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Adult stem cells have therapeutic potential because of their intrinsic capacity for self-renewal, especially for bone regeneration. The present study demonstrates the utility of ex vivo modified mesenchymal stem cells (MSC) to enhance bone density in an immunocompetent mouse model of osteopenia. MSC were transduced ex vivo with a recombinant adeno-associated virus 2 (rAAV) expressing BMP-2 under the transcriptional control of collagen type-1α promoter. To enrich bone homing in vivo, the cells were further modified to transiently express the mouse α-4 integrin. The modified MSC were systemically administered to ovariectomized, female C57BL/6 mice. Effects of the therapy were determined by dual energy x-ray absorptiometry, 3D micro-CT, histology, and immunohistochemistry for up to six months. Results indicated that mice transplanted with MSC expressing BMP-2 showed significant increase in bone mineral density and bone mineral content(p<0.001) with relatively better proliferative capabilities of bone marrow stromal cells and higher osteocompetent pool of cells compared to control animals. Micro-CT analysis of femora and other bone histomorphometric analyses indicated more trabecular bone following MSC-BMP-2 therapy. Results obtained by transplanting genetically modified MSC from GFP transgenic mouse suggested that production of BMP2 from transplanted MSC also influenced the mobilization of endogenous progenitors for new bone formation.
Advancement in cell and gene therapy approaches to provide long-term phenotypic correction may become a clinical possibility by genetic modifications of stem cells in vitro prior to transplantation.1–3 Adult mesenchymal stem cells (MSC) represent an ideal source for ex-vivo therapy for the treatment of genetic, age-related, and segmental bone defects to increase bone mass.4 Recent studies have demonstrated that pluripotent MSC can home to the bone marrow and participate in new bone formation, raising the possibility that MSC-based therapies may be developed for effective intervention of bone loss.5–7 One of the approaches to utilize MSC for osteoinductive therapy is genetic modification of the cells ex vivo to function as continuous source for providing progeny osteoblasts. Induction of endogenous cytokine, and growth factor production by exogenous stimuli from implanted MSC may further mobilize progenitor cells and improve tissue repair.8 MSC, cultured ex vivo with appropriate growth factors under controlled conditions can retain multi-lineage potential and can differentiate into adipocytes, osteocytes, chondrocytes or myocytes. MSC can be utilized to perform a variety of roles in tissue repair models, as tissue protective cells in brain injury such as acute cerebral infarction 9 and as drug delivery vehicle for tumor-stromal cells, targeting invasive and metastatic malignant tumor cells.10,11 MSC migration to the sites of injury and their differentiation to replace existing damaged tissues are not clearly defined processes and key effectors that control these restricted processes are being elucidated. Despite the potential of delivered MSC in vivo, site-specific differentiation and integration of the injected cells into existing tissues are still being optimized. Nonetheless, potential mobilization of progenitors from the bone marrow is associated with exogenous stimuli secreted by the MSC to stimulate endogenous progenitors.12
Recombinant viruses remain are efficient for gene transfer into many cell types including MSC. Among the vectors currently being tested, adeno-associated virus (AAV) is a non-pathogenic, replication defective vector capable of providing long-term gene expression with very low vector immunity. We recently demonstrated efficient transduction of MSC by rAAV2 and expression of transgenes during multi-stage osteogenic differentiation.13,14 In the present study, we describe the potential of MSC, transduced ex vivo by a rAAV2 encoding BMP2 in long-term correction of osteopenia in a mouse model of osteoporosis. Bone morphogenetic protein (BMP) signaling regulates body axis determination, morphogenesis in limb bud, apoptosis in finger development and differentiation of cells in both ectodermal and mesenchymal origin.15–18 However, the molecules involved in BMP regulation of biological events have not been fully understood. Molecular analyses of the mechanisms of biological events induced by the BMP-2 have identified key regulatory transcription factors to be the target.19–21 Bone cell development is also regulated by BMPs, which induces osteogenesis when implanted in ectopic sited via stimulation of the differentiation of mesenchymal cells into osteoblastic cells.22–25 BMP actions are also under the influence of the signals triggered by the interaction between osteoblastic cells and extracellular matrix.26,27
Despite the potential of MSC in ex vivo therapy, a major limitation of using genetically-modified MSC is bone-specific homing upon autologous transfer. To overcome this, we employed a novel strategy to transiently express mouse α-4 integrin ectopically on these cells prior to in vivo transfer. Heterodimerization of the mouse α-4 integrin with endogenous β1 integrin was confirmed to mediate enhanced bone homing of the MSC, and induction of new bone formation as determined by several bone parameters.28 Further, BMP2 expression by transplanted MSC was found to effect therapeutic gain also by mobilization of endogenous progenitors.
The phenotype of MSC isolated and cultured from low density bone marrow cells of C57BL/6 mice was determined by flow cytometry. The cells were negative for CD34 and CD45 surface markers and positive for CD29, CD44, Sca1 and CD106 (Supplemental Figure 1). Multilineage property of MSC was confirmed by differentiating the cells into adipocytes, chondrocytes, osteoblasts or myocytes as before (Supplemental Figure 2).28
To demonstrate the expression of BMP2 following rAAV2 transduction, mouse MSC were transduced with rAAV2-BMP2 at an MOI of 1000. To determine the influence of α4 integrin on the expression of BMP2 in MSC, cells were transiently transfected with the mouse α4 integrin plasmid. Expression of BMP2 protein in MSC was detected by immunoblotting with an anti-mouse BMP2 monoclonal antibody and a goat anti-mouse IgG, conjugated to horseradish peroxidase. The secreted BMP2 protein by rAAV2-BMP2 transfected MSC was detected in culture media after concentration and immunoblot analysis but not in rAAV2-GFP infected MSC. The molecular mass of the secreted BMP2 as 21 kDa protein indicated that it is in mature form (Figure 1). Results, shown in Figure 1, indicate that transient expression of α4 integrin does not affect BMP2 expression in mouse MSC.
By ELISA, we determined the amount of BMP2 in rAAV-BMP2 transduced MSC to be 3268 ± 591pg/ml/106 cells in non osteogenic medium. However, the level of BMP2 increased several fold to 47926 ± 8165 pg/ml when the cells were cultured in osteogenic medium.
To determine the efficacy of MSC expressing α4 integrin in bone homing in vivo, the cells were intravenously injected through tail vein in C57BL/6 mice after transient transfection with the mouse α4 integrin expression plasmid. A total of 2×106 MSC were administered per mouse in five consecutive days by i.v. injection. All animals survived after the MSC infusion. In situ hybridization was performed in decalcified bone sections of long bones (femur and tibia) with Y-chromosome specific probe (Supplemental Figure 3 and in situ method).28 For the quantification of positive signal in the region of interest (ROI), below the growth plate of each femur section, at least five random fields were counted in a microscope (Supplemental Figure 3).28 Results indicated enriched homing of MSC to the long bones, as a result of expression of α4 integrin on these MSC similar to our findings in recently published studies.28 There was no significant difference in the bone homing of MSC between untransduced MSC and rAAV2-BMP2 transduced MSC injection in the transplanted mice (Supplemental Figure 3). Semi-quantitative real-time PCR analysis for the biodistribution of donor cells in different tissues indicated a significant increase in homing of the transplanted cells to bone marrow when the cells were transfected to express α4 integrin prior to transplantation. This was also accompanied by reduction in the number of cells homing to other tissues including lung, liver, kidney and lymph node (Supplemental Figure 4). We were able to amplify Y-specific PCR from total DNA isolated from harvested bone marrow of transplanted animals after four months of transplantation (data not shown), which indicates long term survival of transplanted MSC.
No significant differences were observed in the circulating BMP2 level in the serum samples collected from MSC transplanted animals, although there was moderately higher levels was determined in the serum samples from MSC transplanted animals compared to naïve control until 5 weeks (Table 1).
All experimental animals were kept on a phytoestrogen-free diet 13 and their baseline DXA was obtained prior to the start of in vivo MSC injection experiment. Six week-old ovariectomized female C57BL/6 mice were intravenously injected with rAAV2-BMP2 transduced MSC, rAAV2-GFP transduced MSC, unmodified MSC and transiently transfected with mouse α4 integrin plasmid. Quantitative analysis of BMD data by ANOVA indicated a significant difference between the group of animals transplanted with MSC-rAAV-BMP2+α4 integrin treated group compared to other MSC transplanted control groups (p<0.001). Non-invasive DXA analysis performed after 5 weeks post transplantation revealed a significant increase in total bone mass and trabecular BMD in mice treated with AAV2-BMP2 transduced MSC and transiently transfected with α4 integrin plasmid compared to mice treated with rAAV2-GFP transduced MSC and transiently transfected with α4 integrin plasmid, rAAV2-BMP2 transduced MSC, MSC transiently transfected with α4 integrin plasmid, unmodified MSC (p<0.001) or naïve group (p<0.0001). Increase in the BMD values were found to be statistically significant compared to other control MSC transplanted groups (p<0.001) and untreated control animals (Figure 2, p<0.0001). An increase of 21.5 ± 2.5% in BMD values, in group of mice injected with MSC transduced with rAAV2-BMP2 and transiently transfected with α4 integrin plasmid was observed at week 5 compared to the other MSC transplanted controls (All comparisons, p<0.001). This increase in BMD compared to the controls was maintained until 15 weeks (Figure 2). Statistical analysis of BMD in the group of animal transplanted with MSC-rAAV-GFP+α4 integrin, MSC+α4 integrin, MSC-rAAV-BMP2 or MSC only had statistically significant increase in BMD compared to untreated control group of animal (Figure 2, p<0.05). We also tested intra-muscular injection of 2×1011 vg/mouse rAAV-Col.I:BMP2 virus and monitored the BMD for ten weeks but did not observe significant bone growth in the animals (Figure 2).
Figure 2 data suggests that increase in BMD reached a plateau after week 5 and did not increase during weeks 10 and 15. To explain this observation, we looked into the osteoblasts/osteoclasts ratio in the bone sections from different treatment groups and determined no significant differences (p>0.05), indicating that a coupled bone formation and remodeling process was maintained after the initial increase in bone formation activity.
Cohorts of MSC transplanted mice were sacrificed at regular time points and their femur bones were analyzed by micro-CT. Results of this analysis, shown in Figure 3A, indicated that increase in total BMD in femora of BMP2-expressing MSC treated mice was accompanied by a corresponding decrease in endosteal circumference. Meanwhile, there was no effect on periosteal circumference (data not shown), suggesting that MSC expressing BMP2 exerted the increased bone formation on the endosteal bone surface. Several micro architectural parameters were collected from the micro-CT analysis. Results of these analyses, shown in Figure 3B, indicated that the group of mice transplanted with rAAV2-BMP2 transduced MSC had significant bone growth compared to the controls. The observed increase was seen in both bone mass (bone volume) and bone activity (as reflected by the parameters for bone surface and bone surface-to-volume ratio). Significant increase in bone structural parameters of trabecular bones, bone volume by tissue volume (BV/TV), ratio of bone surface to bone volume (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular connectivity density (ConnD) and degree on anisotrophy (DA) were found in AAV2-BMP2 transduced MSC group compared to controls (p<0.03).
Results of bone architecture by DXA and micro-CT were further supported by histological analysis. At 5, 10 and 15 weeks after the MSC transplantation, mice from each group were sacrificed and bone tissues were examined by histology. Standard trichrome staining confirmed an increase in the bone mass in mice treated with rAAV2-BMP2 transduced MSC. Trabecular bone areas extended from the endosteal surface of the cortical bone into the marrow cavity of the femur (Figure 4A). Many cuboidal osteoblasts were found lining the surfaces of the trabecular bone. Evidence of trabecular bone growth was minimal in control femur sections. Next, we sought to determine whether the transplanted MSC expressing BMP2 mobilizes endogenous progenitors to the site of new bone formation. To this end, donor MSC were derived from syngeneic, GFP transgenic male mice and transplanted to 6-week-old ovariectomized female recipients. Cohorts of mice were sacrificed periodically and bone sections stained with GFP antibody. Results shown in Figures 4B and indicated that there was a decline in the number of GFP-positive cells in the bone sections after 5 weeks. However, trabecular bone with the number of osteoblasts lining the remodeling area and osteocytes remained in a steady increase indicating their mobilization from endogenous progenitors. Quantitative PCR analysis using donor specific DNA sequences in bone marrow cells of mice also corroborated with the results of immunohistochemistry indicating a decrease in the number of donor cells over time, despite a steady-state increase in new bone formation (Figure 4C). We detected 10.58%, 3.62% and 1.18% GFP positive cells in the bone marrow of MSC+rAAV-BMP2+α4 integrin transplanted group compared to 1.33%, 0.92% and 0.045% GFP positive cells in the MSC transplanted group on week 1, 5 and 10 respectively (Figure 4C). Similar to our earlier observation, expression of α4 integrin on MSC leads to several fold increase in bone-specific homing although, we were able to detect presence of transplanted cells in other tissues like lung, liver, pancreas, kidney and fat tissues, comparatively in lower amounts.28
MSC isolated from bone marrow of animals transplanted with rAAV-BMP2 and transfected with α4 integrin plasmid did show comparatively higher proliferation capabilities in an in vitro cell proliferation assay compared to all other control groups (p<0.05) (Figure 5A). MSC included for these studies were GFP negative and represented only endogenous MSC. In vitro proliferation was higher in bone marrow stroma harvested from rAAV-BMP2 treated mice at days 2, 4, 6 and 8 of culture (Figure 5A). These data are consistent with the observed increase in the bone mineral contents (BMC) in the MSC- AAV-BMP2 transplanted group (data not shown).
FACS analysis to determine the osteo-competent population pool of cells in the bone marrow stroma harvested from naïve group, animals transplanted with MSC or animals transplanted with MSC transduced with rAAV-BMP2 and transfected with α4 integrin plasmid was done using MSC specific, PE-labeled CD29 and FITC labeled Runx2 antibodies. FACS results in Figure 5B show several fold increase in immature pre-osteoblast marker Runx2 expressing MSC in the bone marrow stroma harvested from MSC-AAV-BMP2 transplanted group compared to other control groups. In FACS analysis, 25.4% of the cultured bone marrow stromal cells harvested from animals transplanted with MSC-AAV-BMP2 did show double-positive cells for MSC marker (CD29) and immature pre-osteoblast marker (Runx2 ) compared to 4.15% of double positive cells from MSC group and 2.68% double-positive cells from naïve group (Figure 5B).
Bone histology and histomorphometry following the rAAV-BMP2 therapy showed more cuboidal-shaped osteoblasts lining the mineralized trabecular bones. Counting of the number of osteoblasts in five separate fields indicated a significant increase in the number of osteoblast in the MSC-rAAV-BMP2 group compared to the naïve or MSC transplanted group (Figure 5C).
Taken together these data showing more proliferative capabilities of harvested bone marrow stroma, having higher osteo-competent pool of cells with increased number of osteoblasts in the bone sections indicates involvement of the pro-osteoblastic process inside bone in the presence of MSC expressing BMP2 in the bone marrow microenvironment.
Immunohistochemistry in bone sections from OVX mice transplanted with BMP-transduced MSC from GFP transgenic mice indicated more Runx2-positive cells around the trabecular area compared to non-specific control vector-transduced MSC (Figure 6A). When this region was stained with GFP antibody, there was no significant numbers of positive cells in the new bone forming area, which showed positive staining for Runx2. By including bone sections from control GFP transgenic mouse, we confirmed that the lack of GFP staining is not because of the inability of antibody to detect GFP in Runx2-positive cells in this region. Quantification of the Runx2 positive cells in the bone sections from each of the experimental groups indicated that AAV-BMP2-transduced, and transplanted MSC have significant contribution in new bone growth by enriching endogenous progenitors, mediated by BMP2 expression from the transplanted MSC (Figure 6B). Three-point bending analysis as a functional testing for the quality of bones demonstrated a significant increase in peak load, stiffness and toughness of femur and tibia of BMP2 therapy group compared to other controls (data not shown).
The plasticity of MSC for multilineage differentiation has opened new prospects for clinical applications.29,30 Transplantation of genetically modified MSC has the potential to correct disorders of bone, cartilage and muscle.31–33 A continuous source of osteoblast recruitment for bone growth, remodeling and fracture repair is ensured by mesenchymal progenitor cells, which have been identified in the bone marrow and other tissues.34,35 Migration of bone forming cells is an important event during various physiological and pathological situations and their differentiation to cell lineages is controlled by bioactive factors found in the local micro-environment or secreted by the engineered cells. Thus, in principle, ex vivo therapy to increase bone formation may be accomplished by intravenous injections of MSC expressing therapeutic transgenes. However, engraftment potential of ex vivo modified MSC remained a limiting factor.36 Use of an ectopic bone homing signal in MSC in the present study was found to significantly increase bone homing of genetically modified MSC. The α4β1 integrin is a heterodimer present in bone homing hematopoietic stem cells and metastatic cancer cells.37,38 Since the homing signal is required only during the initial phase of MSC repopulation, it was adequate to transiently express α4 integrin using a plasmid vector.
Since in autologous stem cell therapy using MSC, cells are transduced ex vivo and maintained in culture until optimal transgene expression has occurred, vector structural proteins are greatly diminished and virtually no immune reactions against the vector would be expected. In addition, rAAV do not encode for vector proteins, the only transgene expressed will be BMP2 or any such therapeutic transgene. In the present study, MSC from GFP transgenic mice served as a means for long-term in vivo cell tracking. While we can not definitely exclude the possibility of down-regulation of GFP expression with terminal differentiation in vivo or possibly by promoter silencing.39 Based on the results, in vivo osteoblast differentiation by transplanted MSC alone is unlikely to have a significant contribution to the therapeutic benefit observed in this study. MSC have been shown to augment tissue repair primarily through actions of trophic mediators in a variety of injury models.40–43 It is therefore likely that transplanted MSC after homing to bone sites induced the expression of growth factors in the local micro-environment, which recruited endogenous progenitors to promote osteogenesis. This is corroborated by both quantitative PCR and immuno-histochemistry in the present study. BMP2 expression from AAV-BMP2 was using a 2.3 kb collagen type I promoter, specifically expressed in MSC 44, restricted the transgene expression in the bone marrow micro environment in response to the existing local stimuli.
Results of the present study demonstrated that mice injected with MSC expressing BMP2 improve bone growth beginning week 5 as indicated by increased BMD compared with only MSC injected mice or control ovariectomized mice. There was significant increase in BMD in the group of mice injected with rAAV2-BMP2 transduced and transiently transfected with mouse α4-integrin plasmid compared to the rAAV2-BMP2 group, possibly because α4 expressing MSC homed significantly more in the bone sites exerting better therapeutic effect. Although the precise mechanism by which this effect occurred remains to be elucidated, increased proliferation capabilities of harvested bone marrow stroma from transplanted animals, presence of immature pre-osteoblast marker Runx2 positive cells in bone marrow stroma and the observed augmentation in osteoblast number combined with increased trabecular bone is suggestive of a pro-osteoblastic mechanism. The robust trabecular bone growth observed in mice injected with MSC expressing BMP2, however, is unlikely to be due to osteoblast differentiation of the transplanted MSC alone as bone growth was sustained even after 15 weeks, when there was very little identification of donor MSC within the bone sections. During early time point, some GFP positive cells were found in close proximity of bone remodeling area near trabecular bones.28 Thus, BMP2 production by the transplanted MSC may have induced paracrine effects for pro-osteoblastic local bone growth milieu by influencing the existing endogenous progenitors inside the bone marrow, either by cell recruitment and/or cell proliferation. Experimental data from this study indicates that there were more osteo-competent pool of cells in the AAV-BMP2 MSC group, which may be derivatives of osteoprogenitor cells as a result of either active proliferation of available progenitors or active recruitment of osteo- progenitors, since they did not stained positive with GFP antibody.
Progenitor cell-based therapy offers certain physiologic advantages compared to direct application of viral vectors in vivo by gene therapy, even though improved bone growth has been observed with other modalities.45–47 Integration of the transgene into the host chromosome is not required and supraphysiological over expression of transgene is necessarily avoided. Instead, the transplanted host cellular machinery regulates the production of cellular growth factors in response to the immediate need to the appropriate microenvironment. Additionally, application of protein therapy requires multiple administration of products, while genetically modified cell-based strategies can theoretically continue to produce a protein of interest until the cell is cleared from the transplanted subject.
Our data support the therapeutic potential of MSC in the restoration of bone growth in an osteopenia model. These effects appear to be also mediated by the creation of a pro-osteoblastic environment in the presence of MSC expressing BMP2 and not only by direct incorporation of transplanted MSC. Indirect mechanisms such as cell recruitment and paracrine effects of growth factors and cytokines produced by MSC are likely to be responsible for the observed bone growth improvement. Thus, the potential of this therapy may also be utilized in pathological situations requiring short-term benefits, such as multiple fractures and osteolytic bone damage in cancer.
HEK293, was purchased from ATCC and maintained in DMEM supplemented with 10% newborn calf serum. Restriction endonucleases and other modifying enzymes were purchased from either NEB (Beverly, MA) or Promega (Madison, WI). BMP-2 and α4 integrin antibodies were purchased from R&D systems (Minneapolis, MN) and e-biosciences (San Diego, CA) respectively. Recombinant BMP2 was purchased from R&D Systems.
All AAV2 plasmids were constructed using pSub201 as the backbone. cDNA encoding rat BMP2 was kindly provided by Dr. Chen (University of Texas at San Antonio, TX) and a cDNA encoding mouse α4 integrin was kindly provided by Dr. David Rowe (University of Connecticut Health Science Center, Farmington, CT). The coding sequence of rat BMP-2 was subcloned under collagen type I promoter and mouse α4 integrin was subcloned under the control of CMV promoter.48 Recombinant AAV2 encoding GFP was described earlier.48 Packaging of rAAV2 was done in an adenovirus-free system as described.48 Purification of the virions was done in a discontinuous iodixanol gradient centrifugation followed by heparin affinity chromatography. Particle titers of the purified virions were determined by quantitative slot blot analysis as described.48
Ovariectomized C57BL/6 mice were purchased from the National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD), and GFP transgenic mice (C57BL/6-Tg(ACTbEGFP)1Osb/J) were purchased from the Jackson Laboratories (Barr Harbor, ME). All animal protocols were approved by IACUC. To obtain bone marrow stromal cells, 4–6 week-old male mice were sacrificed and bone marrow was flushed from the femur and tibia, and the marrow mononuclear cells were purified by ficoll gradient.28 Bone marrow stromal cells were grown in stemline MSC expansion medium (Sigma) supplemented with 10 % FBS, 10−9 M FGF2 to maintain the cells in pluripotent and undifferentiated state.49 Residual macrophages from the MSC culture were removed by IMAC using anti-mouse CD11b beads (cat # 558013 BD IMag, BD Biosciences, San Diego, CA). After 14 days, the adherent stromal cells were split prior to attaining confluence to avoid possible onset of differentiation. The cells were routinely prepared and used for in vitro and in vivo studies as low passage cultures (passage 4–8).
Undifferentiated MSC were transduced with 1000 multiplicity of infection (MOI; 1 MOI = 50 genomic particles) of rAAV2-BMP2 or rAAV2-GFP i.e. an MOI of 1000 requires 50,000 viral genomic particles. Virus infection was performed in Opti-MEM for 2 h at 37°C following which complete medium with FGF2 was added. The cells were grown for 10 more days before transplantation into mice.
To enrich MSC engraftment to bone, we transiently expressed the α4 integrin by plasmid transfection.28 A 3.5 kb full-length cDNA fragment of the murine α4 integrin subunit was cloned into pcDNA3.1 under the transcriptional control of CMV promoter for transient transfection. Ten μg of plasmid was used for transient transfection in 175 mm culture flask using FuGENE 6 (Roche Diagnostics, Indianapolis, IN) transfection reagent following the manufacturer’s instructions. Transfection efficiency was found to be between 70–80%. Ectopic expression of α4 was confirmed by staining the cells using PE -conjugated α4 integrin antibody and FITC-conjugated β1 integrin antibody. Co-localization of the two subunits on cell membrane was confirmed by merging the images. Forty-eight hours after transient transfection, cells were trypsinized and sorted by magnetic cell separator (IMAC) in sterile condition based on α4-integrin positivity. The α4-integrin positive cell population was used in transplantation experiments in vivo.
The recipient animals were 6-week-old ovariectomized female C57BL/6 mice were grouped in 10mice each. Mock-transduced or rAAV2 (GFP or Col1-BMP2)-transduced MSC were resuspended in a volume of 100 μl Saline and i.v. administered into recipient mice through tail vein. Prior to in vivo administration, the cells were transiently transfected with the plasmid encoding the mouse α4 integrin. Cohorts of mice received a total of 2×106 MSC in five consecutive days (4×105cells/injection) by i.v. injection, each of untransduced, AAV2-GFP transduced or AAV2-BMP2 transduced MSC. Five weeks after transplantation, animals were subjected for DXA and micro-CT analysis. Identification of homed MSC from the donor male mice was performed by semi-quantitative real-time PCR using Y-chromosome specific primers.28
BMP2 level in the mouse serum collected from the animals at different time point was determined by BMP2 Quantikine kit (R&D Systems, Minneapolis, MN). A standard curve was generated using purified rhBMP2 protein.
For DXA analysis, animals were briefly anesthetized with isoflurane (2%) and placed in a prostrate position on the imaging plate. Bone mineral density (BMD), bone mineral content (BMC) and other body composition was assessed in vivo by DXA (GE-Lunar PIXImus, software version 1.45, GE-Lunar) periodically. To assess bone density, bone mass, geometry and bone microarchitecture, intact right femur from each mouse was scanned using high-resolution micro-CT imaging system (μCT40, SCANCO Medical). Histomorphometric parameters including bone volume, trabecular connectivity, trabecular thickness, trabecular separation and degree of anisotropy were evaluated.
Formalin-fixed tissues were decalcified in EDTA solution for two weeks and embedded in paraffin. Longitudinal sections of 5 μm thicknesses were cut from paraffin embedded blocks of frontal sections of tibia, using a Leica 2265 microtome. For immunohistochemistry, 5-μm sections from each block were mounted onto treated slides. Following rehydration, antigen retrieval was performed in a high-steam cooker for 10 minutes; then slides were treated with 3% H2O2 for 5 minutes to quench the endogenous peroxidase activity, blocked with preimmune goat serum (1%) for 20 minutes and incubated with anti-GFP antibody for 1 hr. A delete was included in each staining procedure for each slide by omitting the primary antibody from the staining procedure. All slides were than incubated with secondary antibody conjugated to horseradish peroxidase (HRP) for 30 minutes. Finally, slides were developed with the substrate 3,3′-diaminobenzidine (DAB) for visualization of antigen-antibody complex and counterstained lightly with hematoxylin.
Quantitation of donor cells that homed to different organs was determined by real-time PCR using genomic DNA isolated from tissues from recipient mice. Total DNA was isolated from different tissues. Quantitative real-time PCR was performed using GFP-specific primers in a Biorad icycler (Optical Module) using SYBR Green I dye kit (BIORAD) following manufacturer’s instructions. PCR products were subjected to melting curve analysis using the light cycler system to exclude amplification of non-specific sequences. Values obtained from amplification of vector-specific sequences from each sample were normalized to copy number of GAPDH gene amplification from the same sample to derive the relative vector copy number.
To distinguish transplanted MSC from endogenous MSC in target tissues and to determine whether transplanted MSC increase bone density directly or indirectly, donor cells were derived from transgenic, GFP-positive mice and were transplanted into ovariectomized syngeneic female recipients. A total of 2×106 MSC were administered per mouse in five consecutive days of i.v. injection. Cohorts of mice were sacrificed at weeks 1, 5 and 15 following MSC transplantation and long bones were fixed in 10% buffered-formalin, followed by decalcification and mounting. Five μm sections were prepared and stained with Runx2 and GFP antibodies to localize donor MSC in different areas of the bone. The slides were also stained by trichrome method to determine the location of GFP-positive donor MSC. The number of Runx2-positive cells in each area was quantified microscopically.
Bone marrow from the animals after 5 weeks, which were previously transplanted with MSC, were flushed from the long bones and cultured for a week. MSC cells were seeded in 96-well plates (Falcon 3072, Becton- Dickinson, Lincoln Park, NJ) at a density of 5000 cells per well and incubated at 37°C in MSC medium supplemented with 10% FBS (Sigma, Mesenchymal stem cell expansion medium Cat # 1569-1L, St. Louis, MO) for four days, cell growth was measured by using a non-radioactive cell proliferation assay kit (CellTiter 96RAqueous from PROMEGA, Madison, WI). On day 2, 4, 6 and 8 optical density was determined at 490 nm in a multi-well plate reader (BioTek Synergy 2, Vermont, USA). Background absorbance of the medium in the absence of cells was subtracted. All samples were assayed in triplicate, and the mean for each experiment was calculated.
To determine the phenotypic characteristics of MSC isolated and cultured from the experimental animals were trypsinized and washed twice with FACS buffer (PBS containing 2% FBS, 0.1% sodium azide). MSC were stained with PE labeled CD29 (e-biosciences, San Diego, CA) and FITC labeled Runx2 antibodies (R&D systems, Minneapolis, MN) and analyzed by FACS Aria flow cytometer (BD Biosciences, San Diego, CA).
All data are reported as mean ± standard deviation (SD). Comparison of differences between two variables was performed using the two-tailed, two-sample with equal variances, independent t test. Bone mineral density (BMD), bone mineral contents (BMC), lean tissue mass and fat tissue mass were analyzed using ANOVA. Nonparametric data were analyzed using Tukey’s studentized range (HSD) test and Krushal-Wallis test. Results were considered significant when p< 0.05.
Financial support of the National Institutes of Health grant AR50251 and the U.S. Army Department of Defense grants BC044440 and PC050949 are gratefully acknowledged.