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Deep BIOmodeling of human CARDiogenesis

Periodic Reporting for period 2 - BIOCARD (Deep BIOmodeling of human CARDiogenesis)

Reporting period: 2020-03-01 to 2021-08-31

Cardiovascular disease has been the leading cause of death and a major cause of disability worldwide for the last 20 years, yet the development of new cardiac therapies has remained very limited compared to other clinical areas. This is explained in part by a lack of precise preclinical models of human heart physiology and disease.
A highly promising tool for the development of human cardiac models are human pluripotent stem cells, which have the ability to self-renew indefinitely and to give rise to any cell type of the body. Importantly, so-called induced pluripotent stem cells (iPSCs) can be produced from somatic cells of healthy individuals or patients, circumventing the need for embryonic stem cells and allowing the study of disease mechanisms in defined genetic contexts. Although protocols have been designed to differentiate human iPSCs (hiPSCs) into cardiac cell types such as cardiomyocytes and vessel components, it remains a challenge to precisely control the generation of their precursors, cardiac progenitor cells (CPCs). In addition to being a powerful cell source for tissue engineering and regenerative medicine, CPCs hold the potential to model key aspects of human cardiogenesis by self-organizing into complex 3D structures called organoids, but this has not yet been achieved.
The objectives of BIOCARD are to 1) Gain a deeper understanding of human CPC specification and differentiation using genetically engineered hiPSC reporter lines, 2) Study the contribution of human CPCs to various compartments of the heart in chimeric pig models and 3) Develop advanced models of cardiac development and disease in the form of hiPSC-derived cardiac organoids and 3D engineered heart tissue.
The human heart is composed of highly specialized and diverse cell types arising from three main CPC populations – the first heart field (FHF), second heart field (SHF) and proepicardial organ (PEO). We are currently generating reporter hiPSC lines which, combined with appropriate differentiation protocols we have now optimized, will allow us to closely follow the specification of FHF, SHF and PEO CPCs and their differentiation into the cardiac lineages in vitro.
Working towards the generation of chimeric pig models of human cardiogenesis, we have established ex vivo embryo culture platforms that enable appropriate development of mouse and pig embryos from the late bud until the heart looping stage. We could already validate this system by demonstrating that FHF and SHF human CPCs injected in the early cardiac crescent specifically allocate at and contribute to distinct heart compartments.
In parallel, we were able to utilize our understanding of the key signaling pathways controlling human cardiac development to generate novel hiPSC-derived cardiac organoids containing all cardiovascular cell types and showing a significantly higher degree of self-organization than previously reported.
We have also optimized the generation of hiPSC-derived 3D engineered heart tissue based on decellularized ventricular scaffolds and its cultivation in a biomimetic chamber system promoting tissue maturation by continuous mechanical and electrical stimulation. This allowed us to uncover a failure of electromechanical maturation and abnormal cell-cycle progression as underlying mechanisms of hypoplastic left heart syndrome.
Finally, in the perspective of precise modeling of genetically inherited diseases, we have expanded our gene editing expertise by establishing novel methods of gene correction, both in vitro in hiPSCs and their derivatives and in vivo in live pigs.
The novel in vitro and ex vivo model systems we have established, together with advanced techniques in gene editing and molecular analysis, allow us to reach an unprecedented level of precision in modeling human cardiogenesis, from CPC specification to tissue-level morphogenesis and functional maturation processes.
After reaching the envisioned milestones of BIOCARD, we will have provided the cardiovascular community with advanced platforms and multidisciplinary frameworks for disease modeling, drug discovery, and regenerative cell therapy suitable for a wide range of cardiac disorders.