BACKGROUND
cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system .
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  <cit> .
therefore, it is clear that additional therapies, like cell transplantation, might be needed to further enhance restoration of brain function following primary  and secondary  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  <cit> .
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 , with the potential to enhance survival of existing neurons and endogenous neuroregeneration  <cit> .
this approach is currently well-described by several research groups including ours  <cit> .
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  and magnetic resonance imaging  have been proposed as suitable non-invasive methodologies for the follow-up of cell implants in the cns of rodents  <cit> .
while images created by mri have a high spatial resolution, cells need to be loaded with contrast agents, like super paramagnetic iron oxides , 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 , 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  <cit> .
methods
animals
homozygous rosa26-l-s-l-luciferase transgenic mice  were obtained via jackson laboratories  and further bred in the specific pathogen free animal facility of the university of antwerp  <cit> .
male offspring  were used for bone marrow-derived stromal cell  culture, cell implantation experiments and/or elispot analysis.
for all experiments, mice were kept in normal day-night cycle  with free access to food and water.
all experimental procedures were approved by the ethics committee for animal experiments of the university of antwerp .
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.
<cit> .
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  and the total cell population obtained was plated in a t <dig> culture flask  in  <dig> ml 'complete isolation medium' , consisting of rpmi- <dig> medium  supplemented with 8% horse serum , 8% fetal calf serum ,  <dig> u/ml penicillin ,  <dig> mg/ml streptomycin , and  <dig>  mg/ml amphotericin b .
following  <dig> hours of culture, non-adherent cells were removed and  <dig> ml fresh cim was added to the cultures.
for a period of two weeks, cim was replaced every  <dig> to  <dig> days.
next, cultured cells were harvested using trypsin-edta  treatment and replated in a new t <dig> culture flask in  <dig> ml cim.
stromal cell outgrowth in this culture was termed passage  <dig> and further expanded in 'complete expansion medium' , consisting of iscove modified dulbecco's medium  supplemented with 8% fcs, 8% hs,  <dig> u/ml penicillin,  <dig> mg/ml streptomycin and  <dig>  mg/ml amphotericin b. for routine cell culture, bmsc cultures were split 1: <dig> every  <dig> to  <dig> days.
in addition, clonal cultures of luciferase-expressing stromal cells were obtained by limiting dilution.
flow cytometry
immunophenotyping of bmsc cultures derived from rosa26-l-s-l-luciferase transgenic mice was performed using the following monoclonal antibodies: fluorescein-isothiocyanate -labelled anti-mouse cd <dig> , fitc-labelled anti-mouse cd <dig> , fitc-labelled anti-mouse cd <dig> , fitc-labelled anti-mouse sca- <dig> , fitc-labelled anti-mouse mhc-i , phycoerythrin -labeled anti-mouse cd <dig> , and pe-labelled anti-mouse mhc-ii .
immunostaining for a2b <dig> was performed using an unconjugated mouse-anti-mouse a2b <dig> monoclonal antibody  followed by staining with pe-labelled rat-anti-mouse secondary antibody .
before staining, harvested cells were washed twice with pbs supplemented with 1% fcs  and resuspended in pbs* at a concentration of  <dig> ×  <dig> cells/ml.
for antibody staining,  <dig> μg of antibody was added to  <dig> μl of cell suspension for  <dig> min at 4°c.
following incubation, cells were washed once with pbs*, resuspended in  <dig> ml pbs*, and analysed using an epics xl-mcl analytical flow cytometer .
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  to the cell suspension immediately before flow cytometric analysis.
at least  <dig>  cells per sample were analysed per sample and flow cytometry data were analysed using flowjo software.
messenger rna electroporation
messenger rna encoding the enhanced green fluorescent protein  and the cre recombinase protein was prepared as described previously  <cit> .
prior to electroporation of bmsc populations, cells were washed twice with serum-free optimem medium  and resuspended at a final concentration of 5– <dig> ×  <dig> cells/ml in serum-free optimem medium.
subsequently,  <dig> μl of the cell suspension was mixed with  <dig> μg of mrna and electroporated in a  <dig> mm electroporation cuvette at 300v and  <dig> μf using a gene pulser xcell electroporation device .
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  was measured using the commercial bright-glo luciferase assay system , according to the manufacturer's instructions.
lentiviral construction
construction of the pchmws-egfp-ires-pac vector was performed in two consecutive steps using standard cloning techniques.
first, the puromycin resistance gene  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  <cit> .
lentiviral transduction
lentiviral vector production was performed as described earlier by geraerts et al., with minor modifications  <cit> .
filtered vector particles were concentrated using vivaspin  <dig> columns , aliquoted and stored at -80°c.
for transduction experiments, cells were seeded in a 24-well plate at  <dig>  cells per well.
the next day, cells were transduced with vector expressing the egfp-ires-pac cassette  in cem medium.
after  <dig> hrs of incubation, the vectors were washed from the cells and medium was replaced.
cells were subcultured at least  <dig> 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  were resuspended at a concentration of  <dig> ×  <dig> cells/ml in pbs for intracerebral cell implantation or at a concentration of  <dig> ×  <dig> 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  + xylazin  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  <dig> mm on the right side of the midline.
thereafter, an automatic micro-injector pump  with a  <dig> μl hamilton syringe was positioned above the exposed dura.
a 30-gauge needle , attached to the syringe, was stereotactically placed through the intact dura to a depth of  <dig> mm.
after  <dig> minutes of pressure equilibration,  <dig> ×  <dig> bmsc-luc or bmsc-luc/egfp/pac in  <dig> μl pbs were injected at  <dig>  μl/min.
the needle was retracted after another  <dig> minutes to allow pressure equilibration and to prevent backflow of the injected cell suspension.
next, the skin was sutured, a  <dig> % 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  + n <dig>  + o <dig>  gas mixture.
directly thereafter,  <dig> ×  <dig> bmsc-luc or bmsc-luc/egfp/pac in  <dig> μl pbs were injected in the right pelvic limb muscles.
in vivo bioluminescence imaging
at different time points between day  <dig> and week  <dig> after cell implantation, mice were analysed by real-time in vivo bioluminescence imaging  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  + xylazin  mixture, followed by an intraperitoneal  or intravenous  injection of d-luciferin .
immediately after luciferin administration, mice were imaged for  <dig> minutes using an in vivo real-time φ-imager system .
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  <dig> or week  <dig> post-implantation, mice were deeply anaesthetized in an induction chamber by inhalation of an isofluorane , oxygen  and nitrogen  mixture for  <dig> minutes, followed by cervical dislocation.
whole brains were surgically removed and fixed in 4% paraformaldehyde for  <dig> hours.
histological analysis
fixed brains were dehydrated in sucrose gradients , frozen in liquid nitrogen and stored at -80°c until further processing.
consecutive  <dig> μm-thick cryosections were cut using a microm hm <dig> cryostat and stained with haematoxylin-eosin  to locate the transplantation site.
further immunohistochemical analysis was performed using a biotin-labeled anti-mouse sca- <dig> antibody  for bmsc identification, and a biotin-labeled anti-mouse cd11b antibody  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  <dig> min incubation with methanol containing 1% hydrogen peroxide.
next, slides were washed with water and washing buffer, followed by incubation with normal rat serum  for  <dig> hour at room temperature.
subsequently, slides were incubated for  <dig> hours with the biotin-labeled primary antibody at room temperature.
following this, slides were rinsed with washing buffer, and incubated for  <dig> hour at room temperature with a streptavidin-horse-radish-peroxidase complex .
visualization for all slides was carried out after staining with diaminobenzidine , according to manufacturer's instructions, and nuclei were counterstained with carazzi's haematoxylin.
bright-field immunohistochemical analysis was done using an olympus bx <dig> microscope equipped with an olympus dp <dig> camera.
olympus dp software was used for image collection.
elispot analysis
a murine ifn-γ elispot assay  was performed according to manufacturer's instructions.
in brief, spleens were dissected from cell-transplanted rosa26-l-s-l-luciferase transgenic mice  at  <dig> weeks post-injection.
next, after dissociation of the spleens over a  <dig> μm nylon filter, mononuclear cells were enriched following a density-based centrifugation step .
magnetic isolation of cd8+ t-cells was done using anti-cd <dig> macs microbeads , according to the manufacturer's instructions.
isolated cd8+ t-cells  were plated on elispot plates at  <dig> ×  <dig> cells/well in imdm supplemented with 10% fbs, penicillin/streptomycin and amphotericin b. cells were then cultured for  <dig> hours either:  un-stimulated,  stimulated with  <dig> ×  <dig> parental bmsc , or  stimulated with  <dig> ×  <dig> bmsc-luc or bmsc-luc/egfp/pac .
all experiments were performed in quadruplicate per mouse.
the elispot plates were analysed using an aid elispot reader .
data are presented as ifn-γ spot-forming cells  per  <dig> ×  <dig> cd8+ responder t-cells.
statistical analysis
results are expressed as mean ± standard deviation.
comparisons were validated using student's t-test.
a p-value <  <dig>  was considered to be statistically significant.
RESULTS
culture and characterisation of a clonal luciferase-expressing bone marrow-derived stromal cell line from rosa26-l-s-l-luciferase transgenic mice
rosa26-l-s-l-luciferase transgenic mice were originally described as a mouse reporter strain for non-invasive monitoring of in vivo cre-recombination by bioluminescence imaging <cit> .
in these mice, a luciferase reporter gene, which is preceded by a loxp-flanked neomycin resistance gene, was integrated into the rosa <dig> genomic locus by homologous recombination .
in this study, we first attempted to culture bone marrow-derived stromal cells  starting from bone marrow of 3-week old rosa26-l-s-l-luciferase transgenic mice, as described in detail in the materials and methods section.
at passage  <dig>  when cultures became homogenous for cells with bmsc morphology , cell cultures  were analysed by flow cytometry for the presence and absence of typical membrane proteins characteristic for defining murine bmsc  <cit> .
flow cytometric analysis indicated that the cultured cell populations displayed uniform expression of sca- <dig>  v-cam, and mhc-i, without detectable expression of haematopoietic , endothelial  and neural  membrane proteins .
one parental bmsc line was then chosen ad random and used for further experiments described in this manuscript.
in order to allow expression of the luciferase protein in bmsc derived from rosa26-l-s-l-luciferase transgenic mice, a "floxed" neomycin resistance cassette needs to be excised by the cre recombinase protein.
we previously described a methodology for efficient cre-mediated excision of target sequences following electroporation of cells with messenger rna encoding cre recombinase  <cit> .
following this strategy, we first evaluated whether murine bmsc were susceptible for mrna-based gene transfer.
flow cytometric analysis of bmsc electroporated with mrna encoding the enhanced green fluorescent protein  indicated efficient transgene expression in up to 80% of cells at  <dig> hours post-electroporation .
next, parental bmsc were electroporated with mrna encoding the cre recombinase protein in order to activate luciferase expression.
a polyclonal luciferase-expressing bmsc line was obtained, which displayed stable expression of luciferase protein for at least  <dig> passages, as demonstrated by standard in vitro luminescence assays .
next, multiple clonal luciferase-expressing bmsc lines were obtained by limiting dilution and screened for high luciferase activity by standard in vitro luminescence assay.
one clonal line was chosen and used for further characterisation and transplantation experiments described below.
this clonal luciferase-expressing bmsc line  was further characterised in vitro based on  bmsc morphology  and immune phenotype , for which no difference was observed with parental bmsc derived from rosa26-l-s-l-luciferase transgenic mice, and based on  luciferase activity, which was increased as compared to the described polyclonal luciferase-expressing bmsc cells and remained stable over at least  <dig> passages as demonstrated by standard in vitro luminescence assays .
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
in order to investigate whether our cultured clonal bmsc-luc derived from rosa26-l-s-l-luciferase transgenic mice can survive intrinsically and/or immunologically upon implantation into the central nervous system  of immune competent mice,  <dig> ×  <dig> cells were grafted in the cns of syngeneic rosa26-l-s-l-luciferase mice , following procedures described in detail in the materials and methods section.
in this experimental context, only the luciferase protein produced by the implanted bmsc-luc can be seen as a potential immunogenic antigen in the cns.
next, survival of bmsc-luc implants was monitored by real-time in vivo bioluminescence imaging  until day  <dig>  week  <dig>  week  <dig>  week  <dig> or week  <dig> post-implantation.
only mice showing a clear bli signal on day  <dig> post-implantation , indicating successful cell implantation in the cns for bli imaging, were included for long-term follow-up by bli.
for these mice, a clear bli signal was detected in 17/ <dig> mice analysed at week  <dig> post-implantation, in 17/ <dig> mice analysed at week  <dig> post-implantation, in 6/ <dig> mice analysed at week  <dig> post-implantation, and in 5/ <dig> mice analysed at week  <dig> post-implantation.
these results suggest both intrinsic and immunological survival of bmsc-luc cell implants in the cns of syngeneic immunocompetent mice , despite potential immunogenicity of the luciferase transgene.
in addition, at different time-points post-implantation, randomly chosen animals  were sacrificed and dissected brains were analysed for cell graft survival and inflammatory responses.
as shown in figure 2b, cell implants are clearly visible at week  <dig> and week  <dig> post-implantation following haematoxylin-eosin  staining of brain slices.
these results also validate the observed in vivo bli results described above.
moreover, immunohistochemical staining for sca- <dig> clearly identifies the bmsc origin of the cell implants observed .
in addition, we investigated whether the implantation of autologous  bmsc-luc in the cns of mice triggers inflammatory responses.
for this, brain sections were stained for the presence of cd11b+ activated microglia in the surroundings of grafted bmsc-luc .
results indicate that at an early time point post-implantation  microglial activation does occur, however, this inflammatory response is only temporary as the presence of these cd11b+ activated microglial cells is highly diminished at a later time point post-implantation .
in contrast to the above-described results, intramuscular bmsc-luc cell implants did not survive beyond week  <dig> post-implantation as demonstrated by in vivo bli .
for the latter, a clear bli signal was detected in 8/ <dig> mice analysed at day  <dig> post-implantation, in 4/ <dig> mice analysed at week  <dig> post-implantation, in 0/ <dig> mice analysed at week  <dig> post-implantation and in 0/ <dig> mice analysed at week  <dig> post-implantation.
survival of bmsc genetically modified with multiple reporter genes following implantation in the central nervous system of syngeneic immunocompetent mice
in order to investigate whether the immunological survival of bmsc-luc in the cns of syngeneic immunocompetent mice was due to low immunogenicity of the luciferase protein in the cns, we introduced additional xenogeneic reporter genes into our cultured bmsc-luc.
for this, bmsc-luc were transduced with a lentivirus encoding the enhanced green fluorescence protein  and the puromycin resistance gene .
following puromycin selection and single clone selection, a clonal luciferase-, egfp- and pac-expressing bmsc line was obtained .
flowcytometric analysis of bmsc-luc/egfp/pac demonstated egfp transgene expression in > 95% of the cells , which remained stable in culture for at least  <dig> passages.
in addition, phenotypical properties  and luciferase activity  were not influenced following lentiviral-transduction of bmsc-luc.
next, in order to investigate the in vivo immunological survival of bmsc genetically-modified with multiple reporter genes in immunocompetent mice,  <dig> ×  <dig> bmsc-luc/egfp/pac were implanted in the cns of syngeneic rosa26-l-s-l-luciferase transgenic mice .
survival of grafted bmsc-luc/egfp/pac was then monitored by in vivo bli on day  <dig>  week  <dig> and week  <dig> post-implantation .
a clear bli signal was observed in 8/ <dig> mice analysed at day  <dig> post-implantation, in 6/ <dig> mice analysed at week  <dig> post-implantation and in 6/ <dig> mice analysed at week  <dig> post-implantation.
in addition, histological analysis  at week  <dig> post-implantation confirmed:  the presence of egfp-expressing bmsc-luc/egfp/pac implants, and  a limited number of activated cd11b+ microglia surrounding bmsc-luc/egfp/pac implants, indicating survival of bmsc-luc/egfp/pac cell implants in the cns of syngeneic immunocompetent mice.
in contrast, intramuscular bmsc-luc/egfp/pac implants did not survive beyond week  <dig> post-implantation  as demonstrated by in vivo bli.
in these experiments, a clear bli signal was detected in 4/ <dig> mice at day  <dig> post-implantation, in 3/ <dig> mice at week  <dig> post-implantation and in 0/ <dig> mice at week  <dig> post-implantation.
induction of bmsc-specific cd8+ t-cell responses following intramuscular, but not intracerebral, cell implantation in syngeneic immunocompetent mice
in order 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, we evaluated the presence of reporter gene-specific interferon -γ-producing cd8+ t-cells by elispot analysis.
for this, spleen cd8+ t-cells were isolated from control mice and from bmsc-luc or bmsc-luc/egfp/pac cell implanted mice  at week  <dig> post-implantation.
then, isolated cd8+ t-cells were un-stimulated, re-stimulated with parental bmsc, or re-stimulated with bmsc-luc or bmsc-luc/egfp/pac in an ifn-γ elispot assay .
interestingly, no significant number of ifn-γ-producing cd8+ t-cells directed against bmsc-luc  versus intracerebral bmsc-luc graft  – p =  <dig> ) or bmsc-luc/egfp/pac  versus intracerebral bmsc-luc/egfp/pac graft  – p =  <dig> ) cell implants was detected upon intracerebral cell implantation.
however, a large number of reactive ifn-γ-producing cd8+ t-cells directed against bmsc-luc  – p <  <dig> ) or bmsc-luc/egfp/pac  – p <  <dig> ) was observed upon intramuscular cell implantation.
these results demonstrate that bmsc-luc and bmsc-luc/egfp/pac specific ifn-γ-producing t-cells are efficiently induced upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating potential immunogenicity of our reporter gene-modified bmsc for the peripheral immune system, but not for the cns immune system.
in an attempt to demonstrate that the observed ifn-γ-producing t-cells were specific for luciferase, egfp or pac, isolated cd8+ t-cells were also re-stimulated with parental bmsc.
surprisingly, cd8+ t-cells isolated from intramuscular bmsc-luc or bmsc-luc/egfp/pac cell implanted mice were equally reactive against parental non-modified bmsc   the latter indicates that the non-survival of intramuscular bmsc-luc or bmsc-luc/egfp/pac cell implants – in our experimental model – is not mediated by reporter gene-specific t-cells, although it is unclear at the moment which antigens were responsible for initiating t-cell activation against our cultured bmsc.
discussion
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 , 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  <cit> .
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  <cit> .
second, cre recombination in cells derived from rosa26-l-s-l-luciferase transgenic mice allows removal of a floxed neomycin resistance gene , 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  <cit> .
immunophenotypic analysis  clearly demonstrated the uniform expression of mesenchymal markers  without detectable expression of endothelial , haematopoietic  or neural  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  <cit> .
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  reporter gene .
the latter is of importance when transient protein expression is desired and introduction of dna sequences  should be avoided  <cit> .
next, our cultured bmsc populations were electroporated with mrna encoding the cre recombinase protein, following previously described procedures  <cit> .
although luciferase expression was induced , the culture of a clonal luciferase-expressing bmsc was necessary in order to obtain a pure population expressing high levels of the luciferase protein .
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   <cit> .
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  <dig> ×  <dig> cells in order to obtain a clear signal for in vivo bioluminescence imaging .
further experiments revealed a minimum of  <dig> ×  <dig> cells to be required for obtaining a minimum signal above background .
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- <dig> weeks by real-time bli , while intramuscular bmsc-luc implants did not survive during the same follow-up period .
also, when the same bmsc population was implanted in the cns of immunocompetent allogeneic c57/bl <dig> mice  or when c57/bl <dig> bmsc were implanted in the cns of immunocompetent rosa26-l-s-l-luciferase transgenic mice , 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- <dig> weeks, we did not observe a significant increase of in vivo bioluminescence signal over time.
the latter, although further investigation will be needed , might become a tool to exclude tumour formation following cell implantation  <cit> .
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 .
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 .
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 , which are from an immunological point of view a foreign antigens, are tolerated in the cns.
several explanations can be hypothesised for this:  some cell populations, among them bmsc, have been ascribed immune modulatory properties  <cit> , or  immune surveillance mechanisms in the cns are not properly activated  <cit> , 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  <dig> post-implantation, the presence of these cns immune cells was highly diminished by week  <dig> post-implantation, indicating immunological acceptance of autologous bmsc-luc  or bmsc-luc/egfp/pac .
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– <dig> post-implantation .
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 , 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:  due to the use of fetal calf serum and horse serum for in vitro bmsc expansion, xenogeneic serum components  might have induced cellular immunogenicity, or  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.
CONCLUSIONS
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.
authors' contributions
ib carried out cell culture, flow cytometry, cell transplantations, bioluminescence imaging, histological analysis, elispot analysis, data collection, data interpretation and drafted the manuscript.
ndv carried out cell culture, cell transplantations and bioluminescence imaging, bt carried out histological analysis, elispot analysis, and assisted with data interpretation, jv assisted with bioluminescence imaging, kr assisted with cell culture, jd carried out mrna preparation, cell transfections and assisted with cell culture and flow cytometry.
ai carried out lentiviral transduction experiments.
vfivt acquired funding.
sc acquired funding and assisted in evaluation of histological analysis.
hg acquired funding.
pgj acquired funding and assisted in study design.
vb provided expertise and support for lentiviral transduction experiments.
dy provided support for animal experiments.
evm provided support for histological analysis.
zb acquired funding and assisted in study design and data interpretation.
avdl acquired funding and assisted in study design and data interpretation.
pp acquired funding, carried out study design, data collection, data interpretation and drafted the manuscript.
all authors have read and approved the manuscript.
supplementary material
additional file 1
in vitro bioluminescence of cultured luciferase-expressing bone marrow-derived stromal cells using the biospacein vivo bioluminescence camera.
additional data showing detection sensitivity of in vitro bioluminescence by luciferase-expressing bone marrow-derived stromal cells using the biospace in vivo bioluminescence camera.
click here for file
additional file 2
histological analysis of luciferase-expressing rosa26-l-s-l-luc bone marrow-derived stromal cells following allogeneic transplantation in c57bl/ <dig> mice.
additional data showing immunological rejection of luciferase-expressing rosa26-l-s-l-luc bone marrow-derived stromal cells following allogeneic transplantation in c57bl/ <dig> mice.
click here for file
acknowledgements
we acknowledge helpful assistance from august van laer  with animal handling and surgical procedures, and from frank rylant  with histological analysis.
this work was supported by research grants  <dig> .03n  and g. <dig>   of the fund for scientific research-flanders , by research grants bof-kp  <dig> , bof-kp  <dig> , bof-noi  <dig> , id-bof  <dig>  from the antwerp university, by research grant brainstim of the flemish institute for science and technology , in part by a methusalem research grant from the flemish government , in part by the ec fp6-project dimi  and the ec fp6-project emil , and by the fund for cell therapy from the antwerp university hospital.
peter ponsaerts is a post-doctoral fellow of the fwo-vlaanderen.

 cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system. 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. therefore, it is clear that additional therapies, like cell transplantation, might be needed to further enhance restoration of brain function following primary and secondary 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. 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, with the potential to enhance survival of existing neurons and endogenous neuroregeneration. this approach is currently well-described by several research groups including ours. 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 and magnetic resonance imaging have been proposed as suitable non-invasive methodologies for the follow-up of cell implants in the cns of rodents. while images created by mri have a high spatial resolution, cells need to be loaded with contrast agents, like super paramagnetic iron oxides, 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, 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. methods animals homozygous rosa26-l-s-l-luciferase transgenic mice were obtained via jackson laboratories and further bred in the specific pathogen free animal facility of the university of antwerp. male offspring were used for bone marrow-derived stromal cell culture, cell implantation experiments and/or elispot analysis. for all experiments, mice were kept in normal day-night cycle with free access to food and water. all experimental procedures were approved by the ethics committee for animal experiments of the university of antwerp. 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 and the total cell population obtained was plated in a t culture flask in ml'complete isolation medium', consisting of rpmi- medium supplemented with 8% horse serum, 8% fetal calf serum, u/ml penicillin, mg/ml streptomycin, and mg/ml amphotericin b. following hours of culture, non-adherent cells were removed and ml fresh cim was added to the cultures. for a period of two weeks, cim was replaced every to days. next, cultured cells were harvested using trypsin-edta treatment and replated in a new t culture flask in ml cim. stromal cell outgrowth in this culture was termed passage and further expanded in'complete expansion medium', consisting of iscove modified dulbecco's medium supplemented with 8% fcs, 8% hs, u/ml penicillin, mg/ml streptomycin and mg/ml amphotericin b. for routine cell culture, bmsc cultures were split 1: every to days. in addition, clonal cultures of luciferase-expressing stromal cells were obtained by limiting dilution. flow cytometry immunophenotyping of bmsc cultures derived from rosa26-l-s-l-luciferase transgenic mice was performed using the following monoclonal antibodies: fluorescein-isothiocyanate -labelled anti-mouse cd, fitc-labelled anti-mouse cd, fitc-labelled anti-mouse cd, fitc-labelled anti-mouse sca-, fitc-labelled anti-mouse mhc-i, phycoerythrin -labeled anti-mouse cd, and pe-labelled anti-mouse mhc-ii. immunostaining for a2b was performed using an unconjugated mouse-anti-mouse a2b monoclonal antibody followed by staining with pe-labelled rat-anti-mouse secondary antibody. before staining, harvested cells were washed twice with pbs supplemented with 1% fcs and resuspended in pbs* at a concentration of × cells/ml. for antibody staining, μg of antibody was added to μl of cell suspension for min at 4°c. following incubation, cells were washed once with pbs*, resuspended in ml pbs*, and analysed using an epics xl-mcl analytical flow cytometer. 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 to the cell suspension immediately before flow cytometric analysis. at least cells per sample were analysed per sample and flow cytometry data were analysed using flowjo software. messenger rna electroporation messenger rna encoding the enhanced green fluorescent protein and the cre recombinase protein was prepared as described previously. prior to electroporation of bmsc populations, cells were washed twice with serum-free optimem medium and resuspended at a final concentration of 5– × cells/ml in serum-free optimem medium. subsequently, μl of the cell suspension was mixed with μg of mrna and electroporated in a mm electroporation cuvette at 300v and μf using a gene pulser xcell electroporation device. 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 was measured using the commercial bright-glo luciferase assay system, according to the manufacturer's instructions. lentiviral construction construction of the pchmws-egfp-ires-pac vector was performed in two consecutive steps using standard cloning techniques. first, the puromycin resistance gene 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 transduction lentiviral vector production was performed as described earlier by geraerts et al., with minor modifications. filtered vector particles were concentrated using vivaspin columns, aliquoted and stored at -80°c. for transduction experiments, cells were seeded in a 24-well plate at cells per well. the next day, cells were transduced with vector expressing the egfp-ires-pac cassette in cem medium. after hrs of incubation, the vectors were washed from the cells and medium was replaced. cells were subcultured at least 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 were resuspended at a concentration of × cells/ml in pbs for intracerebral cell implantation or at a concentration of × 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 + xylazin 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 mm on the right side of the midline. thereafter, an automatic micro-injector pump with a μl hamilton syringe was positioned above the exposed dura. a 30-gauge needle, attached to the syringe, was stereotactically placed through the intact dura to a depth of mm. after minutes of pressure equilibration, × bmsc-luc or bmsc-luc/egfp/pac in μl pbs were injected at μl/min. the needle was retracted after another minutes to allow pressure equilibration and to prevent backflow of the injected cell suspension. next, the skin was sutured, a % 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 + n + o gas mixture. directly thereafter, × bmsc-luc or bmsc-luc/egfp/pac in μl pbs were injected in the right pelvic limb muscles. in vivo bioluminescence imaging at different time points between day and week after cell implantation, mice were analysed by real-time in vivo bioluminescence imaging 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 + xylazin mixture, followed by an intraperitoneal or intravenous injection of d-luciferin. immediately after luciferin administration, mice were imaged for minutes using an in vivo real-time φ-imager system. 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 or week post-implantation, mice were deeply anaesthetized in an induction chamber by inhalation of an isofluorane, oxygen and nitrogen mixture for minutes, followed by cervical dislocation. whole brains were surgically removed and fixed in 4% paraformaldehyde for hours. histological analysis fixed brains were dehydrated in sucrose gradients, frozen in liquid nitrogen and stored at -80°c until further processing. consecutive μm-thick cryosections were cut using a microm hm cryostat and stained with haematoxylin-eosin to locate the transplantation site. further immunohistochemical analysis was performed using a biotin-labeled anti-mouse sca- antibody for bmsc identification, and a biotin-labeled anti-mouse cd11b antibody 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 min incubation with methanol containing 1% hydrogen peroxide. next, slides were washed with water and washing buffer, followed by incubation with normal rat serum for hour at room temperature. subsequently, slides were incubated for hours with the biotin-labeled primary antibody at room temperature. following this, slides were rinsed with washing buffer, and incubated for hour at room temperature with a streptavidin-horse-radish-peroxidase complex. visualization for all slides was carried out after staining with diaminobenzidine, according to manufacturer's instructions, and nuclei were counterstained with carazzi's haematoxylin. bright-field immunohistochemical analysis was done using an olympus bx microscope equipped with an olympus dp camera. olympus dp software was used for image collection. elispot analysis a murine ifn-γ elispot assay was performed according to manufacturer's instructions. in brief, spleens were dissected from cell-transplanted rosa26-l-s-l-luciferase transgenic mice at weeks post-injection. next, after dissociation of the spleens over a μm nylon filter, mononuclear cells were enriched following a density-based centrifugation step. magnetic isolation of cd8+ t-cells was done using anti-cd macs microbeads, according to the manufacturer's instructions. isolated cd8+ t-cells were plated on elispot plates at × cells/well in imdm supplemented with 10% fbs, penicillin/streptomycin and amphotericin b. cells were then cultured for hours either: un-stimulated, stimulated with × parental bmsc, or stimulated with × bmsc-luc or bmsc-luc/egfp/pac. all experiments were performed in quadruplicate per mouse. the elispot plates were analysed using an aid elispot reader. data are presented as ifn-γ spot-forming cells per × cd8+ responder t-cells. statistical analysis results are expressed as mean ± standard deviation. comparisons were validated using student's t-test. a p-value < 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 rosa26-l-s-l-luciferase transgenic mice were originally described as a mouse reporter strain for non-invasive monitoring of in vivo cre-recombination by bioluminescence imaging. in these mice, a luciferase reporter gene, which is preceded by a loxp-flanked neomycin resistance gene, was integrated into the rosa genomic locus by homologous recombination. in this study, we first attempted to culture bone marrow-derived stromal cells starting from bone marrow of 3-week old rosa26-l-s-l-luciferase transgenic mice, as described in detail in the materials and methods section. at passage when cultures became homogenous for cells with bmsc morphology, cell cultures were analysed by flow cytometry for the presence and absence of typical membrane proteins characteristic for defining murine bmsc. flow cytometric analysis indicated that the cultured cell populations displayed uniform expression of sca- v-cam, and mhc-i, without detectable expression of haematopoietic, endothelial and neural membrane proteins. one parental bmsc line was then chosen ad random and used for further experiments described in this manuscript. in order to allow expression of the luciferase protein in bmsc derived from rosa26-l-s-l-luciferase transgenic mice, a "floxed" neomycin resistance cassette needs to be excised by the cre recombinase protein. we previously described a methodology for efficient cre-mediated excision of target sequences following electroporation of cells with messenger rna encoding cre recombinase. following this strategy, we first evaluated whether murine bmsc were susceptible for mrna-based gene transfer. flow cytometric analysis of bmsc electroporated with mrna encoding the enhanced green fluorescent protein indicated efficient transgene expression in up to 80% of cells at hours post-electroporation. next, parental bmsc were electroporated with mrna encoding the cre recombinase protein in order to activate luciferase expression. a polyclonal luciferase-expressing bmsc line was obtained, which displayed stable expression of luciferase protein for at least passages, as demonstrated by standard in vitro luminescence assays. next, multiple clonal luciferase-expressing bmsc lines were obtained by limiting dilution and screened for high luciferase activity by standard in vitro luminescence assay. one clonal line was chosen and used for further characterisation and transplantation experiments described below. this clonal luciferase-expressing bmsc line was further characterised in vitro based on bmsc morphology and immune phenotype, for which no difference was observed with parental bmsc derived from rosa26-l-s-l-luciferase transgenic mice, and based on luciferase activity, which was increased as compared to the described polyclonal luciferase-expressing bmsc cells and remained stable over at least passages as demonstrated by standard in vitro luminescence assays. 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 in order to investigate whether our cultured clonal bmsc-luc derived from rosa26-l-s-l-luciferase transgenic mice can survive intrinsically and/or immunologically upon implantation into the central nervous system of immune competent mice, × cells were grafted in the cns of syngeneic rosa26-l-s-l-luciferase mice, following procedures described in detail in the materials and methods section. in this experimental context, only the luciferase protein produced by the implanted bmsc-luc can be seen as a potential immunogenic antigen in the cns. next, survival of bmsc-luc implants was monitored by real-time in vivo bioluminescence imaging until day week week week or week post-implantation. only mice showing a clear bli signal on day post-implantation, indicating successful cell implantation in the cns for bli imaging, were included for long-term follow-up by bli. for these mice, a clear bli signal was detected in 17/ mice analysed at week post-implantation, in 17/ mice analysed at week post-implantation, in 6/ mice analysed at week post-implantation, and in 5/ mice analysed at week post-implantation. these results suggest both intrinsic and immunological survival of bmsc-luc cell implants in the cns of syngeneic immunocompetent mice, despite potential immunogenicity of the luciferase transgene. in addition, at different time-points post-implantation, randomly chosen animals were sacrificed and dissected brains were analysed for cell graft survival and inflammatory responses. as shown in figure 2b, cell implants are clearly visible at week and week post-implantation following haematoxylin-eosin staining of brain slices. these results also validate the observed in vivo bli results described above. moreover, immunohistochemical staining for sca- clearly identifies the bmsc origin of the cell implants observed. in addition, we investigated whether the implantation of autologous bmsc-luc in the cns of mice triggers inflammatory responses. for this, brain sections were stained for the presence of cd11b+ activated microglia in the surroundings of grafted bmsc-luc. results indicate that at an early time point post-implantation microglial activation does occur, however, this inflammatory response is only temporary as the presence of these cd11b+ activated microglial cells is highly diminished at a later time point post-implantation. in contrast to the above-described results, intramuscular bmsc-luc cell implants did not survive beyond week post-implantation as demonstrated by in vivo bli. for the latter, a clear bli signal was detected in 8/ mice analysed at day post-implantation, in 4/ mice analysed at week post-implantation, in 0/ mice analysed at week post-implantation and in 0/ mice analysed at week post-implantation. survival of bmsc genetically modified with multiple reporter genes following implantation in the central nervous system of syngeneic immunocompetent mice in order to investigate whether the immunological survival of bmsc-luc in the cns of syngeneic immunocompetent mice was due to low immunogenicity of the luciferase protein in the cns, we introduced additional xenogeneic reporter genes into our cultured bmsc-luc. for this, bmsc-luc were transduced with a lentivirus encoding the enhanced green fluorescence protein and the puromycin resistance gene. following puromycin selection and single clone selection, a clonal luciferase-, egfp- and pac-expressing bmsc line was obtained. flowcytometric analysis of bmsc-luc/egfp/pac demonstated egfp transgene expression in > 95% of the cells, which remained stable in culture for at least passages. in addition, phenotypical properties and luciferase activity were not influenced following lentiviral-transduction of bmsc-luc. next, in order to investigate the in vivo immunological survival of bmsc genetically-modified with multiple reporter genes in immunocompetent mice, × bmsc-luc/egfp/pac were implanted in the cns of syngeneic rosa26-l-s-l-luciferase transgenic mice. survival of grafted bmsc-luc/egfp/pac was then monitored by in vivo bli on day week and week post-implantation. a clear bli signal was observed in 8/ mice analysed at day post-implantation, in 6/ mice analysed at week post-implantation and in 6/ mice analysed at week post-implantation. in addition, histological analysis at week post-implantation confirmed: the presence of egfp-expressing bmsc-luc/egfp/pac implants, and a limited number of activated cd11b+ microglia surrounding bmsc-luc/egfp/pac implants, indicating survival of bmsc-luc/egfp/pac cell implants in the cns of syngeneic immunocompetent mice. in contrast, intramuscular bmsc-luc/egfp/pac implants did not survive beyond week post-implantation as demonstrated by in vivo bli. in these experiments, a clear bli signal was detected in 4/ mice at day post-implantation, in 3/ mice at week post-implantation and in 0/ mice at week post-implantation. induction of bmsc-specific cd8+ t-cell responses following intramuscular, but not intracerebral, cell implantation in syngeneic immunocompetent mice in order 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, we evaluated the presence of reporter gene-specific interferon -γ-producing cd8+ t-cells by elispot analysis. for this, spleen cd8+ t-cells were isolated from control mice and from bmsc-luc or bmsc-luc/egfp/pac cell implanted mice at week post-implantation. then, isolated cd8+ t-cells were un-stimulated, re-stimulated with parental bmsc, or re-stimulated with bmsc-luc or bmsc-luc/egfp/pac in an ifn-γ elispot assay. interestingly, no significant number of ifn-γ-producing cd8+ t-cells directed against bmsc-luc versus intracerebral bmsc-luc graft – p = ) or bmsc-luc/egfp/pac versus intracerebral bmsc-luc/egfp/pac graft – p = ) cell implants was detected upon intracerebral cell implantation. however, a large number of reactive ifn-γ-producing cd8+ t-cells directed against bmsc-luc – p < ) or bmsc-luc/egfp/pac – p < ) was observed upon intramuscular cell implantation. these results demonstrate that bmsc-luc and bmsc-luc/egfp/pac specific ifn-γ-producing t-cells are efficiently induced upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating potential immunogenicity of our reporter gene-modified bmsc for the peripheral immune system, but not for the cns immune system. in an attempt to demonstrate that the observed ifn-γ-producing t-cells were specific for luciferase, egfp or pac, isolated cd8+ t-cells were also re-stimulated with parental bmsc. surprisingly, cd8+ t-cells isolated from intramuscular bmsc-luc or bmsc-luc/egfp/pac cell implanted mice were equally reactive against parental non-modified bmsc the latter indicates that the non-survival of intramuscular bmsc-luc or bmsc-luc/egfp/pac cell implants – in our experimental model – is not mediated by reporter gene-specific t-cells, although it is unclear at the moment which antigens were responsible for initiating t-cell activation against our cultured bmsc. discussion 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, 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. 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. second, cre recombination in cells derived from rosa26-l-s-l-luciferase transgenic mice allows removal of a floxed neomycin resistance gene, 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 clearly demonstrated the uniform expression of mesenchymal markers without detectable expression of endothelial, haematopoietic or neural 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. 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 reporter gene. the latter is of importance when transient protein expression is desired and introduction of dna sequences should be avoided. next, our cultured bmsc populations were electroporated with mrna encoding the cre recombinase protein, following previously described procedures. although luciferase expression was induced, the culture of a clonal luciferase-expressing bmsc was necessary in order to obtain a pure population expressing high levels of the luciferase protein. 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. 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 × cells in order to obtain a clear signal for in vivo bioluminescence imaging. further experiments revealed a minimum of × cells to be required for obtaining a minimum signal above background. 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- weeks by real-time bli, while intramuscular bmsc-luc implants did not survive during the same follow-up period. also, when the same bmsc population was implanted in the cns of immunocompetent allogeneic c57/bl mice or when c57/bl bmsc were implanted in the cns of immunocompetent rosa26-l-s-l-luciferase transgenic mice, 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- weeks, we did not observe a significant increase of in vivo bioluminescence signal over time. the latter, although further investigation will be needed, 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. 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. 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, which are from an immunological point of view a foreign antigens, are tolerated in the cns. several explanations can be hypothesised for this: some cell populations, among them bmsc, have been ascribed immune modulatory properties, or 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 post-implantation, the presence of these cns immune cells was highly diminished by week post-implantation, indicating immunological acceptance of autologous bmsc-luc or bmsc-luc/egfp/pac. 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– post-implantation. 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, 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: due to the use of fetal calf serum and horse serum for in vitro bmsc expansion, xenogeneic serum components might have induced cellular immunogenicity, or 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. authors' contributions ib carried out cell culture, flow cytometry, cell transplantations, bioluminescence imaging, histological analysis, elispot analysis, data collection, data interpretation and drafted the manuscript. ndv carried out cell culture, cell transplantations and bioluminescence imaging, bt carried out histological analysis, elispot analysis, and assisted with data interpretation, jv assisted with bioluminescence imaging, kr assisted with cell culture, jd carried out mrna preparation, cell transfections and assisted with cell culture and flow cytometry. ai carried out lentiviral transduction experiments. vfivt acquired funding. sc acquired funding and assisted in evaluation of histological analysis. hg acquired funding. pgj acquired funding and assisted in study design. vb provided expertise and support for lentiviral transduction experiments. dy provided support for animal experiments. evm provided support for histological analysis. zb acquired funding and assisted in study design and data interpretation. avdl acquired funding and assisted in study design and data interpretation. pp acquired funding, carried out study design, data collection, data interpretation and drafted the manuscript. all authors have read and approved the manuscript. supplementary material additional file 1 in vitro bioluminescence of cultured luciferase-expressing bone marrow-derived stromal cells using the biospacein vivo bioluminescence camera. additional data showing detection sensitivity of in vitro bioluminescence by luciferase-expressing bone marrow-derived stromal cells using the biospace in vivo bioluminescence camera. click here for file additional file 2 histological analysis of luciferase-expressing rosa26-l-s-l-luc bone marrow-derived stromal cells following allogeneic transplantation in c57bl/ mice. additional data showing immunological rejection of luciferase-expressing rosa26-l-s-l-luc bone marrow-derived stromal cells following allogeneic transplantation in c57bl/ mice. click here for file acknowledgements we acknowledge helpful assistance from august van laer with animal handling and surgical procedures, and from frank rylant with histological analysis. this work was supported by research grants.03n and g. of the fund for scientific research-flanders, by research grants bof-kp, bof-kp, bof-noi, id-bof from the antwerp university, by research grant brainstim of the flemish institute for science and technology, in part by a methusalem research grant from the flemish government, in part by the ec fp6-project dimi and the ec fp6-project emil, and by the fund for cell therapy from the antwerp university hospital. peter ponsaerts is a post-doctoral fellow of the fwo-vlaanderen.
cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system. 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. in this study, we performed cell implantation experiments in the cns of immunocompetent mice using autologous luciferase-expressing bone marrow-derived stromal cells cultured from rosa26-l-s-l-luciferase transgenic mice, and bmsc-luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein and the puromycin resistance gene. 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. 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 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. 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 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.
 cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system. 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 luciferase-expressing bone marrow-derived stromal cells cultured from rosa26-l-s-l-luciferase transgenic mice, and bmsc-luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein and the puromycin resistance gene. 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 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. 