VEGF-expressing mesenchymal stem cells for improved angiogenesis in regenerative medicine : a bone tissue engineering approach

Rapid vascularization of tissue-engineered grafts is a major bottleneck in the development of regenerative medicine approaches. In order to overcome this limitation, we aimed to develop a bone tissue engineering strategy combining cell therapy with pro-angiogenic gene therapy. Vascular Endothelial Growth Factor (VEGF) is the master regulator of physiological vascular growth and is commonly used as a therapeutic transgene for the induction of angiogenesis. However, uncontrolled and high levels of VEGF expression can lead to aberrant vascular growth. To achieve controlled expression in vivo, a high-throughput flow cytometry-based method has previously been developed in our group. Linking the VEGF cDNA to a cell-surface marker (a truncated version of CD8a) in a bicistronic construct enabled the rapid purification of genetically modified myoblasts secreting a desired VEGF level, using FACS sorting based on the intensity of CD8 expression in each cell. Controlled VEGF expression in skeletal muscle, achieved by implantation of these FACS-purified myoblast populations, induced only normal, stable and functional vascular networks and avoided any aberrant angiogenesis. The aims of this thesis were to adapt this method to human adipose tissue- and bone marrow-derived mesenchymal stromal/stem cells (ASC and BMSC), and to apply these in a bone tissue engineering approach to increase the vascularization potential of osteogenic grafts. As MSC gradually loose their regenerative potential during in vitro expansion, we first optimized our genetic engineering method for MSC, so as to enable high transduction efficiency and FACS-purification with minimal in vitro manipulation. Chapter 2 describes the generation of an optimized protocol allowing routine transduction efficiencies of > 90% of primary human ASC and BMSC already during the first plating, as well as flow cytometry purification of transduced cells at the time of the first passage. In addition we demonstrated that it was possible to FACS-purify specific sub-populations of transduced MSC homogeneously producing desired VEGF doses. Neither retroviral vector transduction, FACS-purification, nor the expression of the transgenes VEGF and CD8 impaired MSC proliferation and in vitro differentiation potential. Transgene expression was not lost during in vitro differentiation. In Chapter 3, proof-of-principle was obtained by applying this platform to a bone tissue engineering approach. Human BMSC, transduced and rapidly FACS-purified to eliminate non-expressing cells, were seeded onto hydroxyapatite granules to generate non-critically sized constructs, and were implanted subcutaneously in nude rats. In vivo vascularization potential was significantly increased in VEGF-expressing BMSC. Although VEGF expression was heterogeneous, no aberrant angiogenesis was observed. Indeed, orderly vascular beds were induced, with flow-conducting arterioles feeding into extensive capillary networks, where metabolic exchanges can take place efficiently. The improvement in vascularization was not diminished by extensive in vitro expansion of the transduced BMSC up to 35 population doublings, showing that genetic modification conferred a stable angiogenic potential. As expected, these expanded BMSC lost their osteogenic potential. However, their sustained capacity to induce vascularization could be useful in other applications, where effective expansion of the vascular bed is required, but not progenitor differentiation, such as in cell-based approaches for therapeutic angiogenesis in peripheral or coronary artery diseases. By minimizing cell expansion, both naïve and control transduced MSC generated abundant bone tissue in vivo. However, VEGF over-expression specifically caused a strong reduction in bone formation. This correlated with an increased recruitment of TRAP-positive osteoclasts specifically in VEGF-expressing constructs. These data suggest that VEGF over-expression might impair bone formation by disrupting the balance between bone formation and resorption towards excessive degradation. To fully understand the underlying mechanism, further experiments will be needed. The method described in chapter 2 provides a general platform to generate populations of genetically modified MSC, expressing specific levels of a therapeutic transgene, already at the time of the first passage. Therefore, it has the potential to be applied in other fields of regenerative medicine, beyond bone tissue engineering. We briefly describe two recently initiated projects, based on the results described in this thesis, which aim at either promoting or inhibiting angiogenesis in order to improve cardiac function after myocardial infarction, or cartilage tissue formation, respectively

Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling

Chondrogenic differentiation of bone marrow-derived mesenchymal stromal/stem cells (MSCs) can be induced by presenting morphogenetic factors or soluble signals but typically suffers from limited efficiency, reproducibility across primary batches, and maintenance of phenotypic stability. Considering the avascular and hypoxic milieu of articular cartilage, we hypothesized that sole inhibition of angiogenesis can provide physiological cues to direct in vivo differentiation of uncommitted MSCs to stable cartilage formation. Human MSCs were retrovirally transduced to express a decoy soluble vascular endothelial growth factor (VEGF) receptor-2 (sFlk1), which efficiently sequesters endogenous VEGF in vivo, seeded on collagen sponges and immediately implanted ectopically in nude mice. Although naïve cells formed vascularized fibrous tissue, sFlk1-MSCs abolished vascular ingrowth into engineered constructs, which efficiently and reproducibly developed into hyaline cartilage. The generated cartilage was phenotypically stable and showed no sign of hypertrophic evolution up to 12 weeks. In vitro analyses indicated that spontaneous chondrogenic differentiation by blockade of angiogenesis was related to the generation of a hypoxic environment, in turn activating the transforming growth factor-β pathway. These findings suggest that VEGF blockade is a robust strategy to enhance cartilage repair by endogenous or grafted mesenchymal progenitors. This article outlines the general paradigm of controlling the fate of implanted stem/progenitor cells by engineering their ability to establish specific microenvironmental conditions rather than directly providing individual morphogenic cues.; Chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs) is typically targeted by morphogen delivery, which is often associated with limited efficiency, stability, and robustness. This article proposes a strategy to engineer MSCs with the capacity to establish specific microenvironmental conditions, supporting their own targeted differentiation program. Sole blockade of angiogenesis mediated by transduction for sFlk-1, without delivery of additional morphogens, is sufficient for inducing MSC chondrogenic differentiation. The findings represent a relevant step forward in the field because the method allowed reducing interdonor variability in MSC differentiation efficiency and, importantly, onset of a stable, nonhypertrophic chondrocyte phenotype

Generation of human adult mesenchymal stromal/stem cells expressing defined xenogenic vascular endothelial growth factor levels by optimized transduction and flow cytometry purification

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

FACS-purified myoblasts producing controlled VEGF levels induce safe and stable angiogenesis in chronic hind limb ischemia

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-A(164) (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 × 10(6) 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

Controlled angiogenesis in the heart by cell-based expression of specific vascular endothelial growth factor levels

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 FACS-purified 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

IL-13 as Target to Reduce Cholestasis and Dysbiosis inAbcb4Knockout Mice

The Th2 cytokine IL-13 is involved in biliary epithelial injury and liver fibrosis in patients as well as in animal models. The aim of this study was to investigate IL-13 as a therapeutic target during short term and chronic intrahepatic cholestasis in anAbcb4-knockout mouse model (Abcb4(-/-)). Lack of IL-13 protectedAbcb4(-/-)mice transiently from cholestasis. This decrease in serum bile acids was accompanied by an enhanced excretion of bile acids and a normalization of fecal bile acid composition. InAbcb4(-/-)/IL-13(-/-)double knockout mice, bacterial translocation to the liver was significantly reduced and the intestinal microbiome resembled the commensal composition in wild type animals. In addition, 52-week-oldAbcb4(-/-)IL-13(-/-)mice showed significantly reduced hepatic fibrosis.Abcb4(-/-)mice devoid of IL-13 transiently improved cholestasis and converted the composition of the gut microbiota towards healthy conditions. This highlights IL-13 as a potential therapeutic target in biliary diseases