Fatty acid-based monolayer culture to promote in vitro neonatal rat cardiomyocyte maturation.

The development of functional and reliable in vitro cardiac models composed of fully mature cardiomyocytes is essential for improving drug screening test quality, therefore, the success of clinical trial outcomes. In their lifespan, cardiomyocytes undergo a dynamic maturation process from the fetal to adult stage, radically changing their metabolism, morphology, contractility and electrical properties. Before employing cells of human origin, in vitro models often use neonatal rat cardiomyocytes (NRCM) to obtain key proof-of-principles. Nevertheless, NRCM monolayers are prone to de-differentiate when maintained in culture. Supplementation of free fatty acids (FFA), the main energy source for mature cardiomyocytes, and co-culture with fibroblasts are each by itself known to promote the shift from fetal to adult cardiomyocytes. Using a co-culture system, our study investigates the effects of FFA on the cardiomyocyte phenotype in comparison to glucose as typical fetal energy source, and to 10% serum used as standard control condition. NRCM decreased their differentiation status and fibroblasts increased in number after 7days of culture in the control condition. On the contrary, both glucose- and FFA-supplementation better preserved protein expression of myosin-light-chain-2v, a marker of mature cardiomyocytes, and the fibroblast number at levels similar to those found in freshly isolated NRCM. Nevertheless, compared to glucose, FFA resulted in a significant increase in sarcomere striation and organization. Our findings constitute an important step forward towards the definition of the optimal culture conditions, highlighting the possible benefits of a further supplementation of specific FFA to promote CM maturation in a co-culture system with FB.

Polo-Like Kinase 2 is Dynamically Regulated to Coordinate Proliferation and Early Lineage Specification Downstream of Yes-Associated Protein 1 in Cardiac Progenitor Cells.

BACKGROUND: Recent studies suggest that adult cardiac progenitor cells (CPCs) can produce new cardiac cells. Such cell formation requires an intricate coordination of progenitor cell proliferation and commitment, but the molecular cues responsible for this regulation in CPCs are ill defined. METHODS AND RESULTS: Extracellular matrix components are important instructors of cell fate. Using laminin and fibronectin, we induced two slightly distinct CPC phenotypes differing in proliferation rate and commitment status and analyzed the early transcriptomic response to CPC adhesion (<2 hours). Ninety-four genes were differentially regulated on laminin versus fibronectin, consisting of mostly downregulated genes that were enriched for Yes-associated protein (YAP) conserved signature and TEA domain family member 1 (TEAD1)-related genes. This early gene regulation was preceded by the rapid cytosolic sequestration and degradation of YAP on laminin. Among the most strongly regulated genes was polo-like kinase 2 (Plk2). Plk2 expression depended on YAP stability and was enhanced in CPCs transfected with a nuclear-targeted mutant YAP. Phenotypically, the early downregulation of Plk2 on laminin was succeeded by lower cell proliferation, enhanced lineage gene expression (24 hours), and facilitated differentiation (3 weeks) compared with fibronectin. Finally, overexpression of Plk2 enhanced CPC proliferation and knockdown of Plk2 induced the expression of lineage genes. CONCLUSIONS: Plk2 acts as coordinator of cell proliferation and early lineage commitment in CPCs. The rapid downregulation of Plk2 on YAP inactivation marks a switch towards enhanced commitment and facilitated differentiation. These findings link early gene regulation to cell fate and provide novel insights into how CPC proliferation and differentiation are orchestrated.

Scaffold Composition Determines the Angiogenic Outcome of Cell-Based Vascular Endothelial Growth Factor Expression by Modulating Its Microenvironmental Distribution.

Delivery of genetically modified cells overexpressing Vascular Endothelial Growth Factor (VEGF) is a promising approach to induce therapeutic angiogenesis in ischemic tissues. The effect of the protein is strictly modulated by its interaction with the components of the extracellular matrix. Its therapeutic potential depends on a sustained but controlled release at the microenvironmental level in order to avoid the formation of abnormal blood vessels. In this study, it is hypothesized that the composition of the scaffold plays a key role in modulating the binding, hence the therapeutic effect, of the VEGF released by 3D-cell constructs. It is found that collagen sponges, which poorly bind VEGF, prevent the formation of localized hot spots of excessive concentration, therefore, precluding the development of aberrant angiogenesis despite uncontrolled expression by a genetically engineered population of adipose tissue-derived stromal cells. On the contrary, after seeding on VEGF-binding egg-white scaffolds, the same cell population caused aberrantly enlarged vascular structures after 14 d. Collagen-based engineered tissues also induced a safe and efficient angiogenesis in both the patch itself and the underlying myocardium in rat models. These findings open new perspectives on the control and the delivery of proangiogenic stimuli, and are fundamental for the vascularization of engineered tissues/organs.

Engineering of an angiogenic niche by perfusion culture of adipose-derived stromal vascular fraction cells.

In vitro recapitulation of an organotypic stromal environment, enabling efficient angiogenesis, is crucial to investigate and possibly improve vascularization in regenerative medicine. Our study aims at engineering the complexity of a vascular milieu including multiple cell-types, a stromal extracellular matrix (ECM), and molecular signals. For this purpose, the human adipose stromal vascular fraction (SVF), composed of a heterogeneous mix of pericytes, endothelial/stromal progenitor cells, was cultured under direct perfusion flow on three-dimensional (3D) collagen scaffolds. Perfusion culture of SVF-cells reproducibly promoted in vitro the early formation of a capillary-like network, embedded within an ECM backbone, and the release of numerous pro-angiogenic factors. Compared to static cultures, perfusion-based engineered constructs were more rapidly vascularized and supported a superior survival of delivered cells upon in vivo ectopic implantation. This was likely mediated by pericytes, whose number was significantly higher (4.5-fold) under perfusion and whose targeted depletion resulted in lower efficiency of vascularization, with an increased host foreign body reaction. 3D-perfusion culture of SVF-cells leads to the engineering of a specialized milieu, here defined as an angiogenic niche. This system could serve as a model to investigate multi-cellular interactions in angiogenesis, and as a module supporting increased grafted cell survival in regenerative medicine.

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.

Beating heart on a chip: a novel microfluidic platform to generate functional 3D cardiac microtissues.

In the past few years, microfluidic-based technology has developed microscale models recapitulating key physical and biological cues typical of the native myocardium. However, the application of controlled physiological uniaxial cyclic strains on a defined three-dimension cellular environment is not yet possible. Two-dimension mechanical stimulation was particularly investigated, neglecting the complex three-dimensional cell-cell and cell-matrix interactions. For this purpose, we developed a heart-on-a-chip platform, which recapitulates the physiologic mechanical environment experienced by cells in the native myocardium. The device includes an array of hanging posts to confine cell-laden gels, and a pneumatic actuation system to induce homogeneous uniaxial cyclic strains to the 3D cell constructs during culture. The device was used to generate mature and highly functional micro-engineered cardiac tissues (µECTs), from both neonatal rat and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), strongly suggesting the robustness of our engineered cardiac micro-niche. Our results demonstrated that the cyclic strain was effectively highly uniaxial and uniformly transferred to cells in culture. As compared to control, stimulated µECTs showed superior cardiac differentiation, as well as electrical and mechanical coupling, owing to a remarkable increase in junction complexes. Mechanical stimulation also promoted early spontaneous synchronous beating and better contractile capability in response to electric pacing. Pacing analyses of hiPSC-CM constructs upon controlled administration of isoprenaline showed further promising applications of our platform in drug discovery, delivery and toxicology fields. The proposed heart-on-a-chip device represents a relevant step forward in the field, providing a standard functional three-dimensional cardiac model to possibly predict signs of hypertrophic changes in cardiac phenotype by mechanical and biochemical co-stimulation.

Three dimensional multi-cellular muscle-like tissue engineering in perfusion-based bioreactors.

Conventional tissue engineering strategies often rely on the use of a single progenitor cell source to engineer in vitro biological models; however, multi-cellular environments can better resemble the complexity of native tissues. Previous described co-culture models used skeletal myoblasts, as parenchymal cell source, and mesenchymal or endothelial cells, as stromal component. Here, we propose instead the use of adipose tissue-derived stromal vascular fraction cells, which include both mesenchymal and endothelial cells, to better resemble the native stroma. Percentage of serum supplementation is one of the crucial parameters to steer skeletal myoblasts toward either proliferation (20%) or differentiation (5%) in two-dimensional culture conditions. On the contrary, three-dimensional (3D) skeletal myoblast culture often simply adopts the serum content used in monolayer, without taking into account the new cell environment. When considering 3D cultures of mm-thick engineered tissues, homogeneous and sufficient oxygen supply is paramount to avoid formation of necrotic cores. Perfusion-based bioreactor culture can significantly improve the oxygen access to the cells, enhancing the viability and the contractility of the engineered tissues. In this study, we first investigated the influence of different serum supplementations on the skeletal myoblast ability to proliferate and differentiate during 3D perfusion-based culture. We tested percentages of serum promoting monolayer skeletal myoblast-proliferation (20%) and differentiation (5%) and suitable for stromal cell culture (10%) with a view to identify the most suitable condition for the subsequent co-culture. The 10% serum medium composition resulted in the highest number of mature myotubes and construct functionality. Co-culture with stromal vascular fraction cells at 10% serum also supported the skeletal myoblast differentiation and maturation, hence providing a functional engineered 3D muscle model that resembles the native multi-cellular environment.

Facile fabrication of egg white macroporous sponges for tissue regeneration.

The availability of 3D sponges combining proper biochemical, biophysical, and biomechanical properties with enhanced capacity of in vivo engraftment and vascularization is crucial in regenerative medicine. A simple process is developed to generate macroporous scaffolds with a well-defined architecture of interconnected pores from chicken egg white (EW), a material with protein- and growth factor-binding features which has not yet been employed in regenerative medicine. The physicomechanical properties and degradation rates of the scaffold are finely tuned by using varying concentrations of the cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, without alteration of the biochemical traits. In vitro, EW scaffolds supported active metabolism, proliferation, and migration of human dermal fibroblasts, thereby generating uniform cellular constructs. In vivo, subcutaneous implantation in mice reveals negligible immune reaction and efficient cell and tissue ingrowth. Angiogenesis into EW scaffolds is enhanced as compared to standard collagen type I sponges used as reference material, likely due to significantly higher adsorption of the proangiogenic factor vascular endothelial growth factor. In summary, a material is presented derived by facile processing of a highly abundant natural product. Due to the efficient subcutaneous engraftment capacity, the sponges can find utilization for soft tissue regeneration.