Long-Term Severe In Vitro Hypoxia Exposure Enhances the Vascularization Potential of Human Adipose Tissue-Derived Stromal Vascular Fraction Cell Engineered Tissues.

The therapeutic potential of mesenchymal stromal/stem cells (MSC) for treating cardiac ischemia strongly depends on their paracrine-mediated effects and their engraftment capacity in a hostile environment such as the infarcted myocardium. Adipose tissue-derived stromal vascular fraction (SVF) cells are a mixed population composed mainly of MSC and vascular cells, well known for their high angiogenic potential. A previous study showed that the angiogenic potential of SVF cells was further increased following their in vitro organization in an engineered tissue (patch) after perfusion-based bioreactor culture. This study aimed to investigate the possible changes in the cellular SVF composition, in vivo angiogenic potential, as well as engraftment capability upon in vitro culture in harsh hypoxia conditions. This mimics the possible delayed vascularization of the patch upon implantation in a low perfused myocardium. To this purpose, human SVF cells were seeded on a collagen sponge, cultured for 5 days in a perfusion-based bioreactor under normoxia or hypoxia (21% and <1% of oxygen tension, respectively) and subcutaneously implanted in nude rats for 3 and 28 days. Compared to ambient condition culture, hypoxic tension did not alter the SVF composition in vitro, showing similar numbers of MSC as well as endothelial and mural cells. Nevertheless, in vitro hypoxic culture significantly increased the release of vascular endothelial growth factor (p < 0.001) and the number of proliferating cells (p < 0.00001). Moreover, compared to ambient oxygen culture, exposure to hypoxia significantly enhanced the vessel length density in the engineered tissues following 28 days of implantation. The number of human cells and human proliferating cells in hypoxia-cultured constructs was also significantly increased after 3 and 28 days in vivo, compared to normoxia. These findings show that a possible in vivo delay in oxygen supply might not impair the vascularization potential of SVF- patches, which qualifies them for evaluation in a myocardial ischemia model.

Neonatal Nav1.5 Protein Expression in Human Colorectal Cancer: Immunohistochemical Characterization and Clinical Evaluation.

Voltage-gated Na+ channels (VGSCs) are expressed widely in human carcinomas and play a significant role in promoting cellular invasiveness and metastasis. However, human tissue-based studies and clinical characterization are lacking. In several carcinomas, including colorectal cancer (CRCa), the predominant VGSC is the neonatal splice variant of Nav1.5 (nNav1.5). The present study was designed to determine the expression patterns and clinical relevance of nNav1.5 protein in human CRCa tissues from patients with available clinicopathological history. The immunohistochemistry was made possible by the use of a polyclonal antibody (NESOpAb) specific for nNav1.5. The analysis showed that, compared with normal mucosa, nNav1.5 expression occurred in CRCa samples (i) at levels that were significantly higher and (ii) with a pattern that was more delineated (i.e., apical/basal or mixed). A surprisingly high level of nNav1.5 protein expression also occurred in adenomas, but this was mainly intracellular and diffuse. nNav1.5 showed a statistically significant association with TNM stage, highest expression being associated with TNM IV and metastatic status. Interestingly, nNav1.5 expression co-occurred with other biomarkers associated with metastasis, including hERG1, KCa3.1, VEGF-A, Glut1, and EGFR. Finally, univariate analysis showed that nNav1.5 expression had an impact on progression-free survival. We conclude (i) that nNav1.5 could represent a novel clinical biomarker ('companion diagnostic') useful to better stratify CRCa patients and (ii) that since nNav1.5 expression is functional, it could form the basis of anti-metastatic therapies including in combination with standard treatments.

Myocardial infarction stabilization by cell-based expression of controlled Vascular Endothelial Growth Factor levels.

Vascular Endothelial Growth Factor (VEGF) can induce normal or aberrant angiogenesis depending on the amount secreted in the microenvironment around each cell. Towards a possible clinical translation, we developed a Fluorescence Activated Cell Sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a desired specific VEGF level from heterogeneous primary populations. Here, we sought to induce safe and functional angiogenesis in ischaemic myocardium by cell-based expression of controlled VEGF levels. Human adipose stromal cells (ASC) were transduced with retroviral vectors and FACS purified to generate two populations producing similar total VEGF doses, but with different distributions: one with cells homogeneously producing a specific VEGF level (SPEC), and one with cells heterogeneously producing widespread VEGF levels (ALL), but with an average similar to that of the SPEC population. A total of 70 nude rats underwent myocardial infarction by coronary artery ligation and 2 weeks later VEGF-expressing or control cells, or saline were injected at the infarction border. Four weeks later, ventricular ejection fraction was significantly worsened with all treatments except for SPEC cells. Further, only SPEC cells significantly increased the density of homogeneously normal and mature microvascular networks. This was accompanied by a positive remodelling effect, with significantly reduced fibrosis in the infarcted area. We conclude that controlled homogeneous VEGF delivery by FACS-purified transduced ASC is a promising strategy to achieve safe and functional angiogenesis in myocardial ischaemia.

Control of angiogenesis and host response by modulating the cell adhesion properties of an Elastin-Like Recombinamer-based hydrogel.

The control of the in vivo vascularization of engineered tissue substitutes is essential in order to obtain either a rapid induction or a complete inhibition of the process (e.g. in muscles and hyaline-cartilage, respectively). Among the several polymers available, Elastin-Like Recombinamers (ELR)-based hydrogel stands out as a promising material for tissue engineering thanks to its viscoelastic properties, non-toxicity, and non-immunogenicity. In this study, we hypothesized that varying the cell adhesion properties of ELR-hydrogels could modulate the high angiogenic potential of adipose tissue-derived stromal vascular fraction (SVF) cells, predominantly composed of endothelial/mural and mesenchymal cells. Human SVF cells, embedded in RGD-REDV-bioactivated or unmodified ELR-hydrogels, were implanted in rat subcutaneous pockets either immediately or upon 5-day-culture in perfusion-bioreactors. Perfusion-based culture enhanced the endothelial cell cord-like-organization and the release of pro-angiogenic factors in functionalized constructs. While in vivo vascularization and host cell infiltration within the bioactivated gels were highly enhanced, the two processes were strongly inhibited in non-functionalized SVF-based hydrogels up to 28 days. ELR-based hydrogels showed a great potential to determine the successful integration of engineered substitutes thanks to their capacity to finely control the angiogenic/inflammation process at the recipient site, even in presence of SVF cells.

Engineered mesenchymal cell-based patches as controlled VEGF delivery systems to induce extrinsic angiogenesis.

UNLABELLED: Therapeutic over-expression of Vascular Endothelial Growth Factor (VEGF) by transduced progenitors is a promising strategy to efficiently induce angiogenesis in ischemic tissues (e.g. limb muscle and myocardium), but tight control over the micro-environmental distribution of the dose is required to avoid induction of angioma-like tumors. Therapeutic VEGF release was achieved by purified transduced adipose mesenchymal stromal cells (ASC) that homogeneously produce specific VEGF levels, inducing only normal angiogenesis after injection in non-ischemic tissues. However, the therapeutic potential of this approach mostly in the cardiac field is limited by the poor cell survival and the restricted area of effect confined to the cell-injection site. The implantation of cells previously organized in vitro in 3D engineered tissues could overcome these issues. Here we hypothesized that collagen sponge-based construct (patch), generated by ASC expressing controlled VEGF levels, can function as delivery device to induce angiogenesis in surrounding areas (extrinsic vascularization). A 7-mm-thick acellular collagen scaffold (empty), sutured beneath the patch, provided a controlled and reproducible model to clearly investigate the ongoing angiogenesis in subcutaneous mice pockets. VEGF-expressing ASC significantly increased the capillary in-growth inside both the patch itself and the empty scaffold compared to naïve cells, leading to significantly improved survival of implanted cells. These data suggest that this strategy confers control (i) on angiogenesis efficacy and safety by means of ASC expressing therapeutic VEGF levels and (ii) over the treated area through the specific localization in an engineered collagen sponge-based patch. STATEMENT OF SIGNIFICANCE: Development of efficient pro-angiogenic therapies to restore the micro-vascularization in ischemic tissues is still an open issue. Although extensively investigated, the promising approach based on injections of progenitors transduced to over-express Vascular Endothelial Growth Factor (VEGF) has still several limitations: (i) need of a tight control over the microenvironmental VEGF dose to avoid angioma-like tumor growth; (ii) poor implanted cell survival; (iii) effect area restricted mainly to the injection sites. Here, we aimed to overcome these drawbacks by generating a novel cell-based controlled VEGF delivery device. In particular, transduced mesenchymal cells, purified to release a sustained, safe and efficient VEGF dose, were organized in three-dimensional engineered tissues to improve cell survival and provide a uniform vascularization throughout both the mm-thick implanted constructs themselves and the surrounding area.

A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.