Messenger (m)RNA encoding the enhanced green fluorescent protein (eGFP) and the Cre recombinase protein was prepared as described previously [22, 23]. Prior to electroporation of BMSC populations, cells were washed twice with serum-free OptiMem medium (Invitrogen) and resuspended at a final concentration of 5–10 × 106 cells/ml in serum-free OptiMem medium. Subsequently, 200 μl of the cell suspension was mixed with 20 μg of mRNA and electroporated in a 4 mm electroporation cuvette at 300V and 150 μF using a Gene Pulser Xcell electroporation device (Bio-Rad). After electroporation, fresh complete medium was added to the cell suspension and cells were further cultured as described above.
In vitro bioluminescence assay
Luciferase activity in cultured BMSC, BMSC-Luc and BMSC-Luc/eGFP/Pac cell populations (1 × 105 cells per assay) was measured using the commercial Bright-Glo luciferase assay system (Promega), according to the manufacturer's instructions.
Construction of the pCHMWS-eGFP-IRES-Pac vector was performed in two consecutive steps using standard cloning techniques. First, the puromycin resistance gene (Pac) was inserted downstream of an IRES element and the resulting IRES-Pac clone was amplified by PCR and cloned after the eGFP in the pCHMWS-eGFP vector .
Lentiviral vector production was performed as described earlier by Geraerts et al., with minor modifications . Filtered vector particles were concentrated using Vivaspin 15 columns (Vivascience, Hannover, Germany), aliquoted and stored at -80°C. For transduction experiments, cells were seeded in a 24-well plate at 50,000 cells per well. The next day, cells were transduced with vector expressing the eGFP-IRES-Pac cassette (2.86 105pg p24/well) in CEM medium. After 48 hrs of incubation, the vectors were washed from the cells and medium was replaced. Cells were subcultured at least 4 times and transduction efficiency was determined by flow cytometry. In addition, a clonal line was obtained by limiting dilution for use in further cell implantation experiments.
Cell preparation for implantation experiments
Following harvesting of BMSC-Luc and BMSC-Luc/eGFP/Pac cell populations via trypsin/EDTA treatment, cells were washed twice with PBS. Next, cells (mean viability of cell populations was 90–95%) were resuspended at a concentration of 100 × 106 cells/mL in PBS for intracerebral cell implantation or at a concentration of 5 × 106 cells/mL in PBS for intramuscular cell implantation. Cell preparations were kept on ice until intracerebral cell implantation.
Cell transplantation experiments
For cell implantation in the CNS, mice were anaesthetized by an intraperitoneal injection of a ketamin (80 mg/kg) + xylazin (16 mg/kg) mixture and placed in a stereotactic frame. Next, a midline scalp incision was made and a hole was drilled in the skull using a dental drill burr at an equal distance between RCS and lambda and at 2 mm on the right side of the midline. Thereafter, an automatic micro-injector pump (kdScientific) with a 10 μl Hamilton Syringe was positioned above the exposed dura. A 30-gauge needle (Hamilton), attached to the syringe, was stereotactically placed through the intact dura to a depth of 2 mm. After 2 minutes of pressure equilibration, 2 × 105 BMSC-Luc or BMSC-Luc/eGFP/Pac in 2 μl PBS were injected at 0.7 μl/min. The needle was retracted after another 3 minutes to allow pressure equilibration and to prevent backflow of the injected cell suspension. Next, the skin was sutured, a 0.9% NaCl solution was administered subcutaneously in order to prevent dehydration and mice were placed under a heating lamp to recover. For intramuscular cell injection, mice were anaesthetized in an induction chamber using an isoflurane (3%) + N2 (1 L/min) + O2 (0,5 L/min) gas mixture. Directly thereafter, 5 × 105 BMSC-Luc or BMSC-Luc/eGFP/Pac in 100 μl PBS were injected in the right pelvic limb muscles.
In vivo bioluminescence imaging
At different time points between day 1 and week 4 after cell implantation, mice were analysed by real-time in vivo bioluminescence imaging (BLI) in order to determine the presence or absence of viable cell implants in the CNS. For this, mice were anaesthetized by intraperitoneal injection of a ketamin (80 mg/kg) + xylazin (16 mg/kg) mixture, followed by an intraperitoneal (brain BLI) or intravenous (muscle BLI) injection of D-luciferin (150 mg/kg body weight dissolved in PBS, Synchem). Immediately after luciferin administration, mice were imaged for 20 minutes using an in vivo real-time φ-imager system (Biospace). At the end of every acquisition a photographic image was obtained. The data were analysed with Photovision software, which superimposes the bioluminescence signal on the photographic image. The most intense bioluminescence signal detected is shown in red, while the weakest signal is shown in blue.
Brain dissection for histological analysis
At week 1 or week 3 post-implantation, mice were deeply anaesthetized in an induction chamber by inhalation of an isofluorane (4%), oxygen (0,5 L/min) and nitrogen (1 L/min) mixture for 2 minutes, followed by cervical dislocation. Whole brains were surgically removed and fixed in 4% paraformaldehyde for 2 hours.
Fixed brains were dehydrated in sucrose gradients (5%, 10% and 20%), frozen in liquid nitrogen and stored at -80°C until further processing. Consecutive 10 μm-thick cryosections were cut using a Microm HM5000 cryostat and stained with haematoxylin-eosin (HE) to locate the transplantation site. Further immunohistochemical analysis was performed using a biotin-labeled anti-mouse Sca-1 antibody (eBioscience 13-5981-85) for BMSC identification, and a biotin-labeled anti-mouse CD11b antibody (eBioscience 13-0112-85) for detection of activated microglia at the site of cell implantation. In brief, slides were rinsed with a washing buffer and endogenous peroxidase was blocked following 30 min incubation with methanol containing 1% hydrogen peroxide. Next, slides were washed with water and washing buffer, followed by incubation with normal rat serum (Jackson Immuno Research 012-000-120) for 1 hour at room temperature. Subsequently, slides were incubated for 3 hours with the biotin-labeled primary antibody at room temperature. Following this, slides were rinsed with washing buffer, and incubated for 1 hour at room temperature with a streptavidin-horse-radish-peroxidase complex (Dako 00032671). Visualization for all slides was carried out after staining with diaminobenzidine (DAB, Dako), according to manufacturer's instructions, and nuclei were counterstained with Carazzi's haematoxylin. Bright-field immunohistochemical analysis was done using an Olympus Bx41 microscope equipped with an Olympus DP50 camera. Olympus DP Software was used for image collection.
A murine IFN-γ ELISPOT assay (Diaclone, 862.031.010.S) was performed according to manufacturer's instructions. In brief, spleens were dissected from cell-transplanted ROSA26-L-S-L-Luciferase transgenic mice (both intramuscular and intracerebral) at 2 weeks post-injection. Next, after dissociation of the spleens over a 100 μm nylon filter, mononuclear cells were enriched following a density-based centrifugation step (Ficoll-Paque Plus, GE Healthcare). Magnetic isolation of CD8+ T-cells was done using anti-CD8 MACS MicroBeads (Miltenyi Biotec, 130-049-401), according to the manufacturer's instructions. Isolated CD8+ T-cells (= responder cells) were plated on ELISPOT plates at 1 × 105 cells/well in IMDM supplemented with 10% FBS, penicillin/streptomycin and amphotericin B. Cells were then cultured for 16 hours either: (i) un-stimulated, (ii) stimulated with 1 × 104 parental BMSC (= non stimulator cells), or (III) stimulated with 1 × 104 BMSC-Luc or BMSC-Luc/eGFP/Pac (= stimulator cells). All experiments were performed in quadruplicate per mouse. The ELISPOT plates were analysed using an AID ELISPOT Reader (Autoimmun Diagnostika GmbH). Data are presented as IFN-γ spot-forming cells (SFC) per 1 × 105 CD8+ responder T-cells.
Results are expressed as mean ± standard deviation. Comparisons were validated using Student's t-test. A p-value < 0.01 was considered to be statistically significant.
Culture and characterisation of a clonal luciferase-expressing bone marrow-derived stromal cell line from ROSA26-L-S-L-Luciferase transgenic mice
Survival of luciferase-expressing BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice following implantation in the central nervous system of syngeneic immunocompetent mice
Survival of BMSC genetically modified with multiple reporter genes following implantation in the central nervous system of syngeneic immunocompetent mice
Induction of BMSC-specific CD8+ T-cell responses following intramuscular, but not intracerebral, cell implantation in syngeneic immunocompetent mice
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. In this study, we first aimed to investigate whether luciferase-expressing bone marrow-derived stromal cells (BMSC), derived from ROSA26-L-S-L-Luciferase transgenic mice, can be implanted and survive in the CNS of immunocompetent syngeneic luciferase-negative ROSA26-L-S-L-Luciferase transgenic mice, despite the potential immunogenicity of the luciferase protein [19, 20]. The choice of ROSA26-L-S-L-Luciferase transgenic mice for performing these experiments has two reasons. First, we assumed that the epigenetic stability of luciferase expression would be much higher when cell populations were derived from a well-characterised luciferase-expressing transgenic mouse strain, as compared to ex vivo transgenesis using plasmid DNA or viruses [11, 26]. Second, Cre recombination in cells derived from ROSA26-L-S-L-Luciferase transgenic mice allows removal of a floxed neomycin resistance gene (Figure 1A), resulting in luciferase protein expression without additional selection markers. Following this strategy, i.e. derivation of cell populations from ROSA26-L-S-L-Luciferase transgenic mice followed by Cre-recombination in order to activate luciferase expression, autologous transplantation experiments can be performed in syngeneic luciferase-negative ROSA26-L-S-L-Luciferase transgenic mice with only the luciferase protein as potential immunogen. In this context, we derived BMSC cultures from ROSA26-L-S-L-Luciferase transgenic mice and characterised these BMSC populations as described by Peister et al . Immunophenotypic analysis (Figure 1E) clearly demonstrated the uniform expression of mesenchymal markers (Sca-1 and V-CAM) without detectable expression of endothelial (CD31), haematopoietic (c-kit, CD45 and MHC-II) or neural (A2B5) markers.
Next, in order to allow expression of the luciferase protein in BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice, a floxed neomycine resistance cassette needs to be excised by the Cre recombinase protein. We previously described a non-viral non-DNA gene transfer methodology for highly efficient protein expression in a variety of cell types, including human BMSC, based on electroporation of messenger RNA [27–30]. In this study, following these previous reports, we also describe for the first time highly efficient mRNA-based gene transfer in murine BMSC using the enhanced green fluorescent protein (eGFP) reporter gene (Figure 1C). The latter is of importance when transient protein expression is desired and introduction of DNA sequences (either by plasmid DNA or viruses) should be avoided [31, 32]. Next, our cultured BMSC populations were electroporated with mRNA encoding the Cre recombinase protein, following previously described procedures [22, 23]. Although luciferase expression was induced (Figure 1D, BMSC-Luc polyclonal), the culture of a clonal luciferase-expressing BMSC was necessary in order to obtain a pure population expressing high levels of the luciferase protein (Figure 1D, BMSC-Luc clonal). The fact that recombination efficiency was rather low in cultured BMSC following electroporation with Cre recombinase mRNA, despite the observation that electroporation with EGFP mRNA resulted in high levels of transfection efficiency, can be ascribed to variations in Cre recombinase activity in different cell types (published and unpublished data) [22, 23].
In our transplantation model, i.e. autologous implantation of BMSC-luc derived from ROSA26-L-S-L-Luciferase transgenic mice in the CNS of syngeneic luciferase-negative ROSA26-L-S-L-Luciferase transgenic mice, we routinely transplant 2 × 105 cells in order to obtain a clear signal for in vivo bioluminescence imaging (BLI). Further experiments revealed a minimum of 5 × 104 cells to be required for obtaining a minimum signal above background (data not shown). However, this detection limit might be different when using BMSC derived from another luciferase-expressing transgenic mouse or following lentiviral transduction with the luciferase reporter protein. Following cell transplantation in this model, we did not observe immune-mediated rejection of BMSC-Luc implants in the CNS during a follow-up period of 3-4 weeks by real-time BLI (Figure 2A), while intramuscular BMSC-Luc implants did not survive during the same follow-up period (Figure 2C). Also, when the same BMSC population was implanted in the CNS of immunocompetent allogeneic C57/BL6 mice (see Additional file 2) or when C57/BL6 BMSC were implanted in the CNS of immunocompetent ROSA26-L-S-L-Luciferase transgenic mice (data not shown), no survival of grafted cells was observed during the same follow-up period. These results suggest that BMSC-Luc derived from ROSA26-L-S-L-Luciferase transgenic mice can indeed survive immunologically in the CNS of immunocompetent luciferase-negative ROSA26-L-S-L-Luciferase transgenic mice, despite the potential immunogenicity of the luciferase protein. In addition, during the observation period of 3-4 weeks, we did not observe a significant increase of in vivo bioluminescence signal over time. The latter, although further investigation will be needed (e.g. quantitative analysis), might become a tool to exclude tumour formation following cell implantation .
In order to further investigate the tolerogenic properties of the CNS with regard to reporter gene-modified BMSC implants, we further genetically engineered our BMSC-Luc cells using a lentivirus encoding eGFP and the puromycin resistance gene (Figure 3A). Following transplantation of these BMSC-Luc/eGFP/Pac in the CNS of syngeneic immunocompetent mice, a similar degree of cell survival was observed as compared to BMSC-Luc implants (Figure 3C and 3E). Again, no cell survival was observed upon intramuscular BMSC-Luc/eGFP/Pac implantation. These results demonstrate that reporter gene-modified BMSC can survive immunologicaly in the CNS of syngeneic immunocompetent mice. Currently, we do not know why expression of reporter proteins (in this study Luc, eGFP and Pac), which are from an immunological point of view a foreign antigens, are tolerated in the CNS. Several explanations can be hypothesised for this: (1) some cell populations, among them BMSC, have been ascribed immune modulatory properties , or (2) immune surveillance mechanisms in the CNS are not properly activated , both possibly leading to immunological acceptance of the neo-expressed reporter proteins in the CNS. In this context, we investigated whether inflammatory responses occur following cell implantation in the CNS. Although histological analysis of cell-implanted brains indicated the presence of activated CD11b+ microglial cells surrounding the cell graft at week 1 post-implantation, the presence of these CNS immune cells was highly diminished by week 3 post-implantation, indicating immunological acceptance of autologous BMSC-Luc (Figure 2B) or BMSC-Luc/eGFP/Pac (Figure 3E). However, the observed immune tolerance of the CNS for reporter gene-modified BMSC does not imply an absolute immune tolerance of the CNS. In contrast, allogeneic cell implantation in the CNS of immunocompetent mice leads to a sustained activation of microglia and rejection of cell implants by week 2–4 post-implantation (see Additional file 2).
Finally, we aimed to investigate whether the non-survival of intramuscular BMSC-Luc and BMSC-Luc/eGFP/Pac cell implants was mediated by the host's immune system. Although the presence of reactive IFN-γ-producing CD8+ T-cells was clearly demonstrated following intramuscular, but not intracerebral, BMSC-Luc and BMSC-Luc/eGFP/Pac cell implantation (Figure 4), surprisingly these immune reactive T-cell response were not specific for the introduced reporter genes. Although further research will be needed to elucidate the specificity of the induced BMSC-specific IFN-γ-producing CD8+ T-cells, several explanations can be hypothesised for this: (1) due to the use of fetal calf serum and horse serum for in vitro BMSC expansion, xenogeneic serum components (eg. glycolipids) might have induced cellular immunogenicity, or (2) cell culture induced genomic alterations might have resulted in the expression of highly immunogenic neo-antigens, both possibly leading to immunological rejection of our BMSC cultures following intramuscular cell implantation.
While many cell transplantation studies are currently performed under immunosuppressive therapy or in immune-deficient mice, clinical applications of cell therapy will most likely have to deal with immunocompetent patients. In this study, we demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon cell implantation in the CNS of syngeneic immunocompetent mice. The proposed research model thus provides a powerful tool for studying survival and localisation of autologous BMSC implants in the central nervous system of syngeneic mice by real-time bioluminescence imaging and/or histological analysis in the absence of immunosuppressive therapy.
We acknowledge helpful assistance from August Van Laer (Laboratory of Experimental Surgery) with animal handling and surgical procedures, and from Frank Rylant (Laboratory of Pathology) with histological analysis. This work was supported by research grants 7.0004.03N (granted to PJ) and G.0132.07 (granted to ZB) of the Fund for Scientific Research-Flanders (FWO-Vlaanderen, Belgium), by research grants BOF-KP 2005 (granted to PP), BOF-KP 2006 (granted to SC), BOF-NOI 2006 (granted to PP and SC), ID-BOF 2006 (granted to AVDL and PP) from the Antwerp University, by research grant BRAINSTIM of the Flemish Institute for Science and Technology (granted to ZB and AVDL), in part by a Methusalem research grant from the Flemish government (granted to HG), in part by the EC FP6-project DiMI (LSHB-CT-2005-512146 granted to AVDL) and the EC FP6-project EMIL (LSHC-CT-2004-503569 granted to AVDL), and by the Fund for Cell Therapy from the Antwerp University Hospital. Peter Ponsaerts is a post-doctoral fellow of the FWO-Vlaanderen.
- Arvidson A, Collin T, Kirik D, Kokaia Z, Lindvall O: Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med. 2002, 8: 963-970. 10.1038/nm747.View ArticleGoogle Scholar
- Kokaia Z, Lindvall O: Neurogenesis after ischaemic brain insults. Curr Opin Neurobiol. 2003, 13: 127-132. 10.1016/S0959-4388(03)00017-5.View ArticleGoogle Scholar
- Webber DJ, Bradbury EJ, McMahon SB, Minger SL: Transplanted neural progenitor cells survive and differentiate but achieve limited functional recovery in the lesioned adult rat spinal cord. Regen Med. 2007, 2: 929-945. 10.2217/174607188.8.131.529.View ArticleGoogle Scholar
- Li J, Sun CR, Zhang H, Tsang KS, Li JH, Zhang SD, An YH: Induction of functional recovery by co-transplantation of neural stem cells and Schwann cells in a rat spinal cord contusion injury model. Biomed Environ Sci. 2007, 20: 242-249.Google Scholar
- Ormerod BK, Palmer TD, Caldwell MA: Neurodegeneration and cell replacement. Philos Trans R Soc Lond B Biol Sci. 2008, 363: 153-170. 10.1098/rstb.2006.2018.View ArticleGoogle Scholar
- Rosser AE, Zietlow R, Dunnett SB: Stem cell transplantation for neurodegenerative diseases. Curr Opin Neurol. 2007, 20: 688-692.View ArticleGoogle Scholar
- Ronsyn MW, Berneman ZN, Van Tendeloo VF, Jorens PG, Ponsaerts P: Can cell therapy heal a spinal cord injury?. Spinal Cord. 2008, 46: 532-539. 10.1038/sc.2008.13.View ArticleGoogle Scholar
- Lu P, Jones LL, Tuszynski MH: BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury. Exp Neurol. 2005, 191: 344-360. 10.1016/j.expneurol.2004.09.018.View ArticleGoogle Scholar
- Bakshi A, Shimizu S, Keck CA, Cho S, LeBold DG, Morales D, Arenas E, Snyder EY, Watson DJ, McIntosh TK: Neural progenitor cells engineered to secrete GDNF show enhanced survival, neuronal differentiation and improve cognitive function following traumatic brain injury. Eur J Neurosci. 2006, 23: 2119-2134. 10.1111/j.1460-9568.2006.04743.x.View ArticleGoogle Scholar
- Kurozumi K, Nakamura K, Tamiya T, Kawano Y, Ishii K, Kobune M, Hirai S, Uchida H, Sasaki K, Ito Y, Kato K, Honmou O, Houkin K, Date I, Hamada H: Mesenchymal stem cells that produce neurotrophic factors reduce ischemic damage in the rat middle cerebral artery occlusion model. Mol Ther. 2005, 11: 96-104. 10.1016/j.ymthe.2004.09.020.View ArticleGoogle Scholar
- Ronsyn MW, Daans J, Spaepen G, Chatterjee S, Vermeulen K, D'Haese P, Van Tendeloo VF, Van Marck E, Ysebaert D, Berneman ZN, Jorens PG, Ponsaerts P: Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord. BMC Biotechnology. 2007, 7: 90-10.1186/1472-6750-7-90.View ArticleGoogle Scholar
- Keyaerts M, Verschueren J, Bos TJ, Tchouate-Gainkam LO, Peleman C, Breckpot K, Vanhove C, Caveliers V, Bossuyt A, Lahoutte T: Dynamic bioluminescence imaging for quantitative tumour burden assessment using IV or IP administration of D: -luciferin: effect on intensity, time kinetics and repeatability of photon emission. Eur J Nucl Med Mol Imaging. 2008, 35: 999-1007. 10.1007/s00259-007-0664-2.View ArticleGoogle Scholar
- Bradbury MS, Panagiotakos G, Chan BK, Tomishima M, Zanzonico P, Vider J, Ponomarev V, Studer L, Tabar V: Optical bioluminescence imaging of human ES cell progeny in the rodent CNS. J Neurochem. 2007, 102: 2029-2039. 10.1111/j.1471-4159.2007.04681.x.View ArticleGoogle Scholar
- Magnitsky S, Watson DJ, Walton RM, Pickup S, Bulte JW, Wolfe JH, Poptani H: In vivo and ex vivo MRI detection of localized and disseminated neural stem cell grafts in the mouse brain. Neuroimag. 2005, 26: 744-754. 10.1016/j.neuroimage.2005.02.029.View ArticleGoogle Scholar
- Arbab AS, Yocum GT, Wilson LB, Parwana A, Jordan EK, Kalish H, Frank JA: Comparison of transfection agents in forming complexes with ferumoxides, cell labelling efficiency, and cellular viability. Mol Imaging. 2004, 3: 24-32. 10.1162/153535004773861697.View ArticleGoogle Scholar
- Gambotto A, Dworacki G, Cicinnati V, Kenniston T, Steitz J, Tüting T, Robbins PD, DeLeo AB: Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope. Gene Ther. 2000, 7: 2036-2040. 10.1038/sj.gt.3301335.View ArticleGoogle Scholar
- Stripecke R, Carmen Villacres M, Skelton D, Satake N, Halene S, Kohn D: Immune response to green fluorescent protein: implications for gene therapy. Gene Ther. 1999, 6: 1305-1312. 10.1038/sj.gt.3300951.View ArticleGoogle Scholar
- Hakamata Y, Murakami T, Kobayashi E: Firefly rats as an organ/cellular source for long-term in vivo bioluminescent imaging. Transplantation. 2006, 81: 1179-1184. 10.1097/01.tp.0000203137.06587.4a.View ArticleGoogle Scholar
- Vandermeulen G, Staes E, Vanderhaeghen ML, Bureau MF, Scherman D, Préat V: Optimisation of intradermal DNA electrotransfer for immunisation. J Control Release. 2007, 124: 81-87. 10.1016/j.jconrel.2007.08.010.View ArticleGoogle Scholar
- Safran M, Kim WY, Kung AL, Horner JW, DePinho RA, Kaelin WG: Mouse reporter strain for noninvasive bioluminescent imaging of cells that have undergone Cre-mediated recombination. Mol Imaging. 2003, 2: 297-302. 10.1162/153535003322750637.View ArticleGoogle Scholar
- Peister A, Mellad JA, Larson BL, Hall BM, Gibson LF, Prockop DJ: Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood. 2004, 103: 1662-1668. 10.1182/blood-2003-09-3070.View ArticleGoogle Scholar
- Plas Van den D, Ponsaerts P, Van Tendeloo V, Van Bockstaele DR, Berneman ZN, Merregaert J: Efficient removal of LoxP-flanked genes by electroporation of Cre recombinase mRNA. Biochem Biophys Res Commun. 2003, 305: 10-15. 10.1016/S0006-291X(03)00669-7.View ArticleGoogle Scholar
- Ponsaerts P, Brown JP, Plas Van den D, Eeden Van den L, Van Bockstaele DR, Jorens PG, Van Tendeloo VF, Merregaert J, Singh PB, Berneman ZN: Messenger RNA electroporation is highly efficient in mouse embryonic stem cells: successful FLPe- and Cre-mediated recombination. Gene Ther. 2004, 11: 1606-1610. 10.1038/sj.gt.3302342.View ArticleGoogle Scholar
- Baekelandt V, Eggermont K, Michiels M, Nuttin B, Debyser Z: Optimized lentiviral vector production and purification procedure prevents immune response after transduction of mouse brain. Gene Ther. 2003, 10: 1933-1940. 10.1038/sj.gt.3302094.View ArticleGoogle Scholar
- Geraerts M, Michiels M, Baekelandt V, Debyser Z, Gijsbers R: Upscaling of lentiviral vector production by tangential flow filtration. J Gene Med. 2005, 7: 1299-1310. 10.1002/jgm.778.View ArticleGoogle Scholar
- Di Ianni M, Terenzi A, Perruccio K, Ciurnelli R, Lucheroni F, Benedetti R, Martelli MF, Tabilio A: 5-Azacytidine prevents transgene methylation in vivo. Gene Ther. 1999, 6: 703-707. 10.1038/sj.gt.3300848.View ArticleGoogle Scholar
- Van Tendeloo VF, Ponsaerts P, Lardon F, Nijs G, Lenjou M, Van Broeckhoven C, Van Bockstaele DR, Berneman ZN: Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood. 2001, 98: 49-56. 10.1182/blood.V98.1.49.View ArticleGoogle Scholar
- Ponsaerts P, Bosch Van den G, Cools N, Van Driessche A, Nijs G, Lenjou M, Lardon F, Van Broeckhoven C, Van Bockstaele DR, Berneman ZN, Van Tendeloo VF: Messenger RNA electroporation of human monocytes, followed by rapid in vitro differentiation, leads to highly stimulatory antigen-loaded mature dendritic cells. J Immunol. 2002, 169: 1669-1675.View ArticleGoogle Scholar
- Smits E, Ponsaerts P, Lenjou M, Nijs G, Van Bockstaele DR, Berneman ZN, Van Tendeloo VF: RNA-based gene transfer for adult stem cells and T cells. Leukemia. 2004, 18: 1898-1902. 10.1038/sj.leu.2403463.View ArticleGoogle Scholar
- Wiehe JM, Ponsaerts P, Rojewski MT, Homann JM, Greiner J, Kronawitter D, Schrezenmeier H, Hombach V, Wiesneth M, Zimmermann O, Torzewski J: mRNA-mediated gene delivery into human progenitor cells promotes highly efficient protein expression. J Cell Mol Med. 2007, 11: 521-530. 10.1111/j.1582-4934.2007.00038.x.View ArticleGoogle Scholar
- Ponsaerts P, Berneman ZN: Modulation of cellular behavior by exogenous messenger RNA. Leukemia. 2006, 20: 767-769. 10.1038/sj.leu.2404219.View ArticleGoogle Scholar
- Van Tendeloo VF, Ponsaerts P, Berneman ZN: mRNA-based gene transfer as a tool for gene and cell therapy. Curr Opin Mol Ther. 2007, 9: 423-431.Google Scholar
- Ryan JM, Barry FP, Murphy JM, Mahon BP: Mesenchymal stem cells avoid allogeneic rejection. J Inflamm (Lond). 2005, 26: 8-10.1186/1476-9255-2-8.View ArticleGoogle Scholar
- Carson MJ, Doose JM, Melchior B, Schmid CD, Ploix CC: CNS immune privilege: hiding in plain sight. Immunol Rev. 2006, 213: 48-65. 10.1111/j.1600-065X.2006.00441.x.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.