Modelling, design, fabrication and characterization of engineered human myocardium made with melt electrowriting and cardiac cells derived from hiPSCs

Abstract

The adult human heart has evolved to become a highly specialized organ, whose continuous pumping of blood is critical for survival. However, its ability to regenerate or self-repair following injury is very limited, so consequently any event or disease resulting in damage to the heart poses a serious threat to the patient. Moreover, cardiovascular diseases represent one of the most pressing healthcare concerns nowadays, as they are the leading cause of death worldwide, and the number of cases is only expected to increase in the following years. Despite great progress made over the years to treat cardiovascular diseases, to date there is no therapy able to fully cure a heart that has been damaged. In consequence, there is a dire need to generate new strategies to repair the heart damage and restore the lost cardiac function, as well as to develop accurate modelling platforms to advance in the understanding of disease progression and assess the effectiveness of new drugs. Since its advent, cardiac tissue engineering and regenerative medicine has been regarded as a promising candidate to realise this enormous challenge. Given its interdisciplinary nature, scientific breakthroughs in different areas such as cellular reprogramming, polymer chemistry, and additive manufacturing technologies have resulted in the advancement of cardiac tissue engineering and regenerative medicine over the years. One of such cornerstone discoveries was the generation of induced pluripotent stem cells and subsequent differentiation to different cardiac phenotypes, and the present Thesis revolves around their application to generate patient-specific cardiac disease models and humanised engineered functional cardiac minitissues. Firstly, we reprogrammed peripheral blood mononuclear cells from a transthyretin amyloid cardiomyopathy patient, resulting in the generation of a new cell line carrying a c.128G>A (p.Ser43Asn) mutation in the transthyretin gene. Experiments demonstrated the efficacy and safety of the approach, confirming the pluripotency of the cells, the presence of the disease-causing mutation, and the removal of reprogramming vectors. This cell line, which is now available in a repository, can be used to investigate disease biology, molecular mechanisms and progression; as well as an advanced cellular model to test novel therapeutic strategies. Secondly, we aimed to generate functional human minitissues by combining human cardiomyocytes derived from induced pluripotent stem cells and tridimensional fibrillar scaffolds generated with the technology of melt electrowriting. Compared to conventional two-dimensional cell culture, the cardiac minitissues demonstrated enhanced maturation, with a significant increase in conduction velocity, presence of connexin 43 and expression of cardiac-associated genes such as MYL2, GJA5 and SCN5A, and isoform ratios MYH7/MYH6 and MYL2/MYL7 after 28 days in culture. When investigating the effect of the scaffold fibres on the cells, we found that cardiomyocytes placed close to the fibre were arranged parallel to it, but that alignment was progressively lost towards the centre of the scaffold pore. We then used these data to develop simulations capable of accurately reproducing the experimental performance. In-depth gauging of the structural disposition and intercellular connectivity allowed us to develop an improved computational model able to predict the relationship between cardiac cell alignment and functional performance. This study lays down the path for advancing in the development of in silico tools to predict cardiac biofabricated tissue evolution after generation, and maps the route towards more accurate and biomimetic tissue manufacture. We next aimed at increasing the biological representativity of the cardiac minitissues, by implementing a few changes in cellular (addition of induced pluripotent stem cell-derived cardiac fibroblasts) and hydrogel (substitution of Matrigel for fibrin) composition. We also sought to control cardiomyocyte behaviour based on melt electrowritten scaffold geometry. For this, we hypothesized that diamond-based scaffolds would induce cardiomyocyte contraction in the direction of least mechanical resistance, i.e., the small diagonal of the diamonds. The characterization of the new cardiac minitissues demonstrated functional maturation consistent with the previous work in terms of gene expression and conduction velocity, although the observed low initial cell retention within the scaffold highlighted the need of new strategies to improve cell seeding efficiency. When comparing contractile dynamics between melt electrowritten scaffolds made with square, rectangular, and diamond-shaped pores, we found that the latter resulted in significantly faster, stronger and aligned contraction in the direction that we had anticipated. The potential use of the cardiac minitissues as therapy agents was tested by implanting the constructs in a murine model of chronic myocardial infarction. Compared to controls, implanted animals showed significant improvement, including higher left ventricular ejection fraction and greater wall thickness. Finally, in another attempt to enhance the biological representativity of the constructs, a proof of concept was made to generate melt electrowritten ellipsoidal scaffolds with controlled pore architecture. In summary, the present Thesis revolves around human induced pluripotent stem cells and melt electrowriting as cornerstone tools for cardiac tissue engineering and regenerative medicine efforts. By combining both and iteratively optimising the design and experimental conditions, we were able to generate human functional cardiac minitissues of increased biological relevance.