Periodic Reporting for period 4 - BIOCARD (Deep BIOmodeling of human CARDiogenesis)
Reporting period: 2023-03-01 to 2024-02-29
In the last decades, progress in clinical development has been too slow to adequately confront the public health challenge posed by cardiovascular disease, which is the leading cause of death worldwide. A major difficulty in developing therapies that are both safe and effective lies in the lack of preclinical models precisely recapitulating human heart physiology. Mouse studies have formed the basis of much of our current knowledge of heart development and disease, but many findings cannot be translated to humans due to interspecies differences in organ size, functionality, and genetics. On the other hand, in vitro cell culture models have long been constrained by limited physiological relevance. Human induced pluripotent stem cells (hiPSCs) have emerged as a promising alternative as they can differentiate into any cell type of the body and faithfully replicate patient-specific phenotypes. However, although protocols exist to differentiate hiPSCs into cardiac cell types such as cardiomyocytes and vessel components, it remains challenging to control the generation of specific cardiac progenitor cells (CPCs), which not only serve as a valuable cell source for tissue engineering and regenerative medicine but also hold the key to understanding the mechanisms of many complex heart disorders. Another coveted goal is to use hiPSCs to generate complex 3D tissue mimics and self-organizing heart organoids, which have the potential to reduce the need for animal models in various preclinical applications.
To tackle these challenges, the BIOCARD project pursued three main objectives:
1) Enhance understanding of human CPC specification and differentiation mechanisms,
2) Investigate the role of human CPCs in different compartments of the heart through chimeric animal models, and
3) Develop advanced models of cardiac development and disease in the form of hiPSC-derived cardiac organoids and 3D engineered heart tissue.
By providing high-resolution analyses of human cardiogenesis, innovative platforms for investigating the fate and function of CPCs in vitro and ex utero, mechanistic insights into hypoplastic left heart syndrome (HLHS), and the first cardiac organoid imitating the molecular and functional patterning of the ventricular wall, it succeeded in breaking new ground in precise modeling of human heart development and disease.
To tackle these challenges, the BIOCARD project pursued three main objectives:
1) Enhance understanding of human CPC specification and differentiation mechanisms,
2) Investigate the role of human CPCs in different compartments of the heart through chimeric animal models, and
3) Develop advanced models of cardiac development and disease in the form of hiPSC-derived cardiac organoids and 3D engineered heart tissue.
By providing high-resolution analyses of human cardiogenesis, innovative platforms for investigating the fate and function of CPCs in vitro and ex utero, mechanistic insights into hypoplastic left heart syndrome (HLHS), and the first cardiac organoid imitating the molecular and functional patterning of the ventricular wall, it succeeded in breaking new ground in precise modeling of human heart development and disease.
In Objective 1, we established new protocols for the differentiation of first heart field (FHF) and second heart field (SHF) CPCs, which allowed us to perform an extensive molecular and functional characterization of CPC (sub)populations at the single-cell level. Using a newly established ex utero culture system, we followed the fate of human CPCs injected into mouse embryos at the cardiac crescent stage, confirming their lineage-specific contribution to developing heart structures. We also identified distinct transcriptional dysregulation in FHF and SHF progenitors derived from HLHS patient hiPSCs (Zawada et al. 2023 Nat. Commun). In parallel, we genetically engineered a reporter hiPSC line suitable for diverse lineage tracing applications and a voltage sensor hiPSC line enabling functional characterization of cardiomyocytes in 2D and 3D (Goedel et al. J Vis Exp. 2018, Zhang et al. Stem Cell Res 2022, Zhang et al. Front. Cell Dev. Biol. 2022). We further characterize the potential of human CPCs for heart regeneration (Poch et al. Nat. Cell Biol. 2022).
In Objective 2, to facilitate the use of pigs in preclinical research, we performed an extensive anatomical and molecular characterization of porcine cardiogenesis based on native fetal hearts and cardiac cells derived from porcine expanded pluripotent stem cells (Rawat et al. Front. Cell Dev. Biol. 2023). We then generated porcine somatic cell donors lacking key cardiac regulators for the future generation of cardiogenesis-disabled porcine embryos through somatic cell nuclear transfer. Moreover, we successfully established CRISPR/Cas9 for somatic gene editing of pig hearts in vivo (Moretti et al. Nat. Med. 2020).
In Objective 3, we optimized the generation of 3D engineered myocardium based on native extracellular matrix (ECM) and used it to investigate HLHS phenotypes, demonstrating a significantly reduced contractile force and a failure of electromechanical maturation in constructs from patient hiPSCs compared to healthy controls (Krane et al. Circulation 2021, Lu et al. Theranostics 2021). Moreover, we successfully established the first self-organized cardiac organoid showing co-development of the myocardium and epicardium and imitating the molecular and functional patterning of the ventricular wall, which we called epicardioids (Meier et al. Nat. Biotechnol. 2023). We notably used epicardioids to gain insights into the fate trajectories of the human epicardial lineage and demonstrated their unique capacity to recapitulate congenital and stress-induced hypertrophy and fibrosis.
In Objective 2, to facilitate the use of pigs in preclinical research, we performed an extensive anatomical and molecular characterization of porcine cardiogenesis based on native fetal hearts and cardiac cells derived from porcine expanded pluripotent stem cells (Rawat et al. Front. Cell Dev. Biol. 2023). We then generated porcine somatic cell donors lacking key cardiac regulators for the future generation of cardiogenesis-disabled porcine embryos through somatic cell nuclear transfer. Moreover, we successfully established CRISPR/Cas9 for somatic gene editing of pig hearts in vivo (Moretti et al. Nat. Med. 2020).
In Objective 3, we optimized the generation of 3D engineered myocardium based on native extracellular matrix (ECM) and used it to investigate HLHS phenotypes, demonstrating a significantly reduced contractile force and a failure of electromechanical maturation in constructs from patient hiPSCs compared to healthy controls (Krane et al. Circulation 2021, Lu et al. Theranostics 2021). Moreover, we successfully established the first self-organized cardiac organoid showing co-development of the myocardium and epicardium and imitating the molecular and functional patterning of the ventricular wall, which we called epicardioids (Meier et al. Nat. Biotechnol. 2023). We notably used epicardioids to gain insights into the fate trajectories of the human epicardial lineage and demonstrated their unique capacity to recapitulate congenital and stress-induced hypertrophy and fibrosis.
The novel in vitro and ex vivo model systems we have established, together with advanced techniques in gene editing and molecular analysis, allowed us to reach an unprecedented level of precision in modeling human cardiogenesis, from CPC specification to tissue-level morphogenesis and functional maturation processes. This was demonstrated by the novel insights gained by transitioning from 2D to 3D hiPSC-based modeling of HLHS, which provided the electromechanical environment needed to reveal the impaired maturation and cell cycle defects contributing to left ventricular hypoplasia, arguing against a solely hemodynamic origin of the defect (Krane et al. Circulation 2021). Another notable achievement was the generation of epicardioids, which are the first cardiac organoid recapitulating the formation of the epicardium, the outer layer of the heart (Meier et al. Nat. Biotechnol. 2023). Although the epicardium is quiescent in the healthy adult heart, it plays key roles during embryonic development and has been shown to orchestrate tissue repair in fetal mammals and in species capable of full heart regeneration throughout adulthood, such as zebrafish. Epicardioids therefore offer exciting new opportunities to study human epicardial development, which occurs too early in pregnancy to observe in native tissues, and to explore the epicardium’s potential for heart regeneration after an injury such as a myocardial infarction. Through lineage tracing in epicardioids, we could already demonstrate that human epicardial cells have the potential to give rise to cardiomyocytes, a long-standing open question in the field. We were also the first to identify the human equivalent of murine juxta-cardiac field (JCF) cells and to verify their dual potential for the myocytic and epicardial lineages, both in 2D and in epicardioids (Zawada et al. Nat. Commun 2023). Finally, although technical hurdles slowed down our progress in the generation of pig-human chimeras, we are confident that our characterization of porcine cardiogenesis and our efforts in generating cardiogenesis-disabled pig embryos not only served to advance cardiac modeling but also open new possibilities of testing interspecies cell complementation approaches ex vivo, which should facilitate the generation of chimeric tissues for xenotransplantation, an ambitious goal in regenerative medicine.