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This study assessed whether simple inhibition of angiogenesis by vascular endothelial growth factor (VEGF) blockade is sufficient to direct in vivo chondrogenic differentiation of implanted human mesenchymal stromal/stem cells (MSCs). MSCs transduced to express sFlk‐1 and directly implanted subcutaneously in nude mice without in vitro preculture spontaneously differentiated into the chondrocytic lineage with a stable phenotype for the observation time‐period of 12 weeks. These findings suggest that VEGF blockade is a robust strategy to enhance cartilage repair by endogenous or grafted mesenchymal progenitors.

Adult mesenchymal stromal/stem cells (MSCs) are a valuable source of multipotent progenitors for tissue engineering and regenerative medicine, but may require to be genetically modified to widen their efficacy in therapeutic applications. For example, overexpression of the angiogenic factor vascular endothelial growth factor (VEGF) at controlled levels is an attractive strategy to overcome the crucial bottleneck of graft vascularization and to avoid aberrant vascular growth. Since the regenerative potential of MSCs is rapidly lost during in vitro expansion, we sought to develop an optimized technique to achieve high-efficiency retroviral vector transduction of MSCs derived from both adipose tissue (adipose stromal cells, ASCs) or bone marrow (BMSCs) and rapidly select cells expressing desired levels of VEGF with minimal in vitro expansion. The proliferative peak of freshly isolated human ASCs and BMSCs was reached 4 and 6 days after plating, respectively. By performing retroviral vector transduction at this time point, >90% efficiency was routinely achieved before the first passage. MSCs were transduced with vectors expressing rat VEGF(164) quantitatively linked to a syngenic cell surface marker (truncated rat CD8). Retroviral transduction and VEGF expression did not affect MSC phenotype nor impair their in vitro proliferation and differentiation potential. Transgene expression was also maintained during in vitro differentiation. Furthermore, three subpopulations of transduced BMSCs homogeneously producing specific low, medium, and high VEGF doses could be prospectively isolated by flow cytometry based on the intensity of their CD8 expression already at the first passage. In conclusion, this optimized platform allowed the generation of populations of genetically modified MSCs, expressing specific levels of a therapeutic transgene, already at the first passage, thereby minimizing in vitro expansion and loss of regenerative potential.

We recently developed a method to control the in vivo distribution of vascular endothelial growth factor (VEGF) by high throughput Fluorescence-Activated Cell Sorting (FACS) purification of transduced progenitors such that they homogeneously express specific VEGF levels. Here we investigated the long-term safety of this method in chronic hind limb ischemia in nude rats. Primary myoblasts were transduced to co-express rat VEGF-A164 (rVEGF) and truncated ratCD8a, the latter serving as a FACS-quantifiable surface marker. Based on the CD8 fluorescence of a reference clonal population, which expressed the desired VEGF level, cells producing similar VEGF levels were sorted from the primary population, which contained cells with very heterogeneous VEGF levels. One week after ischemia induction, 12 × 106 cells were implanted in the thigh muscles. Unsorted myoblasts caused angioma-like structures, whereas purified cells only induced normal capillaries that were stable after 3 months. Vessel density was doubled in engrafted areas, but only approximately 0.1% of muscle volume showed cell engraftment, explaining why no increase in total blood flow was observed. In conclusion, the use of FACS-purified myoblasts granted the cell-by-cell control of VEGF expression levels, which ensured long-term safety in a model of chronic ischemia. Based on these results, the total number of implanted cells required to achieve efficacy will need to be determined before a clinical application.

Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACSpurified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10 7 cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.

Background Recent investigations have provided substantial evidence that distinct molecular and morphological changes in subchondral bone tissue, most notably sclerosis, play an active and important role in the pathogenesis of OA. The cellular and molecular regulation of this pathological process remains poorly understood. Objectives We investigated whether osteoimmunology, the reciprocal signaling between cells from the immune and bone system, is involved in OA subchondral bone sclerosis. Methods Tibial plateaus and informed consent were obtained from patients undergoing total knee arthroplasty due to end-stage OA. Subchondral bone mineralization distribution was analyzed using computed tomography osteoabsoptiometry (CT-OAM) and standardized cryosections of low (non-sclerotic) and high (sclerotic) bone mineralization were prepared (n=18 each). Cartilage degeneration was graded in Safranin-O-stained sections using the Mankin scoring system. The presence of T-lymphocytes, B-cells and macrophages was assessed using immunohistochemical staining of their respective surface markers CD3, CD20 and CD68. Osteoclast activity was visualized by staining of the enzyme marker tartrate-resistant acid phosphatase (TRAP). Cellular characterization of ex vivo subchondral bone outgrowth cultures was performed using alkaline phosphatase (ALP), TRAP staining. Correlation between histological parameters was assessed using Spearman´s rank correlation. Statistical differences were calculated using Wilcoxon signed rank test or paired t-test, where appropriate. Results CT-OAM revealed a heterogeneous distribution of subchondral bone mineralization in OA tibial plateaus, displaying focal areas of sclerosis that overlapped macroscopically with areas of cartilage damage. These data were confirmed at the histological level by a strong correlation between Mankin score and grade of sclerosis (r=0.7, p<0.001). Immunohistochemistry showed that CD20+, but not CD3+, lymphocytes and CD68+ mononuclear (macrophage) and multinucleated (osteoclast) cells were present in subchondral marrow spaces. Notably, the number of CD20+ lymphocytes and CD68+ cells was significantly (p<0.05) increased in sclerotic subchondral bone. Enhanced osteoclast activity was confirmed by a significantly increased (p<0.05) number of multinucleated and mononuclear TRAP+ cells in sclerotic bone. Finally, the number of CD68+ cells was strongly correlated (p<0.001) with Mankin score (r=0.7), grade of sclerosis (r=0.8), CD20+ lymphocytes (r=0.8), and TRAP-positive cells (r=0.9). Outgrowth cultures of subchondral bone showed cells of different morphologies including fibroblast-shaped osteoblasts and macrophage-like cells. Expression of ALP was detected in the prior, while TRAP expression was evident in the latter. Corresponding with histological analyses, the number of TRAP+ cells was increased in ex vivo outgrowth cultures of sclerotic compared to non-sclerotic subchondral bone. Conclusions Together, our data suggest that osteoimmunological mechanisms, specifically the interact...

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