Modelling human cardiac diseases with 3D organoid
Bramasta Nugraha, Michele F. Buono, Maximilian Y. Emmert · European Heart Journal · 2018
The development of 3D cultures to resemble cardiac function and physiology in vitro Drug development for the treatment of human diseases is hampered by the limited availability of clinically relevant tissue samples, robust and translational animal models, and reliable in vitro cultures that are able to represent the complex in vivo (patho)physiological conditions.1 In fact, these challenges are significantly slowing down the progress of drug development and clinical translation (from bench to bedside) and has even led to a complete cessation of numerous clinical trials at the latter phase of evaluation.2 Notably, a large percentage of new drug clinical trials failed due to hepatotoxicity, cardiotoxicity, and/or neurotoxicity.3 Therefore, to address these problems and to increase the testing robustness, safety, and efficacy in drug trials, advanced cellular technology platforms are needed with a particular focus to enhance the mimicry of human organ-specific physiological function in vitro in a highly predictive manner.4 In this regard, just recently, so called three dimensional (3D) organoid cell cultures have evolved into an era where large mimicry of native-like tissue structures and functions can be achieved. By the help of such 3D organoids, such human-like tissues can be efficiently recreated in vitro with the ability to closely resemble physiological and also pathophysiological conditions.4 Ideally, organoids can be generated from pluripotent stem or progenitor cells, which can then self-assemble and -organize into complex native-like organ structures covering the entire range of physiological organ functions, but also pathophysiological conditions mimicking organ diseases. Since its first introduction in 2009, where intestinal microvilli structures were recreated in vitro,5 tremendous progress has been made in this novel field. Besides major advances and refinement in the methodology, the range of tissues that can be produced has been continuously expanded. To date, numerous organ-specific organoids mimicking liver,6 brain,7 prostate,8 small intestine,9 and even tumour10 can be generated in vitro providing the unique opportunity to study a vast range of scientific and clinical challenges in the area of developmental biology, disease modelling, drug development, and precision regenerative and personalized medicine. Various cell types reside within a human heart (cardiomyocytes, cardiac fibroblasts, progenitor cells, vascular cells, and others) and their orchestration and homeostasis is well known to be complex. Hence, native-like resemblance of human heart by organoid technologies has been demonstrated to be a particular challenge and therefore an in-depth understanding of cardiac structure and physiology is mandatory to develop the optimal cardiac organoid.11 Of note, the human heart consists of various cardiomyocyte subpopulations such as sinoatrial nodal cells, atrioventricular cells, atrial cells, ventricular cells, and Purkinje cells.12 These different cell populations are controlled by transcriptional differences during cellular differentiation and each of these cell types can be associated to different diseases. To date, several attempts have been successfully accomplished to create human cardiac organoids. However, so far, such organoids have been mainly generated from cardiomyocytes as the main cell-type,13 and major challenges remain in the future development of cardiac organoids. These include on the one hand the identification of the ideal, age-specific ratio of the various cardiomyocyte subtypes, but also the optimal orchestration with other cardiac cell types. In this context, the use of pluripotent stem cell-derived cardiomyocytes can significantly accelerate the discovery of cardiac drugs and improve drug safety by offering more clinically relevant cell-based models than those currently available. They express ion channels and demonstrate beating and action potentials similar to primary cardiac cells. Figure 1 below depicts an overview where cardiac organoid research utilizes tissue engineering concepts to recreate cardiac structure, function, and physiology in three dimensions. Main pillars of tissue engineering strategies to build mimicry of human myocardium in vitro. Left panel depicts 3D system where human induced pluripotent stem cell-derived cells are embedded in hydrogel. Middle panel shows decellularized native tissue being used as scaffold material to culture human induced pluripotent stem cell-derived cells. And right panel exhibits the utility of 3D printing technology to precisely create mini hearts in vitro with combination of cells and bioinks. Credit to A. Kitterman/Science Translational Medicine. Reprinted with permission from AAAS.12 The rapidly evolving field of human induced pluripotent stem cells provides the unique opportunity to derive cell lines from different cardiac patients and culture them in vitro as cardiac organoids, thus enabling to study patient- and disease-specific conditions in vitro. By obtaining patients’ blood samples or skin biopsies, standard cellular reprogramming can be performed to obtain induced pluripotent stem cells. This approach has been much favoured due to less ethical concerns compared to obtaining embryonic stem cells. The two main global pluripotent stem cell banks such as WiCell (Madison, USA)14 and European Bank for induced Pluripotent Stem Cell (EBiSC)15 hold vast human induced pluripotent stem cell (hiPSCs) collections of both; from healthy patients with a wide age range, different race, and gender, as well as from patients suffering from different diseases or genetic mutations. In particular, in regard to heart diseases, hiPSCs have been collected from patients suffering from Duchenne Muscular Dystrophy (DMD), familial long QT syndrome, prolonged QT interval, arrhythmias, hypertrophy, myocardial infarction, and cardiomyopathy. In addition to these various patient- and disease-specific cell lines, the breakthrough and advancement of the ‘clean’, reduced framed shift mutation genomic editing technology, the so called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) enables on the one hand the creation of hiPSC lines with a desired specific gene mutation and on the other hand, the ability to repair a known gene mutation, thereby potentially reversing the diseased cell into normal physiological function. Therefore, by combining the hiPSC and genomic editing technologies, cardiac organoids can be precisely generated and tailored to achieve specific therapeutic effects including the correction of mutations causing disease and pathological conditions as well as the general removal of deleterious genes from the genome. Two unique treatment opportunities that were completely unimaginable a decade ago. Human heart diseases that would strongly benefit from in vitro modelling using cardiac organoids can be divided into non-genetic and genetic diseases. Non-genetic diseases are not triggered by the presence of an inherent genomic mutation within the cardiomyocytes but are mainly driven by environmental factors, organ toxicity, diets, and aging. Acute myocardial infarction (AMI) represents a prominent example of a non-genetic heart disease in which cardiomyocytes are dying due to ischaemia driven by compromised blood flow due to the complete or partial occlusion of a coronary vessel. In a recent study, Voges and colleagues demonstrated that the condition of AMI could be successfully modelled by help of human cardiac organoids that were exposed to local tissue damage using cryoinjury, while the adjacent cells remained viable, and tissue regeneration was seen over a period of 2 weeks16 (Figure 2, left panel). Human Cardiac Organoid for in vitro Disease Modelling. Left panel: cryoinjury and monitoring regeneration capacity in human cardiac organoids (hCOs). (A) (i) Living human cardiac organoids on mechanical pole. (ii) Green fluorescent protein (GFP) imaging of living cardiomyocytes in uninjured human cardiac organoids control. (iii) GFP imaging of living cardiomyocytes in cryoinjured human cardiac organoids; arrow indicates loss of cardiomyocytes. Scale bars: 2 mm. (B) uninjured human cardiac organoids stained with α-actinin and Hoechst33342. (C) Cryoinjured human cardiac organoids on Day 3 post-cryoinjury; arrow indicates site of injury. Scale bars: 1 mm. GFP images of (D) uninjured human cardiac organoids and (E) cryoinjured human cardiac organoids on Days 0, 3, and 14 post-cryoinjury in culture. On Day 14, there is an indication of effective regeneration, shown by indistinguishable GFP intensity levels from control uninjured human cardiac organoids. Right panel: rescued Duchenne Muscular Dystrophy cardiomyocyte-derived organoid showed enhanced force of contraction. (A) Setup design to create engineered tissue organoid and contractile function analysis. (B–D) Contractile dysfunction in organoid-containing can be rescued by myoediting. Force of contraction normalized to muscle content of each individual organoid in response to increasing extracellular calcium concentrations; n = 8/8/6/4/6/6/4/4; *P < 0.05 by two-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. (E) Maximal cardiomyocyte force of contraction (at 4 mM extracellular calcium) normalized to wild yype (WT). n = 8/8/6/4/6/6/4/4; *P < 0.05 by one-way analysis of variance and Tukey’s multiple comparison test. (F) Titration of corrected cardiomyocytes revealed that 30% of cardiomyocytes needed to be repaired to partially rescue the phenotype, and 50% of cardiomyocytes needed to be repaired to fully rescue the phenotype (100% Del-Cor.) in organoids. Adapted from reference (16) and from Long et al., Sci. Adv. 2018;4:eaap9004. © The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. Distributed under a Creative Commons Attribution Non-Commercial License 4.0 (CC BY-NC) http://creativecommons.org/licenses/by-nc/4.0/.17 On the contrary, genetic diseases are caused by the presence of cardiomyocytes with inherent genomic mutations, leading to abnormal and pathological function resulting in cardiac diseases (i.e. cardiomyopathies). By combining the relevant cells in 3D engineered tissue culture models, to date, several human cardiac genetic diseases could be successfully modelled in vitro including dilated cardiomyopathy (DCM),18 hypertrophic cardiomyopathy,19 Barth syndrome (mitochondrial cardiomyopathy),20 left ventricular hypertrophy-associated with glycogen accumulation,21 and cardiomyopathy-associated with DMD.17 For instance, in DMD, 3000 different mutations in the X-linked dystrophin gene are responsible for the associated lethal degeneration of cardiac and skeletal muscle.17 These mutations are clustered in specific hotspot areas of the dystrophin gene (exons 45 to 55 and exon 2 to 10), thus making genomic correction very challenging. However, in their recent study, Long et al. showed the possibility to correct DMD to normal by help of genomic editing, just by having only 50% of cardiomyocytes being corrected. Importantly, this correction was successfully monitored through analysis of force contraction of cardiac organoids highlighting the usefulness of this technology17 (Figure 2, right panel). The continuously growing field of organoid models to closely resemble human tissues has brought significant advancement in the developmental process of novel and innovative therapies. Indeed, the organoid technology acts as a new biological tool to study tissue regeneration and to model disease states and to capture disease progression which cannot be achieved through standard animal models or human testing. However, these advancements are not without some challenges and the need for further in-depth research: for instance, firstly, the role of maturity of cardiomyocytes derived from pluripotent stem cells and the cells being used for co-culture (i.e. cardiac fibroblast or cardiac endothelial cell) to build cardiac organoids needs to be further elucidated. One should be careful in terms of maturity stage of the cardiomyocytes being used as the cell model, since this plays a role in drug predictability, toxicity, metabolic assay response, apoptosis sensitivity, and regenerative capability. In differentiating human pluripotent stem cells, maturation external factors have to be carefully implemented. Secondly, reproducing organoid production in terms of size, shape, architecture, cellular composition, and production scalability have to be taken care of as important factors. And lastly, another challenge that remains a hassle, is how to perform robust organoid imaging, overcoming the imaging depth, and imaging dye diffusion barrier towards organoid core part. However, these challenges are being progressively overcome with the growing imaging technology with higher throughout and deeper imaging capability, improvement in the development of clearing agents, creation of stable fluorescent reporter lines, and vascularization of cardiac organoids. Overall, the careful development of human cardiac organoids will speed up the cardiac drug discovery and personalized cardiac treatment. 1Institute for Regenerative Medicine (IREM), University of Zurich, Wagistrasse 12, 8952 Schlieren, Switzerland 2Department of Cardiovascular Surgery, Charité Universitätsmedizin Berlin, Berlin, Germany 3Department of Cardiothoracic and Vascular Surgery, German Heart Center Berlin, Berlin, Germany 4Wyss Translational Center Zurich, Moussonstrasse 13, 8044 Zurich, Switzerland Conflict of interest: none declared. References are available as supplementary material at European Heart Journal online.