A heart organoid

Abstract

The invention relates to organoids, and particularly, although not exclusively, to heart organoids. The invention extends to novel methods for producing heart organoids, to their use in in vitro and ex vivo assays, in screening methods, particularly, although not exclusively, during the evaluation of the efficacy, cardiotoxicity, and teratogenicity of pharmaceuticals, as well as to apparatus for use in such screening. In addition, the invention extends to the use of the heart organoids in the study of heart disease and / or pathology.

Description

[0001] A HEART ORGANOID

[0002] The present invention relates to organoids, and particularly, although not exclusively, to heart organoids. The invention extends to novel methods for producing heart organoids, to their use in in vitro and ex vivo assays, in screening methods, particularly, although not exclusively, during the evaluation of the efficacy, cardiotoxicity, and teratogenicity of pharmaceuticals, as well as to apparatus for use in such screening. In addition, the invention extends to the use of the heart organoids in the study of heart disease and / or pathology.

[0003] Cardiogenesis, the formation of new heart tissue, is a highly complex and tightly regulated process, which involves a sequence of differentiation and proliferation events, and requires the physical interaction and paracrine signals from surrounding tissues. For example, endoderm and posterior foregut / hepatic-derived paracrine signals are crucial stimuli for normal heart development. In addition, sympathetic neurons (SN) and parasympathetic neurons (PSN), which compose the autonomic nervous system (ANS) of the heart, are ectoderm-derived cells, which are required to regulate cardiac muscle contraction. SNs and PSNs innervate the sinoatrial cells of the heart, regulating the frequency of heart contraction, whilst the innervation of the working myocardium of the ventricle wall is only ensured by SN, forming the so-called ganglionated plexuses which are located at the epicardial surface. This system regulates physiological cardiac function, suggesting that alterations in excitability, density, distribution, and neurotransmitter content of cardiac sympathetic fibres may cause arrhythmic events, such as those that occur after myocardial infarction (MI).

[0004] Although the role of the ANS in normal heart function is known, the molecular mechanisms involved in the establishment of ventricle myocardium innervation are still poorly understood. It has been shown that sympathetic innervation of myocardium develops in close crosstalk with the coronary vasculature [2]. In addition, epicardium- derived paracrine signals and epicardium-derived cells (EPDCs) are responsible for the establishment of the coronary vasculature of the heart [1]. In particular, neurotrophic factors secreted by epicardium-derived vascular cells have been described to guide the extension of axons through the subepicardium, inducing invasion into the myocardium [2], and promote neurite outgrowth and increased density [3].

[0005] A heart organoid model that incorporates the ANS would open the path to study cardiomyopathies and heart pathologies that may be linked to an unbalanced, degenerate, or absence of myocardium innervation, all of which are registered in cardiac arrhythmias. Cardiac arrhythmias of particular note are atrial fibrillation and ventricular arrhythmias, such as in the case of cardiac sympathetic nerve loss, which is observed in neurodegenerative diseases and familial dysautonomia pathology.

[0006] However, there is a lack of human heart organoid models that recreate, in a physiologically relevant manner, the establishment of heart ventricle innervation. There exist a small number of reported studies which describe simple co-culture strategies between primary ventricular cardiac myocytes (CMs) or human pluripotent stem cell (hPSCs)-derived CMs and SNs and PSNs in two-dimensional monolayer culture systems [4-12]. However, none of these models provide a developmental and spatiotemporal perspective of the heart innervation process according to the in situ developmental signalling blueprint. In particular, these models often lack physiological relevance, due to the absence of three-dimensional tissue architecture and dynamic mechanical cues which are present in the native cardiac microenvironment. In addition, the electrical and mechanical coupling between cardiac cells and neurons is not adequately replicated in these existing models, thus limiting the ability to study functional interactions between the cardiac and nervous systems. Furthermore, the absence of long-term culture systems hinders the investigation of maturation and aging processes, which are crucial for understanding the dynamic nature of cardiac innervation over time.

[0007] There is, therefore, a need for a heart organoid that incorporates the ANS, and thus, is innervated, and that better represents the vasculature and tissue composition of the heart.

[0008] In order to address the above problems, the inventors explored various methods to produce a human heart organoid that is innervated, and ideally which incorporates the ANS. As a result of their extensive experimentation, the inventors have surprisingly produced an innervated human heart organoid, and have discovered a methodology that enables the production of innervated heart organoids in a highly reproducible manner.

[0009] As discussed, myocardial innervation develops in coordination with coronary vasculature, guided by neurotrophic signals from epicardium-derived vascular cells.

[0010] Surprisingly, the inventors have also produced a heart organoid that is vascularised, specifically a heart organoid comprising coronary vascularisation.

[0011] Furthermore, hearts contain adipose tissue; normally, this adipose tissue is found as epicardial adipose tissue or pericardial fat. Epicardial adipose plays critical roles in energy storage, mechanical protection, and secretion of bioactive molecules that can influence heart function.

[0012] Unexpectedly, the inventors have also produced a heart organoid that comprises adipose tissue.

[0013] The reproducibility, innervation, vascularisation, tissue composition, functionality, and thus the consequent applicability of the produced organoids of the invention, is improved over existing models.

[0014] Thus, according to a first aspect of the invention, there is provided a heart organoid that: i) is innervated; ii) is vascularised; and / or iii) comprises adipose tissue.

[0015] In one embodiment, the heart organoid comprises an innervated heart organoid.

[0016] Thus, according to an aspect of the invention, there is provided an innervated heart organoid.

[0017] In another aspect, there is provided a vascularised heart organoid.

[0018] In another aspect, there is provided a heart organoid comprising adipose tissue.

[0019] In one embodiment, the heart organoid of the invention is innervated and vascularised.

[0020] In one embodiment, the heart organoid of the invention is innervated and comprises adipose tissue. In one embodiment, the heart organoid of the invention is vascularised and comprises adipose tissue. Typically, the heart organoid of the invention is innervated, vascularised, and comprises adipose tissue.

[0021] In one embodiment, the heart organoid of the first aspect (which is typically innervated) may comprise a percentage of its surface area covered by sympathetic neurons (SNs).

[0022] In one embodiment, the heart organoid may comprise at least 0.0001%, 0.001%, 0.01%, 0.1%, or 1% of its surface area covered by sympathetic neurons (SNs). In some embodiments, the heart organoid may comprise at least 5%, 10%, 20%, 30%, 40%, or 50% of its surface area covered by sympathetic neurons. In some embodiments, the heart organoid may comprise at least 60%, 70%, 80%, 90%, or 99% of its surface area covered by sympathetic neurons.

[0023] In one embodiment, the heart organoid may comprise a percentage of its surface area covered by parasympathetic neurons (PSNs). In some embodiments, the heart organoid may comprise at least 0.0001%, 0.001%, 0.01%, 0.1%, or 1% of its surface area covered by parasympathetic neurons (PSNs). In some embodiments, the heart organoid may comprise at least 5%, 10%, 20%, 30%, 40%, or 50% of its surface area covered by parasympathetic neurons. In some embodiments, the heart organoid may comprise at least 60%, 70%, 80%, 90%, or 99% of its surface area covered by parasympathetic neurons.

[0024] As such, typically, the heart organoid may comprise a model autonomic nervous system (ANS).

[0025] It will be appreciated that the ANS of the heart is composed of sympathetic neurons (SN) and parasympathetic neurons (PSN). As such, it will be appreciated that the term "model ANS" can mean a model comprising SNs and / or PSNs.

[0026] In one embodiment, the heart organoid may comprise an autonomic nervous system spheroid (ANSS).

[0027] It will be appreciated that an autonomic nervous system spheroid (ANSS) is a spheroid comprising both sympathetic neurons (SNs) and parasympathetic neurons (PSNs). It will also be appreciated that an ANSS can be produced by the fusion of one sympathetic neuron spheroid (SNS) with one parasympathetic neuron spheroid (PSNS), or by mixing SNS- and PSNS- progenitors, thus forming a single spheroid (i.e., an ANSS). An embodiment of the organoid of the invention, comprising an ANSS, is shown in Figure 8.

[0028] In one embodiment, therefore, the ANSS may comprise sympathetic neurons (SNs) and parasympathetic neurons (PSNs).

[0029] Advantageously, the methodology enabling the development of the heart organoid described herein has been shown to recreate, in a physiologically relevant manner, the sequential stages of heart embryogenesis, culminating with a final stage of ventricle myocardium-like tissue innervation. In addition, the organoids of the invention are, favourably, three-dimensional, thus further improving the modelling of the heart. In one embodiment, therefore, the heart organoid may be three-dimensional.

[0030] It will be appreciated that the term "three-dimensional" can mean that the organoid comprises height, width, and depth, and is not limited to a monolayer or single-cell thickness.

[0031] Typically, therefore, the heart organoid may comprise ventricle myocardium-epicardium- like tissue innervation.

[0032] It will be appreciated that the myocardium is the middle, muscular, layer of the heart, positioned between the innermost endocardium layer and the outermost epicardium layer. In addition, the myocardium comprises cardiomyocytes arranged in a layer, and as such, the myocardium is the contraction unit of the heart. It will also be appreciated, therefore, that ventricle myocardium-like tissue can mean tissue comprising cardiomyocytes, which may be arranged in a thick layer, similar to that of an in vivo ventricle.

[0033] Advantageously, the heart organoid of the invention recreates the ventricle wall of the heart. The organoid comprises the outer layer of the wall, the epicardium, that surrounds a myocardium region. At an interface of myocardium and epicardium, the organoid comprises a vascular plexus that resembles the coronary vasculature of the heart. The organoid also comprises SNs located in the epicardial region of the organoid. The myocardium in vivo is divided into a compact region, i.e., compact myocardium, which faces the epicardium, and a trabecular region, i.e., trabecular myocardium, which faces the endocardium. It will be appreciated that the term "divided" can mean more than one distinct region. Each region may be defined by its appearance and immunostaining for region specific cell markers. Accordingly, the myocardium of the organoid is divided into a compact region, which faces the epicardium, and a trabecular region, which faces the endocardium.

[0034] In one embodiment, therefore, the heart organoid of the first aspect (which is typically innervated) may comprise epicardium. In one embodiment, the epicardium may be mesothelial epicardium. The epicardium of the organoid may be defined as the epicardial region. In one embodiment, therefore, the heart organoid may comprise an epicardial region. In another embodiment, the heart organoid may comprise myocardium. The myocardium of the organoid may be defined as the myocardial region. In one embodiment, therefore, the heart organoid may comprise a myocardial region. The epicardium may surround the myocardium region of the organoid. In one embodiment, the heart organoid may comprise a vascular plexus. In one embodiment, the vascular plexus may be located at the interface of the myocardium and the epicardium of the organoid. In another embodiment, the vascular plexus may be located within the myocardium and / or epicardium of the organoid. In one embodiment, the vascular plexus may be a coronary vascular plexus.

[0035] In another embodiment, the heart organoid may comprise at least one SN located at the interface of the myocardium and the epicardium of the organoid. In another embodiment, the heart organoid may comprise at least one SN located within the myocardium and / or epicardium of the organoid.

[0036] In one embodiment, therefore, the heart organoid may comprise myocardium which is divided. In one embodiment, the innervated heart organoid may comprise a compact myocardium. The compact myocardium may face the epicardium. In another embodiment, the heart organoid may comprise a trabecular myocardium. The trabecular myocardium may face the endocardium.

[0037] In particular, the innovative model includes, firstly, the production of a ventricle myocardium organoid that incorporates an epicardium-like layer at the outer surface, followed by a step of coronary-like vascular plexus induction. The epicardium-like layer surrounds the entire surface area of the ventricle myocardium-like region, mimicking the in vivo structure of the ventricle wall.

[0038] It will be appreciated that the epicardium is the outermost layer of the heart, and adheres to the myocardium. It will also be appreciated that the epicardium is predominantly comprised of mesothelial cells, adipocytes, and connective tissue.

[0039] It will also be appreciated that, in vivo, the early coronary vascular plexus is located just beneath the epicardium, and subsequently invades the compact myocardium to form coronary arteries, or remain on the surface to produce veins.

[0040] It will also be appreciated that the epicardium comprises an external mesothelium layer and a subepicardial layer facing the compact myocardium. During embryonic development, a subset of epicardial cells undergoes epithelial-to-mesenchymal transition (EMT) (epicardial-derived cells - EPDCs), which migrate into the subepicardial layer, and progressively invade the developing compact myocardium, giving rise to most of the fibroblasts and mural cells, such as vascular smooth muscle cells and pericytes, present in the heart.

[0041] It will be appreciated that the "subepicardial space" referred to herein may also be referred to as the "subepicardium".

[0042] Referring to Figure 8, the heart organoid of the invention may comprise an autonomic nervous system spheroid (ANSS), and an epicardium-myocardium organoid (EMO) comprising, from the outermost layer to the innermost layer, the mesothelial epicardium comprising innervation, the subepicardial space comprising innervation, coronary vascularisation (endothelial cells and pericytes), and adipose tissue, the compact myocardium comprising coronary vascularisation (endothelial cells and pericytes), innervation, and fibroblasts and smooth muscle cells, and the trabecular myocardium.

[0043] In one embodiment, therefore, the heart organoid may comprise mesothelial epicardium. In one embodiment, the mesothelial epicardium may be innervated. In one embodiment, the heart organoid may comprise a subepicardial space. In one embodiment, the subepicardial space may be innervated. In one embodiment, the subepicardial space may comprise coronary vascularisation. In one embodiment, the subepicardial space may comprise endothelial cells and / or pericytes. In one embodiment, the subepicardial space may comprise adipose tissue. In one embodiment, the heart organoid may comprise compact myocardium. In one embodiment, the compact myocardium may comprise coronary vascularisation. In one embodiment, the compact myocardium may comprise endothelial cells and / or pericytes. In one embodiment, the compact myocardium may comprise fibroblasts and / or smooth muscle cells. In one embodiment, the compact myocardium may comprise innervation. In one embodiment, the heart organoid may comprise trabecular myocardium.

[0044] It will be appreciated that the heart organoid may comprise an inner myocardial region and an outer region.

[0045] It will be appreciated that the regions of compact myocardium and trabecular myocardium may be referred to as an inner myocardial region of the heart organoid. It will also be appreciated that compact myocardium may comprise densely packed CTNT+ myocardial cells (i.e., densely packed cells with minimal extracellular space between them), and that trabecular myocardium may comprise loosely packed CTNT+ myocardial cells (i.e., loosely packed cells with abundant intercellular space between them). It will also be appreciated that the inner myocardial region forms the innermost core of the organoid.

[0046] In one embodiment, therefore, the heart organoid may comprise an inner myocardial region. In one embodiment, the inner myocardial region may comprise compact myocardium and trabecular myocardium. In one embodiment, the compact myocardium forms an outermost layer of the inner myocardial region. In one embodiment, the trabecular myocardium forms an innermost layer of the inner myocardial region.

[0047] In one embodiment, the cell density in the compact myocardium may comprise between 1 x 106cells / mm3and 2 x 106cells / mm3.

[0048] In one embodiment, the cell density in the trabecular myocardium may comprise between, 0.1 x 106cells / mm3and 0.9 x 106cells / mm3.

[0049] In one embodiment, the inner myocardial region comprises the innermost region of the heart organoid.

[0050] In one embodiment, the compact myocardium may comprise a higher proportion of Ki67+ proliferating cardiomyocytes than the trabecular myocardium. In one embodiment, the compact myocardium may comprise a higher proportion of NG2, VIM, and / or extra cellular matrix proteins than the inner trabecular-like layer of myocardium. The extracellular matrix proteins may comprise collagen I, fibronectin, and laminin (fibroblasts).

[0051] It will be appreciated that the regions of epicardium and subepicardial space may be referred to as an outer region of the heart organoid. It will also be appreciated that, as discussed above, the epicardium may be innervated, and that the subepicardial space may be innervated and / or may comprise coronary vascularisation.

[0052] In one embodiment, therefore, the heart organoid may comprise an outer region. In one embodiment, the outer region may comprise epicardium and subepicardial space. In one embodiment, the epicardium forms an outermost layer of the outer region. In one embodiment, the subepicardial space forms an innermost layer of the outer region.

[0053] In one embodiment, the epicardium may comprise KRT18+ mesothelial epicardial cells. In one embodiment, the subepicardial space may comprise VIM+ epicardial-derived cells (EPDCs). In one embodiment, the subepicardial space may comprise adipose tissue, optionally comprising fatty acid positive cells.

[0054] In one embodiment, the outer region may be innervated and / or may comprise coronary vascularisation. In one embodiment, the outer region may comprise a sympathetic nervous system, optionally comprising TH+, PHOX2B, DBH, TUJ1, and / or PR.PH+ cells. In one embodiment, the outer region may comprise a coronary vascular plexus, optionally comprising CD31 + , DACH1+, and / or NR.2F2+ coronary endothelial cells.

[0055] In one embodiment, the coronary vascular plexus may be disposed in the subepicardial space.

[0056] In one embodiment, the sympathetic and / or parasympathetic neurons may be disposed in the outer region of the heart organoid. In one embodiment, the sympathetic and / or parasympathetic neurons may be disposed in the inner myocardial region of the heart organoid.

[0057] In one embodiment, the inner myocardial region of the organoid and the outer region of the organoid may substantially reflect the multilayered structure of the ventricular epicardium and myocardium of a human heart. In one embodiment, the inner myocardial region of the organoid and the outer region of the organoid may substantially reflect the multilayered structure of the ventricular epicardium and myocardium of a human embryonic heart and / or an adult heart.

[0058] It will be appreciated that the term "substantially reflect" can mean that the volume, mass, shape, cell composition, cell architecture, and / or size proportions of the inner myocardial region of the organoid and the outer region of the organoid are substantially the same as those of a mature or embryonic human heart.

[0059] In one embodiment, therefore, the heart organoid of the first aspect (which is typically innervated) may comprise at least one cardiac tissue type. In another embodiment, the heart organoid may comprise more than one cardiac tissue type.

[0060] In one embodiment, the heart organoid may comprise a cardiac tissue type selected from a group of cardiac tissue types consisting of: sympathetic neuron, parasympathetic neuron, epicardium, subepicardium, myocardium, endocardium, coronary vascular plexus, and adipose tissue. In another embodiment, the heart organoid may comprise epicardium, myocardium, and coronary vascular plexus. The epicardium may comprise mesothelial cells, adipocytes, and / or connective tissue. The epicardium may be the outermost layer of the organoid. The myocardium may comprise cardiomyocytes, which may be arranged in a layer. The myocardium may be positioned beneath the epicardium. The myocardium may be adhered to the epicardium. In another embodiment, the heart organoid may comprise epicardium and ventricular myocardium. Typically, the epicardium is adhered to and / or surrounds the ventricular myocardium. The coronary vascular plexus may be positioned beneath the epicardium. The coronary vascular plexus may invade the myocardium. The coronary vascular plexus may invade the compact myocardium. The coronary vascular plexus may invade the compact myocardium to form coronary arteries. The coronary vascular plexus may produce veins.

[0061] In another embodiment, the heart organoid may comprise a subepicardial layer. The subepicardial layer may face the myocardium. The subepicardial layer may face the compact myocardium.

[0062] It will be appreciated that adipose tissue is a specialised connective tissue composed primarily of adipocytes, which function to store energy in the form of lipids. It also serves roles in cushioning, insulation, and endocrine signalling.

[0063] In one embodiment, therefore, the adipose tissue may comprise adipocytes.

[0064] In one embodiment, the adipose tissue may be located between the myocardium and the epicardium.

[0065] In another embodiment, the heart organoid may comprise a subset of at least one epicardial cell which undergoes epithelial-to-mesenchymal transition (EMT). In another embodiment, the heart organoid may comprise at least one epicardial-derived cell (EPDC). The at least one epicardial-derived cell may migrate into the subepicardial layer. The at least one epicardial-derived cell may invade the myocardium. The at least one epicardial-derived cell may invade the compact myocardium. In another embodiment, the heart organoid may comprise at least one fibroblast. In another embodiment, the heart organoid may comprise at least one mural cell. The at least one mural cell may comprise at least one vascular smooth muscle cell or pericyte.

[0066] In one embodiment, the myocardium is innervated. In one embodiment, the myocardium may comprise at least one neuron. The myocardium may be innervated with at least one sympathetic neuron and / or at least one parasympathetic neuron. Typically, however, the myocardium is innervated with at least one sympathetic neuron. The myocardium may comprise a plurality of neurons, for example, SNs and / or PSNs. For example, the myocardium may comprise at least one, 10, 100, 1000, 10,000, 100,000, 500,000, or 1,000,000 neurons.

[0067] In another embodiment, the heart organoid comprises a sinoatrial (SA) node, an atrioventricular (AV) node, an AV bundle / bundle of His, and / or Purkinje fibres.

[0068] Furthermore, the inventors observed, during their experimentation, the production of a subepicardial space in the organoids, comprising mainly extracellular matrix (ECM) proteins, including laminin, fibronectin, and collagen I.

[0069] It will be appreciated that, in vivo, a subepicardial space is an area that forms between the epicardium and the myocardium, and is populated with ECM components. It will also be appreciated that the subepicardial space is the site of coronary vascular plexus establishment and development.

[0070] In one embodiment, therefore, the heart organoid may comprise a subepicardial space. In another embodiment, the sub-epicardial space (subepicardium), may contain an ECM protein. The ECM protein may be selected from a group of ECM proteins consisting of: Aggrecan, Agrin, Aspein, Asporin, Biglycan, BMP binding endothelial regulator, Bone sialoprotein, Brevican, Cartilage oligomeric matrix protein, CCDC80, Chondronectin, Cochlin, collagen I, collagen IV, CRISPLD2, Cysteine rich secretory protein led domain containing 1, Decorin, Dentin sialophosphoprotein, Dermatopontin, DMBT1, DMP1, Elastin, Extracellular matrix protein 1, Extracellular matrix protein 2, Fibromodulin, Fibronectin, FRAS1, HAPLN1, Hemicentin 1, Hemicentin 2, Hyalin, Ladinin 1, Laminin, Matricellular protein, Matrilin, Matrilin-1, Matrilin-2, Matrilin-3, Matrix Gia protein, MEPE, MFAP4, MXRA5, Neurocan, Nidogen, Nidogen-1, Nidogen-2, Osteoglycin, Osteonectin, Osteopontin, Periostin, Perlecan, Pikachurin, Proteoglycan 4, R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, SCO-spondin, SIBLING proteins, SNED1, SPARCL1, Spondin 1, Spondin 2, SRPX, SRPX2, TECTA, TECTB, Tenomodulin, TGFBI, Tsukushi, Versican, Vitronectin, and / or VWA2. Typically, however, the subepicardial space contains laminin, fibronectin, collagen I, and / or collagen IV.

[0071] In another embodiment, the heart organoid may comprise a blood vessel. The heart organoid may comprise a blood vessel that is an artery, an arteriole, a capillary, a venule, and / or a vein. During their experimentation, the inventors assessed the effect of sympathetic neuron stimulation on contraction activity of the myocardial region of the organoids. The inventors identified a significant increase in beating rate upon contact of the organoids with nicotine and epinephrine, which was not observed in organoids without innervation. The spontaneous beating rate of the organoids, i.e., organoids which have not been contacted with a stimulus, was observed by the inventors to be between 7 and 30 beats per minute (BPM). The increase in beating rate of the organoids upon contact of the organoids with nicotine, i.e., a stimulant, observed by the inventors was within a range of a factor of 2 to 10.

[0072] It will be appreciated that one organoid contraction represents one beat.

[0073] In one embodiment, therefore, the heart organoid of the first aspect (which is typically innervated) may be contractile. Typically, the myocardium of the heart organoid is contractile. In another embodiment, the myocardium of the heart organoid may contract. The heart organoid may contract spontaneously. The heart organoid may contract at a rate of between 1 and 100 BPM, between 2 and 80 BPM, between 3 and 60 BPM, between 4 and 40 BPM, between 5 and 35 BPM, or between 6 and 32 BPM. Typically, however, the heart organoid contracts at a rate of between 7 and 30 BPM. In another embodiment, the heart organoid may contract when contacted with a stimulus. The stimulus may be selected from a group of stimuli, consisting of: environmental, chemical, temperature, mechanical force, light, water, pH, pressure, and electrical stimuli. For example, the stimulus may be a chemical compound. Therefore, the stimulus may be selected from a group of stimuli, consisting of: nicotine, boron fluoride, catecholamines, such as adrenaline, noradrenaline, and dopamine, amphetamines, Nifedipine, Verapamil, Ryanodine, Lidocaine, E-4031, Caffeine, Cisapride, Ouabain, Rolipram, Milrinone, and / or Thapsigargin. The heart organoid may contract at an increased rate when contacted with a stimulus. The heart organoid may contract at an increased rate of a factor of between 1.01 and 100, between 1.25 and 50, or between 1.5 and 20, when contacted with a stimulus. Typically, however, the heart organoid contracts at an increased rate of a factor of between 2 and 10 when contacted with a stimulus.

[0074] In one embodiment, the heart organoid may be permeable to dextran particles. In one embodiment, the coronary vascular plexus may be permeable to dextran particles.

[0075] In one embodiment, the heart organoid may be responsive to hypoxic stimuli. In one embodiment, the coronary vascular plexus may be permeable to hypoxic stimuli. The inventors have developed a novel method for producing the heart organoid of the first aspect, one embodiment of which is shown in Figure 1.

[0076] Accordingly, in a second aspect of the invention, there is provided a method of producing a heart organoid, wherein the heart organoid is innervated, is vascularised, and / or comprises adipose tissue, the method comprising :

[0077] (i) differentiating at least one induced pluripotent stem cell (iPSC) to produce at least one pro-epicardium organoid (PEO) and at least one myocardium organoid (MO);

[0078] (ii) contacting the at least one PEO and the at least one MO to produce at least one epicardium-myocardium organoid (EMO);

[0079] (iii) differentiating at least one induced pluripotent stem cell (iPSC) to produce at least one sympathetic neuron spheroid (SNS) and / or parasympathetic neuron spheroid (PSNS); and

[0080] (iv) contacting the at least one MO and / or EMO with the at least one SNS and / or at least one PSNS to produce a heart organoid (HO) that is innervated, is vascularised, and / or comprises adipose tissue.

[0081] In one embodiment, the heart organoid of the second aspect (which is typically innervated) may be derived from at least one animal induced pluripotent stem cell (iPSC), which may be a mammal iPSC. In another embodiment, the heart organoid may be derived from at least one human, pig, or mouse iPSC. In one embodiment, the at least one human, pig, or mouse iPSC may be genetically modified and / or derived from healthy or diseased donors. Typically, the heart organoid is derived from at least one human induced pluripotent stem cell (hiPSC).

[0082] In another embodiment, the heart organoid may be derived from at least one embryonic stem cell. Typically, the heart organoid is derived from a human embryonic stem cell.

[0083] In one embodiment, therefore, the method comprises differentiating at least one animal induced pluripotent stem cell (iPSC), at least one mammal iPSC, at least one pig iPSC, at least one mouse iPSC, and / or at least one human iPSC (hiPSC), to produce at least one pro-epicardium organoid (PEO) and at least one myocardium organoid (MO). Typically, the method comprises differentiating at least one human induced pluripotent stem cell (hiPSC) to produce at least one pro-epicardium organoid (PEO) and at least one myocardium organoid (MO). In one embodiment, the induced pluripotent stem cell (iPSC) cell population may comprise an in vitro iPSC population disposed in cell culture medium.

[0084] In one embodiment, the heart organoid may be derived from at least one, 10, or 100 cells. In one embodiment, the heart organoid may be derived from at least 1,000, 10,000, or 100,000 cells. Typically, however, the heart organoid is derived from at least 4,000, 5,000, 6,000, 7,000, 8,000, or 9,000 hiPSCs.

[0085] In one embodiment, the at least one PEO and the at least one MO may be contacted at a ratio of between 50% and 99.9% : between 50% and 0.1%, respectively, between 60% and 95% : between 40% and 5%, respectively, between 70% and 92% : between 30% and 8%, respectively, or between 80% and 90% : between 20% and 10%, respectively. Typically, however, the at least one PEO and the at least one MO are contacted at a ratio of between 85% and 90% : between 15% and 10%, respectively, or about 87% : 13%, respectively.

[0086] In one embodiment, the at least one EMO may be derived from at least 1, 2, 3, 4, 5, 10, 20, or 25 PEOs and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 MOs. Typically, the at least one EMO may be derived from at least 1 PEO and at least 4 MOs.

[0087] In another embodiment, the at least one PEO and / or the at least one MO may comprise a diameter of between 10 pm and 10,000 pm, between 100 pm and 1,000 pm, between 150 pm and 500 pm, between 200 pm and 450 pm, between 225 pm and 425 pm, or between 240 pm and 410 pm. Typically, however, the at least one PEO and / or the at least one MO comprises a diameter of between 250 pm and 400 pm.

[0088] In another embodiment, the at least one EMO may be derived from PEOs and MOs at a ratio of between 1 : 1 and 1 : 1,000, or between 1 : 1 and 1 :100, or between 1 : 1 and 1 :50, or between 1 : 1 and 1 :25, or between 1 : 1 and 1 :20, or between 1 : 1 and 1 : 10, or between 1 : 1 and 1:5, or between 1 : 1 and 1 :4, or between 1 : 1 and 1 :3, or between 1 : 1 and 1:2. Typically, the at least one HO may be derived from PEOs and MOs at a ratio of 1 :9.

[0089] In one embodiment, the at least one PEO and the at least one MO may be contacted with at least one growth factor.

[0090] The at least one growth factor may be fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), insulin like growth factor binding protein 2 (IGFBP2), platelet- derived growth factor beta (PDGF- 0), and / or vascular endothelial growth factor A (VEGFA). Typically, the at least one PEO and the at least one MO are contacted with FGF2, HGF, IGFBP2, PDGF- 0, and VEGFA.

[0091] With reference to Figure 2A2, it will be appreciated that the process of EMO production comprises a process beginning with the at least one PEO and the at least one MO, and ending with the at least one EMO.

[0092] In one embodiment, therefore, the at least one PEO and the at least one MO may be contacted with FGF2, HGF and IGFBP2 on any one of days 1-10 of EMO production, or with FGF2, HGF and IGFBP2 on any one of days 1-8 of EMO production. Typically, however, the at least one PEO and the at least one MO are contacted with FGF2, HGF and IGFBP2 on any one of days 1-6 of EMO production.

[0093] The at least one PEO and the at least one MO may be contacted with FGF2, HGF and IGFBP2 for at least 1, 2, 3, 4, or 5 days during EMO production. Typically, the at least one PEO and the at least one MO are contacted with FGF2, HGF and IGFBP2 for at least 6 days during EMO production.

[0094] In another embodiment, the at least one PEO and the at least one MO may be contacted with PDGF- 0 and VEGFA on any one of days 1-10 of EMO production, with PDGF-0 and VEGFA on any one of days 2-10 of EMO production, or with PDGF- 0 and VEGFA on any one of days 4-10 of EMO production. Typically, however, the at least one PEO and the at least one MO are contacted with PDGF-0 and VEGFA on any one of days 6-10 of EMO production.

[0095] The at least one PEO and the at least one MO may be contacted with PDGF-0 and VEGFA for at least 1, 2, or 3 days during EMO production. Typically, the at least one PEO and the at least one MO are contacted with PDGF- 0 and VEGFA for at least 4 days during EMO production.

[0096] The inventors have established a differentiation platform methodology to obtain sympathetic neuron spheroids (SNSs), which are used herein as a proof of concept for any neuron spheroid, from human induced pluripotent stem cells (hiPSCs). However, it will be appreciated that the inventors envisage the use of the differentiation platform to obtain SNSs and / or parasympathetic neuron spheroids (PSNSs) from any induced pluripotent stem cell (iPSC). Advantageously, the inventors performed their differentiation protocol in a three-dimensional environment. In one embodiment, the method may comprise, at step (iv) contacting the at least one MO and / or EMO with at least one ANSS to produce a heart organoid (HO) that is innervated, is vascularised, and / or comprises adipose tissue.

[0097] As described above, it will be appreciated that an autonomic nervous system spheroid (ANSS) is a spheroid comprising both sympathetic neurons (SNs) and parasympathetic neurons (PSNs). It will also be appreciated that an ANSS can be produced by the fusion of one sympathetic neuron spheroid (SNS) with one parasympathetic neuron spheroid (PSNS), or by mixing SNS- and PSNS- progenitors, thus forming a single spheroid (i.e., an ANSS). An embodiment of the organoid of the invention, comprising an ANSS, is shown in Figure 8.

[0098] In one embodiment, the method comprises differentiating at least one animal induced pluripotent stem cell (iPSC), at least one mammal iPSC, at least one pig iPSC, at least one mouse iPSC, and / or at least one human iPSC (hiPSC), to produce at least one sympathetic neuron spheroid (SNS) and / or at least one parasympathetic neuron spheroid (PSNS). Typically, the method comprises differentiating at least one human induced pluripotent stem cell (hiPSC) to produce at least one sympathetic neuron spheroid (SNS) and / or parasympathetic neuron spheroid (PSNS).

[0099] It will be appreciated that approximately 400 SNSs or PSNSs may be generated from approximately 600,000 iPSCs, such as hiPSCs. As such, it will also be appreciated that one SNS or PSNS may be generated from approximately 1,500 iPSCs, such as hiPSCs.

[0100] In one embodiment, therefore, the at least one SNS or the at least one PSNS may be derived from about 1,500 iPSCs. Typically, the at least one SNS or the at least one PSNS may be derived from at least 500, 1,000, 1,500, 2,000, or 2,500 iPSCs.

[0101] It will be appreciated that the term "spheroid" can mean a three-dimensional cell culture, where cells self-assemble into sphere-like formations during cell proliferation, promoting cell-cell interactions. The term "spheroid" can also mean a cell model which provides cell migration, polarisation, differentiation, survival, and growth.

[0102] In one embodiment, therefore, the heart organoid of the invention (which is typically innervated) may comprise a spheroid. The heart organoid may comprise a sympathetic neuron spheroid (SNS) and / or a parasympathetic neuron spheroid (PSNS). Typically, the heart organoid comprises a SNS. The spheroid may comprise a plurality of cells. For example, the spheroid may comprise at least one, 10, or 100 cells. The spheroid may comprise at least 1000, 10,000, or 100,000 cells. The spheroid may comprise at least 1,000,000, 10,000,000, or 100,000,000 cells.

[0103] The SNS or PSNS may comprise a diameter of between 2 pm and 2,000 pm. The spheroid may comprise a diameter of between 20 pm and 1,750 pm, or between 40 pm and 1,500 pm, or between 60 pm and 1,250 pm, or between 80 pm and 1,000 pm, or between 100 pm and 750 pm, or between 120 pm and 500 pm, or between 140 pm and 300 pm, or between 160 pm and 250 pm, or between 180 pm and 220 pm, or between 185 pm and 215 pm, or between 190 pm and 210 pm, or between 195 pm and 205 pm, or of between 198 pm and 202 pm. Typically, the spheroid comprises a diameter of about 200 pm.

[0104] It will be appreciated that the diameter of the spheroid may be measured by taking a brightfield image of the spheroid using a brightfield microscope. The spheroid may be visualised as dark objects against a bright background. The size of the spheroid may be measured using the digital image scale bar and an imaging analysis software.

[0105] In another embodiment, the spheroid may comprise at least one cell type. For example, the spheroid may comprise at least one induced pluripotent stem cell (iPSC), such as a human induced pluripotent stem cell (hiPSC), at least one neural stem cell, at least one intermediate progenitor cell, at least one neural progenitor cell, at least one sympathetic neuron, and / or at least one parasympathetic neuron.

[0106] It will be appreciated that the term "three-dimensional environment" can mean that cells are permitted to grow, and move, left and right, up and down, forwards and backwards, and interact with surfaces in all three of these dimensions. Such conditions recreate the three-dimensional physiological settings of an in vivo heart in vitro, thus allowing the heart organoids to mimic heart tissue and its microarchitecture.

[0107] In one embodiment, therefore, the at least one iPSC may be differentiated to produce at least one SNS and / or PSNS in a three-dimensional environment.

[0108] In another embodiment, the at least one iPSC may be contacted with at least one growth factor to produce at least one SNS and / or at least one PSNS. The at least one growth factor may be LDN193189 (LDN), SB431542 (SB), CHIR99021 (CHIR), DAPT, SAG, Bone Morphogenetic Protein 4 (BMP4), brain-derived neurotrophic factor (BDNF), glial cell- derived neurotrophic factor (GDNF), and / or nerve growth factor (NGF). Typically, the at least one iPSC is contacted with LDN193189 (LDN), SB431542 (SB), CHIR99021 (CHIR), DAPT, SAG, BMP4, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor (NGF).

[0109] With reference to Figure 2B, it will be appreciated that the process of SNS production comprises a process beginning with the at least one iPSC, such as a hiPSC, and ending with the at least one SNS. It will also be appreciated that the process of PSNS production also comprises a process beginning with the at least one iPSC, such as a hiPSC, and ending with at least one PSNS.

[0110] In one embodiment, the at least one iPSC may be contacted with LDN on any one of days 0-50 of SNS and / or PSNS production, on any one of days 1-30 of SNS and / or PSNS production, on any one of days 0-10 of SNS and / or PSNS production, or on any one of days 0-5 of SNS and / or PSNS production. Typically, however, the at least one iPSC is contacted with LDN on any one of days 0-3 of SNS and / or PSNS production.

[0111] The at least one iPSC may be contacted with LDN for at least 1, or 2 days during SNS and / or PSNS production. Typically, the at least one iPSC is contacted with LDN for at least 3 days during SNS and / or PSNS production.

[0112] In another embodiment, the at least one iPSC may be contacted with SB on any one of days 0-50 of SNS and / or PSNS production, on any one of days 0-30 of SNS and / or PSNS production, on any one of days 0-10 of SNS and / or PSNS production, or on any one of days 0-5 of SNS and / or PSNS production. Typically, however, the at least one hiPSC is contacted with SB on any one of days 0-4 of SNS and / or PSNS production.

[0113] The at least one iPSC may be contacted with SB for at least 1, 2, or 3 days during SNS and / or PSNS production. Typically, the at least one iPSC is contacted with SB for at least 4 days during SNS and / or PSNS production.

[0114] In a further embodiment, the at least one iPSC may be contacted with CHIR and / or DAPT on any one of days 0-50 of SNS and / or PSNS production, on any one of days 0-30 of SNS and / or PSNS production, on any one of days 0-20 of SNS and / or PSNS production, or on any one of days 1-10 of SNS and / or PSNS production. Typically, however, the at least one iPSC is contacted with CHIR and DAPT on any one of days 2-7 of SNS and / or PSNS production. The at least one IPSC may be contacted with CHIR and / or DAPT for at least 1, 2, 3, or 4 days during SNS and / or PSNS production. Typically, the at least one iPSC is contacted with CHIR and / or DAPT for at least 5 days during SNS and / or PSNS production.

[0115] In a further embodiment, the at least one iPSC may be contacted with SAG on any one of days 0-50 of SNS and / or PSNS production, on any one of days 0-30 of SNS and / or PSNS production, on any one of days 1-20 of SNS and / or PSNS production, or on any one of days 2-15 of SNS and / or PSNS production. Typically, however, the at least one iPSC is contacted with SAG on any one of days 3-12 of SNS and / or PSNS production.

[0116] The at least one iPSC may be contacted with SAG for at least 1, 2, 3, 4, 5, 6, 7, or 8 days during SNS and / or PSNS production. Typically, the at least one iPSC is contacted with SAG for at least 9 days during SNS and / or PSNS production.

[0117] In a further embodiment, the at least one iPSC may be contacted with BMP4 on any one of days 0-50 of SNS and / or PSNS production, on any one of days 2-30 of SNS and / or PSNS production, on any one of days 4-20 of SNS and / or PSNS production, or on any one of days 8-15 of SNS and / or PSNS production. Typically, however, the at least one iPSC is contacted with BMP4 on any one of days 10-12 of SNS and / or PSNS production.

[0118] The at least one iPSC may be contacted with BMP4 for at least 1 day during SNS and / or PSNS production. Typically, the at least one iPSC is contacted with BMP4 for at least 2 days during SNS and / or PSNS production.

[0119] In a further embodiment, the at least one iPSC may be contacted with BDNF, GDNF, and / or NGF on any one of days 0-100 of SNS and / or PSNS production, on any one of days 5-90 of SNS and / or PSNS production, on any one of days 8-75 of SNS and / or PSNS production, or on any one of days 10-60 of SNS and / or PSNS production. Typically, however, the at least one iPSC is contacted with BDNF, GDNF, and NGF on any one of days 12-50 of SNS and / or PSNS production.

[0120] The at least one iPSC may be contacted with BDNF, GDNF, and / or NGF for at least 1, 5, 10, 15, 20, 25, 30, 35, 36, or 37 days during SNS and / or PSNS production. Typically, the at least one iPSC is contacted with BDNF, GDNF, and NGF for at least 38 days during SNS and / or PSNS production. After two weeks of co-culture of EMOs and SNSs, which are used herein as a proof of concept for any neuron spheroid, the inventors assessed the effect of SN stimulation on contraction activity of the myocardial region of the heart organoid. However, it will be appreciated that the inventors envisage the use of their platform to co-culture EMOs and SNSs and / or PSNSs to produce a heart organoid (HO). Advantageously, the inventors surprisingly discovered a significant increase in beating rate upon SN stimulation, which was not observed in EMOs without innervation.

[0121] In one embodiment, the at least one HO may be derived from at least 1, 2, 3, 4, 5, 10, 20, 25, 50, 100, or 1,000 EMOs and at least 1, 2, 3, 4, 5, 10, 20, 25, 50, 100, or 1,000 SNSs and / or PSNSs. Typically, the at least one HO may be derived from at least 1 EMO and at least 1 SNS and / or PSNS.

[0122] In another embodiment, the at least one EMO may comprise a diameter of between 10 pm and 10,000 pm, between 50 pm and 1,000 pm, between 100 pm and 800 pm, between 150 pm and 700 pm, between 200 pm and 600 pm, between 250 pm and 550 pm, or between 275 pm and 525 pm. Typically, however, the at least one PEO and / or the at least one MO comprises a diameter of between 300 pm and 500 pm.

[0123] In another embodiment, the at least one HO may be derived from EMOs and SNSs and / or PSNSs at a ratio of between 1 : 1 and 1 :1,000, or between 1 : 1 and 1 :100, or between 1 : 1 and 1:50, or between 1 : 1 and 1 :25, or between 1 :1 and 1 :20, or between 1 : 1 and 1: 10, or between 1 : 1 and 1 :5, or between 1 :1 and 1 :4, or between 1 :1 and 1 :3, or between 1 : 1 and 1 :2. Typically, the at least one HO may be derived from EMOs and SNSs and / or PSNSs at a ratio of about 1 : 1.

[0124] It will be appreciated that the higher the ratio of SNSs and / or PSNSs:EMOs, the higher the percentage of the surface area of the heart organoid will be covered by sympathetic neurons (SNs) and / or parasympathetic neurons (PSNs).

[0125] In one embodiment, the at least one EMO and the at least one SNS and / or PSNS may be co-cultured with media to produce at least one heart organoid (HO).

[0126] In one embodiment, the at least one EMO and the at least one SNS and / or PSNS may be co-cultured with N2 maturation media and / or DMEM / F12 media to produce at least one heart organoid (HO). Typically, however, the at least one EMO and the at least one SNS and / or PSNS are co-cultured with N2 maturation media and DMEM / F12 media to produce at least one heart organoid (HO). With reference to Figure 2C, it will be appreciated that the process of HO production comprises a process beginning with the at least one EMO and the at least one SNS and / or PSNS, and ending with the at least one HO.

[0127] In one embodiment, the at least one EMO and the at least one SNS and / or PSNS may be contacted with BDNF, GDNF, and / or NGF on any one of days 0-100 of HO production, on any one of days 0-50 of HO production, on any one of days 0-25 of HO production, or on any one of days 0-22 of HO production. Typically, however, the at least one EMO and the at least one SNS and / or PSNS are contacted with BDNF, GDNF, and NGF on any one of days 0-20 of HO production.

[0128] The at least one EMO and the at least one SNS and / or PSNS may be contacted with BDNF, GDNF, and / or NGF for at least 1, 5, 10, 15, 20, 25, 30, 35, 36, or 37 days during SNS and / or PSNS production. Typically, the at least one EMO and the at least one SNS and / or PSNS are contacted with BDNF, GDNF, and NGF for at least 38 days during SNS and / or PSNS production.

[0129] In another embodiment, the at least one EMO and the at least one SNS and / or PSNS may be co-cultured with N2 maturation media and DMEM / F12 media on any one of days 0-50 of HO production, on any one of days 0-40 of HO production, on any one of days 0- 30 of HO production, or on any one of days 0-25 of HO production. Typically, however, the at least one EMO and the at least one SNS and / or PSNS are co-cultured with N2 maturation media and DMEM / F12 media on any one of days 0-20 of HO production.

[0130] In a further embodiment, the N2 maturation media and DMEM / F12 media may be at a ratio of between 10: 1 and 1.1 : 1, or of between 1 : 10 and 1 : 1.1. Typically, however, the N2 maturation media and DMEM / F12 media are at a ratio of 1 :1.

[0131] In a further embodiment, the N2 maturation media and DMEM / F12 media may be changed between every 1-20 days, between every 1-15 days, between every 1-10 days, or between every 2-5 days. Typically, however, the N2 maturation media and DMEM / F12 media are changed every 3 days.

[0132] In one embodiment, steps (i)-(iv) of the second aspect may be performed separately, consecutively, and / or at the same time. In another embodiment, the method of step (i) may be performed prior to step (ii), the method of step (ii) may be performed prior to step (iv), the method of step (iii) may be performed prior to, or subsequent to steps (i) and / or (ii), the method of step (iii) may be performed prior to step (iv), and / or the method of step (iv) may be performed subsequent to steps (i), (ii), and / or (iii).

[0133] The inventors have also surprisingly discovered a media formulation that maintains the heart organoids of the invention.

[0134] Accordingly, in another embodiment, the method may further comprise, subsequent to the method of step (iv):

[0135] (v) maintaining the heart organoid (HO) that is innervated, is vascularised, and / or comprises adipose tissue in media.

[0136] In one embodiment, maintaining the heart organoid may comprise culturing the heart organoid in the media.

[0137] The media for maintenance of the heart organoid may comprise N2 maturation media and / or DMEM / F12 media. Typically, the media for maintenance of the heart organoid comprises N2 maturation media and DMEM / F12 media.

[0138] In one embodiment, the N2 maturation media and DMEM / F12 media may be at a ratio of between 10: 1 and 1.1 : 1, or of between 1 :10 and 1 : 1.1. Typically, however, the N2 maturation media and DMEM / F12 media are at a ratio of 1: 1.

[0139] In one embodiment, the N2 maturation media and DMEM / F12 media may be changed between every 1-20 days, between every 1-15 days, between every 1-10 days, or between every 2-5 days. Typically, however, the N2 maturation media and DMEM / F12 media are changed every 3 days.

[0140] In one embodiment, the heart organoid is maintained in culture for at least one hour, at least five hours, at least 10 hours, at least 15 hours, or at least 20 hours. In one embodiment, the heart organoid is maintained in culture for at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, or at least 96 hours. In one embodiment, the heart organoid is maintained in culture for at least one week, at least two weeks, at least three weeks, or at least four weeks. In one embodiment, the heart organoid is maintained in culture for at least one month, at least two months, at least three months, or at least four months. In one embodiment, the heart organoid is maintained in culture for at least six months, at least 12 months, at least 18 months, or at least 24 months.

[0141] In a third aspect of the invention, there is provided a heart organoid that is innervated, is vascularised, and / or comprises adipose tissue obtained, or obtainable by, the method according to the second aspect.

[0142] The heart organoid of the third aspect may be as defined as the organoid of the first aspect.

[0143] The inventors envisage the heart organoid of the invention to have several applications, including, but not limited to, use in: 1) drug evaluation, such as efficacy, cardiotoxicity, and teratogenicity studies; and 2) disease modelling and heart pathology studies.

[0144] It will be appreciated that efficacy, cardiotoxicity, and teratogenicity studies may be performed in the context of a healthy and / or disease state. It will also be appreciated that efficacy studies may comprise inducing a pathology, and assessing whether the organoid responds as expected to a drug and / or substance. It will also be appreciated that cardiotoxicity studies may comprise selecting at least one drug and / or substance, and assessing the cardiotoxicity of the at least one drug and / or substance. It will also be appreciated that teratogenicity studies may comprise assessing the impact of the exposure of at least one drug and / or substance to the organoid on normal progression of heart organogenesis.

[0145] The inventors believe that the organoid of the first and third aspects of the invention may be used in a screen for new, or existing, therapeutic compounds in the drug discovery, development, and evaluation process, particularly for efficacy, cardiotoxicity and / or teratogenicity analysis of drugs. For example, methods and assays using the organoid may be used for candidate compound screening (e.g., High Throughput Screening, selective library screens, and structure-based design etc.), for identifying hits that may offer effective treatment, as well as secondary assays (e.g., in vitro and ex vivo secondary assays), for identifying cardiotoxicity of candidate drugs, as part of the "hit to lead" development of a pharmaceutical. In another example, methods and assays using the organoid may be used to assess whether the organoid responds as expected to a drug and / or substance, where the organoid may have an induced pathology. In another example, methods and assays using the organoid may be used to assess the impact of the exposure of at least one drug and / or substance to the organoid on normal progression of heart organogenesis. Accordingly, in a fourth aspect of the invention, there is provided use of the heart organoid that is innervated, is vascularised, and / or comprises adipose tissue according to the first or third aspect, or a precursor thereof, in a drug evaluation screen.

[0146] It will be appreciated that a precursor of the heart organoid of the invention may comprise any cell or organoid used or produced during the method of the second aspect, including, but not limited to, a human induced pluripotent stem cell (hiPSC), proepicardium organoid (PEO), myocardium organoid (MO), epicardium-myocardium organoid (EMO), sympathetic neuron spheroid (SNS), and / or parasympathetic neuron spheroid (PSNS).

[0147] In one embodiment, therefore, the precursor of the heart organoid of the invention may comprise a cell or organoid used or produced during the method of the second aspect, human induced pluripotent stem cell (hiPSC), pro-epicardium organoid (PEO), myocardium organoid (MO), epicardium-myocardium organoid (EMO), sympathetic neuron spheroid (SNS), and / or parasympathetic neuron spheroid (PSNS).

[0148] Accordingly, in a fifth aspect of the invention, there is provided a method of carrying out a drug evaluation screen, the method comprising contacting the heart organoid that is innervated, is vascularised, and / or comprises adipose tissue according to the first or third aspect, or a precursor thereof, with a test agent, and analysing the effects of the test agent on the heart organoid, or the precursor thereof.

[0149] The inventors envisage the use of the heart organoid of the first or third aspect in various stages of drug evaluation, but particularly during the pre-clinical phase. These stages of drug evaluation may include efficacy analysis, toxicity analysis, teratogenicity analysis, pharmacokinetics analysis, pharmacodynamics analysis, and functional analysis. Furthermore, the inventors envisage the use of the heart organoid in determining a drug's mechanism of action, the adverse effects of a drug, and / or for assessing drug-induced cardiac injury during preclinical trials.

[0150] In one embodiment, therefore, the heart organoid is used for drug evaluation. The heart organoid may be used for drug efficacy analysis, toxicity analysis, teratogenicity analysis, pharmacokinetics analysis, pharmacodynamics analysis, and / or functional analysis. Typically, however, the heart organoid is used for drug efficacy analysis, toxicity analysis, and / or teratogenicity analysis. It will be appreciated that a teratogen is any agent that interferes with normal embryonic and / or foetal development, and / or causes a congenital disability.

[0151] In one embodiment, therefore, the method may comprise contacting the heart organoid according to the first or third aspect, or a precursor thereof, with a test agent, and analysing the effects of the test agent on the heart organoid, or the precursor thereof.

[0152] The heart organoid (which is typically innervated) may be used during the drug research and discovery stage of drug development, during the preclinical stage of drug development, during the clinical stage of drug development, during the drug review stage of drug development, and / or during the post-market drug safety monitoring stage of drug development. The heart organoid may be used during the drug research and discovery stage of drug development, during the preclinical stage of drug development, and / or during the clinical stage of drug development. Typically, however, the heart organoid is used during the preclinical stage of drug development.

[0153] The heart organoid may be used for assessing drug mechanism of action, adverse effects, and / or drug-induced cardiac injury.

[0154] The heart organoid may be used in an in vitro and / or ex vivo assay. The heart organoid may be used in a preclinical in vitro and / or ex vivo assay. The heart organoid may be used in an in vitro and / or ex vivo assay for assessing drug efficacy. The heart organoid may be used in an in vitro and / or ex vivo assay for assessing drug-induced cardiac injury. The heart organoid may be used in an in vitro and / or ex vivo assay for assessing drug-induced cardiac abnormality. Typically, the heart organoid is used in an in vitro and / or ex vivo assay for assessing drug-induced cardiac injury caused by at least one cardiotoxic drug. Typically, the heart organoid is used in an in vitro and / or ex vivo assay for assessing drug-induced cardiac abnormality caused by at least one teratogenic drug.

[0155] In an embodiment, the method may comprise analysing the efficacy, cardiotoxicity, and / or teratogenicity of a test agent. The method may comprise the steps of:

[0156] (i) contacting, in vitro or ex vivo, the heart organoid according to the first or third aspect, or a precursor thereof, with a test agent; and

[0157] (ii) analysing the heart organoid, or the precursor thereof, to detect any changes caused by the test agent, wherein the changes, as compared to a control heart organoid in the absence of the test agent, are an indicator of the efficacy, cardiotoxicity, and / or teratogenicity of the test agent. The changes caused by the test agent may comprise damage, cell death, abnormal progression of heart organogenesis, abnormal function, abnormal structure, abnormal transcriptome, abnormal proteome, and / or abnormal secretome.

[0158] The changes caused by the test agent may be identified using equipment and / or methodology including : i) a confocal microscope for immunostaining analysis of organoid slices and / or whole mount immunostaining, to study abnormal structure, for example; ii) a microelectrode array (MEA), patch clump, voltage sensitive dyes, and / or calcium transients, such as Ca2+ fluorescent dyes, to study abnormal function, such as action potential profile, calcium efflux profile, and / or contraction profile, for example; iii) Alamar Blue and / or MTT live and / or dead staining, to study toxicity, for example; and / or iv) transcriptomic, proteomic, and / or metabolomic analysis, to study abnormal transcriptome, abnormal proteome, and / or abnormal secretome, for example.

[0159] The heart organoid of the invention, or a precursor thereof, may be analysed by its function and / or structure. The heart organoid of the invention, or a precursor thereof, may be analysed by its function and / or structure before and / or after contact with a test agent.

[0160] The analysis of the heart organoid may comprise a physical integrity assessment, structural organisation assessment, and / or functional assessment.

[0161] The physical integrity assessment may comprise assessment of apoptosis, oxidative stress, genotoxic damage, cell membrane permeability, endoplasmic reticulum integrity, mitochondrial membrane potential, the percentage of apoptotic cells, caspase activity, reactive oxygen species (ROS) activity, whole-cell reactive oxygen detection, mitochondrial superoxide detection, quantification of phosphorylated histone Y-H2AX, and / or fluorescence intensity quantification.

[0162] The structural organisation assessment may comprise assessment of heart organoid development (kinetics and / or structure), cardiac cell function and / or organisation, sarcomere structure, CM morphology and / or activity, myocardium cavity formation, epicardial layer formation, epicardial-derived vascularisation formation, myocardium growth / compaction, coronary vascular network development, migration of sympathetic neurons, vascularisation network structure and function, ECM deposition, cardiac autonomic nervous system function, sarcomere length, sarcomere alignment score, sarcomere distribution / card iomyocyte area, cardiomyocyte size (normal vs enlarged, i.e., hypertrophy), cardiomyocyte roundness, cardiomyocyte bi-nucleation score, cardiomyocyte proliferation, i.e., hyperplasia, mitochondria activity and / or structure, and / or progression of maturation.

[0163] The functional assessment may comprise assessment of action potential profile, calcium efflux profile, contraction profile, conduction velocity, action potential duration, for example at 50% and / or 90% repolarisation (APD50 / APD90), beats per minute (BPM), field potential duration (FPD), field potential amplitude (FPA), peak amplitude, time to 50% calcium decay, and / or calcium transient duration.

[0164] It will be appreciated that the term "damage" can mean pathophysiology, and / or pathological changes to the heart organoid. For example, the damage may include changes in organoid appearance and / or function compared with control organoids that have not been exposed to a test agent. An organoid of the invention may comprise abnormal progression of heart organogenesis if the structure of the organoid is compromised, for example in size, shape, and / or if the different cell layers are not correctly established, or if the organoid comprises a functional defect, for example arrhythmic behaviour, slower and / or faster contraction rate, and / or cessation of contraction. The above described changes in organoid appearance and / or function parameters may be quantified based on bright field and / or fluorescent images, or videos taken throughout the differentiation process compared side-by-side with the control conditions, i.e., control organoids.

[0165] The test agent may be a chemical compound or biological agent. The test agent may be a drug or pharmaceutical. The test agent may be a candidate drug. Typically, however, the test agent is a candidate drug in the preclinical stage of drug evaluation.

[0166] The test agent may be selected from a list of therapeutic classes including : analgesic, antibiotic, anticancer, anticoagulant, antidepressant, antidiabetic, antiepileptic, antipsychotic, antispasmodic, antiviral, depressant, sedative, and stimulant. The test agent may comprise a pesticide, a herbicide, a mycotoxin, an antibiotic, a heavy metal, a nitrate, a nitrite, a phosphate, a bacterium, a virus, a pathogen, an endocrine disruptor, a therapeutic drug, and / or an illicit drug.

[0167] In a sixth aspect, there is provided an apparatus for carrying out a drug evaluation screen, the apparatus comprising :

[0168] (i) a heart organoid according to the first or third aspect, or a precursor thereof; and

[0169] (ii) a vessel configured to permit contacting the heart organoid, or the precursor thereof, and a test agent.

[0170] In another aspect, there is provided an apparatus for identifying the efficacy, cardiotoxicity, and / or teratogenicity of a test agent, the apparatus comprising :

[0171] (i) a heart organoid according to the first or third aspect, or a precursor thereof; and

[0172] (ii) a vessel configured to permit contacting the heart organoid, or the precursor thereof, and a test agent.

[0173] The vessel may be a test tube, a microtube, a well plate, a spot plate, a microplate, a multiwell plate, a microwell plate, a tip, a reagent container, a vial, a microchannel device, and / or a microfluidic chip. Typically, the vessel is a multiwell plate and / or a microfluidic chip.

[0174] It will be appreciated that the heart organoid of the invention (which is typically innervated) can be used to detect drug-induced cardiotoxicity and / or teratogenicity in the apparatus and methods of the invention. Hence, if the heart organoid comprises damage and / or abnormal progression of heart organogenesis in response to the presence of the test agent or compound, then this would indicate that the test agent does not represent a useful candidate therapeutic compound. Conversely, if the heart organoid does not comprise damage and / or abnormal progression of heart organogenesis in the presence of the test compound, then this would indicate that the test compound does represent a useful candidate therapeutic compound, and could be taken forward in subsequent trials. Typically, the methods and apparatus of the invention involve identifying the cardiotoxicity and / or teratogenicity of a therapeutic agent.

[0175] The inventors also believe that the organoid of the first and third aspects of the invention (which is typically innervated) may be used in disease modelling and / or heart pathology studies. Therefore, in a seventh aspect, there is provided use of the heart organoid according to the first or third aspect, or a precursor thereof, to study a heart disease and / or pathology.

[0176] It will be appreciated that the study of a heart disease may comprise disease modelling. It will also be appreciated that disease modelling may comprise creating a disease model which mirrors a disease or embodies some of the disease processes and / or features, to assess the cause of a disease.

[0177] In one embodiment, therefore, the heart organoid, or precursor thereof, may be used for disease modelling. The organoid may be used for disease modelling of a genetic disease.

[0178] In another embodiment, therefore, the heart organoid, or precursor thereof, of the invention may be genetically modified. Typically, the heart organoid, or precursor thereof, may be genome edited. The heart organoid, or precursor thereof, may be genome edited by means of an AAV vector or a lentiviral vector.

[0179] In another embodiment, therefore, the heart organoid may be genome edited for the construction of a disease model, typically a genetic disease model.

[0180] It will also be appreciated that the study of a heart pathology may comprise the study of cardiomyopathies. For example, methods and assays using the organoid may be used for the study of cardiomyopathies that may be linked to an unbalanced, degenerate, or absence of myocardium innervation, all of which are registered in cardiac arrhythmias. Cardiac arrhythmias of particular note are atrial fibrillation and ventricular arrhythmias, such as in the case of cardiac sympathetic nerve loss, which is observed in neurodegenerative diseases and familial dysautonomia pathology.

[0181] In one embodiment, therefore, the heart organoid, or precursor thereof, may be used in an in vitro and / or ex vivo assay to study a heart disease and / or pathology.

[0182] The heart disease and / or pathology may be selected from a group of heart pathologies, consisting of: Congenital heart defects (Septal Defects, Atrial Septal Defects, Ventricular Septal Defects, Valve Defects, Tetralogy of Fallot, Hypoplastic left heart syndrome, and Left Ventricle Non-Compaction), Cardiomyopathies (Dilated Cardiomyopathy, Hypertrophic Cardiomyopathy), Neurodevelopmental Disorder (Rett Syndrome, Thymuti syndrome, Noonan syndrome and Costello syndrome), Myocardial Infarction (hypoxia), Neurodegenerative Disorders (diabetic autonomic neuropathy; Parkinson's disease) and Familial dysautonomia (impaired development of the autonomic nervous system), Arrhythmogenic Cardiomyopathies and atrial fibrillation, an unbalanced, degenerate, and / or absence of myocardium innervation, atresia, abdominal aortic aneurysm, acute coronary syndromes, angina, aortic aneurysm, aortic dissection, aortic rupture, aortic aneurysmal disease, aortic narrowing, atherosclerosis, atrial myxoma, atrial septal defect, brugada syndrome, heart block, cardiac arrest, cardiac sympathetic nerve loss, cardiovascular disease, carotid artery disease, coarctation of the aorta, congenital heart disease, coronary artery spasm, coronary heart disease, endocarditis, heart attack, heart disease, heart failure, heart murmurs, heart transplant, heart valve disease, inherited heart conditions, microvascular angina, microvascular disease, myocardial infarction, myocarditis, palpitations, pericarditis, progressive cardiac conduction defect, rhythm conduction disorders, spontaneous coronary artery dissection, sudden arrhythmic death syndrome, thoracic Aortic aneurysm, vasospastic angina, ventricular septal defect, and / or Wolff-Parkinson-White syndrome (WPW).

[0183] In one embodiment, the heart disease and / or pathology may be a cardiomyopathy. The cardiomyopathy may be selected from a group of cardiomyopathies consisting of: dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, restrictive cardiomyopathy, left ventricular noncompaction, takotsubo cardiomyopathy, valve disease, and / or unclassified cardiomyopathies.

[0184] In another embodiment, the heart disease and / or pathology may be a cardiac arrhythmia. The cardiac arrhythmia may be selected from a group of cardiac arrhythmias, consisting of: irregular heartbeat, long QT syndrome, sinus bradycardia, sinus arrhythmia, sinus tachycardia, premature atrial contractions, wandering atrial pacemaker, atrial tachycardia, multifocal atrial tachycardia, catecholaminergic polymorphic ventricular tachycardia, supraventricular tachycardia, atrial flutter, atrial fibrillation, AV nodal re-entrant tachycardia, AV nodal re-entrant tachycardia, arrhythmogenic right ventricular cardiomyopathy, junctional rhythm, junctional tachycardia, premature junctional contraction, premature ventricular contractions, ventricular bigeminy, non-sustained ventricular tachycardia, ventricular tachycardia, accelerated idioventricular rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, tachybrady syndrome, ventricular fibrillation, torsades de pointes, arrhythmogenic right ventricular dysplasia, re-entry ventricular arrhythmia, first-degree heart block, second-degree heart block, third-degree heart block, and / or sinoatrial block. In another embodiment, the heart disease and / or pathology may be a genetic disease. The genetic disease may be selected from a group of genetic diseases, consisting of: Congenital heart defects (Septal Defect, Atrial Septal Defect, Ventricular Septal Defect, Valve Defects, Tetralogy of Fallot, Hypoplastic left heart syndrome, Left Ventricle NonCompaction), Cardiomyopathies (Dilated Cardiomyopathy, Hypertrophic Cardiomyopathy, Arrhythmogenic cardiomyopathy), Neurodevelopmental Disorder (Rett Syndrome, Thymuti syndrome, Noonan syndrome and Costello syndrome), Myocardial Infarction (hypoxia), Myocardial fibrosis, Neurodegenerative Disorders (diabetic autonomic neuropathy; Parkinson's disease) and Familial dysautonomia (impaired development of the autonomic nervous system), atrial fibrillation, Duchenne Muscular Dystrophy, and cardiometabolic diseases.

[0185] Accordingly, in an eighth aspect of the invention, there is provided a method of carrying out a study of a heart disease and / or pathology, the method comprising :

[0186] (i) producing a diseased heart organoid (HO), or a precursor thereof, either from a cell obtained from a patient with a heart disease and / or pathology, or from a genetically modified cell exhibiting a heart disease and / or pathology;

[0187] (ii) producing a control heart organoid (HO), or a precursor thereof, either from a cell obtained from a patient without a heart disease and / or pathology, or from a genetically modified cell not exhibiting a heart disease and / or pathology; and

[0188] (iii) comparing the diseased heart organoid, or the precursor thereof, and the control heart organoid, or the precursor thereof, to study heart disease and / or pathology.

[0189] It will be appreciated that the genetically modified cell exhibiting a heart disease and / or pathology may be obtained from a genetically modified cell line. It will also be appreciated that the genetically modified cell not exhibiting a heart disease and / or pathology may be obtained from a corrected cell line, optionally wherein the cell line has been originally obtained from a patient with a heart disease and / or pathology and then corrected (e.g. through genome editing, genetic modification or genetic engineering) such that it no longer exhibits heart disease and / or pathology. The diseased HO and the control HO may have different genetic backgrounds. However, in embodiments, the diseased HO and the control HO have the same genetic background, i.e., are isogenic.

[0190] It will be appreciated that a diseased heart organoid (HO) is a HO produced either using at least one induced pluripotent stem cell (iPSC) that is derived from a patient with a heart disease and / or pathology, or using at least one induced pluripotent stem cell (iPSC) that is genetically modified to exhibit a heart disease and / or pathology. It will also be appreciated that the at least one induced pluripotent stem cell (iPSC) that is genetically modified to exhibit a heart disease and / or pathology to produce a diseased heart organoid (HO) may be produced from an iPSC that is genetically modified to have a specific mutation, such as a point mutation, that is present in a cell which is predisposed to a heart disease and / or pathology.

[0191] Accordingly, the at least one induced pluripotent stem cell (iPSC) that is genetically modified to be pre-disposed to exhibit a heart disease and / or pathology may be genetically modified to introduce a genomic mutation. The genomic mutation may be a genetic mutation. The genetic mutation may be a silent mutation, nonsense mutation, missense mutation, insertion, deletion, frameshift mutation, or substitution. The genomic mutation may comprise a genomic mutation that is present in a heart disease and / or pathology.

[0192] Accordingly, the diseased heart organoid (HO) may be produced according to the method of the second aspect, wherein either the at least one induced pluripotent stem cell (iPSC) is derived from a patient with a heart disease and / or pathology, or the at least one induced pluripotent stem cell (iPSC) is genetically modified to exhibit a heart disease and / or pathology.

[0193] It will also be appreciated that a control heart organoid (HO) is a HO produced either using at least one induced pluripotent stem cell (iPSC) that is derived from a patient without a heart disease and / or pathology, or using at least one induced pluripotent stem cell (iPSC) that is genetically modified to not exhibit a heart disease and / or pathology. It will also be appreciated that a control heart organoid (HO) may comprise an isogenic control heart organoid (HO). The term "isogenic control HO" can mean either a HO produced using at least one induced pluripotent stem cell (iPSC) that is derived from a wild type parental cell line, or a HO produced using at least one induced pluripotent stem cell (iPSC) that is derived from a parental cell line carrying at least one mutation that is present in a heart disease and / or pathology, where the at least one mutation has been reverted back to wild type with exactly the same genetic background.

[0194] Accordingly, the control heart organoid (HO) may be produced according to the method of the second aspect, wherein either the at least one induced pluripotent stem cell (iPSC) is derived from a patient without a heart disease and / or pathology, or the at least one induced pluripotent stem cell (iPSC) is genetically modified to not exhibit a heart disease and / or pathology. Accordingly, the control heart organoid (HO) may comprise an isogenic control heart organoid (HO).

[0195] The diseased heart organoid and / or the control heart organoid of the eighth aspect may be as defined as the organoid of the first aspect.

[0196] Accordingly, the method may comprise:

[0197] (i) performing the method of the second aspect, wherein either the at least one induced pluripotent stem cell (iPSC) is derived from a patient with a heart disease and / or pathology, or the at least one induced pluripotent stem cell (iPSC) is genetically modified to exhibit a heart disease and / or pathology to produce a diseased heart organoid (HO);

[0198] (ii) performing the method of the second aspect, wherein the at least one induced pluripotent stem cell (iPSC) is derived from a patient without a heart disease and / or pathology, or the at least one induced pluripotent stem cell (iPSC) is genetically modified to not exhibit a heart disease and / or pathology to produce a control heart organoid (HO); and

[0199] (iii) comparing the diseased heart organoid (HO) and the control heart organoid (HO), to study heart disease and / or pathology.

[0200] The method may comprise performing genomic, transcriptomic, proteomic, and / or metabolomic analysis on the diseased organoid and / or the control organoid. The method may comprise comparing the results of genomic, transcriptomic, proteomic, and / or metabolomic analysis performed on the diseased organoid and the results of genomic, transcriptomic, proteomic, and / or metabolomic analysis performed on the control organoid.

[0201] The method may comprise comparing the at least one diseased organoid and the at least one control organoid to identify the genesis of the heart disease and / or pathology. The method may comprise comparing the at least one diseased organoid and the at least one control organoid to identify a therapeutic agent for use in treating the heart disease and / or pathology.

[0202] In another aspect, there is provided an apparatus for carrying out a study of a heart disease and / or pathology, the apparatus comprising :

[0203] (i) a heart organoid according to any one of claims 1-8 or claim 18, or a precursor thereof according to claim 20; and

[0204] (ii) a vessel configured to permit contacting the heart organoid, or the precursor thereof, and a test agent. The inventors also envisage use of the heart organoid of the first or third aspect in a microfluidic chip.

[0205] Accordingly, in a ninth aspect, there is provided a microfluidic chip comprising the heart organoid according to the first or third aspect.

[0206] The microfluidic chip may comprise a chamber in which the heart organoid is disposed.

[0207] The microfluidic chip may comprise a media inlet in fluid communication with the chamber. The microfluidic chip may also comprise a media outlet in fluid communication with the chamber.

[0208] All of the features described herein (including any accompanying claims, abstracts, and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some features and / or steps are mutually exclusive.

[0209] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:-

[0210] Figure 1 illustrates one embodiment of a method for producing a heart organoid, in this embodiment an innervated heart organoid, wherein the production of EMOs from hiPSC spheroids, and the production of SNSs from SN progenitors is shown.

[0211] Figure 2 illustrates further details of the method shown in Figure 1, as well as details concerning the production of HOs (e.g., iHOs) from EMOs and SNSs.

[0212] Figure 3 illustrates an experimental design for drug exposure and redouts acquisition using the innervated organoid of the invention.

[0213] Figure 4 shows an illustration of a cross-section of an embodiment of an epicardiummyocardium organoid (EMO) of the invention, and an embodiment of the process for producing an EMO. Figure 5 shows representative images of immunofluorescent stained D10CC epicardiummyocardium organoids (EMOs) of the embodiment depicted in Figure 4, focused on vascularisation and myocardium composition.

[0214] Figure 6 shows representative images of immunofluorescent stained D10CC epicardiummyocardium organoids (EMOs) of the embodiment depicted in Figure 4, focused on fibroblasts and adipose tissue.

[0215] Figure 7 shows an illustration of a cross-section of an embodiment of a heart organoid (HO) (e.g., an innervated heart organoid (iHO)) of the invention, and an embodiment of the process for producing a HO (e.g., an iHO).

[0216] Figure 8 shows bright-field / fluorescent images of heart organoids (HOs) (e.g., innervated heart organoids (iHOs)) of the embodiment depicted in Figure 7 (CD31 - Endothelial cells, TUJ1 - neurons, cTnT - cardiomyocytes).

[0217] Figure 9 shows a strip plot for the effect of sympathetic neuron stimulation on contraction activity of the heart organoids (HOs) (e.g., innervated heart organoids (iHOs)) of the invention.

[0218] Examples

[0219] Through substantial experimentation, the inventors have developed a novel method that enables the reproducible production of innervated human heart organoids. The process includes a stepwise methodology that recreates, in a physiologically relevant manner, the sequential stages of heart embryogenesis, culminating with a final stage of ventricle myocardium-like tissue innervation. Specifically, the inventors have demonstrated, firstly, the production of a ventricle myocardium organoid that incorporates an epicardium-like layer at the outer surface, followed by a step of coronary-like vascular plexus induction.

[0220] Broadly, the inventors have exemplified a four-step method for the production of heart organoids, comprising :

[0221] (i) differentiating human induced pluripotent stem cells (hiPSCs) to produce proepicardium organoids (PEOs) and myocardium organoids (MOs);

[0222] (ii) aggregating the PEOs and the MOs to produce an epicardium-myocardium organoid (EMO);

[0223] (iii) differentiating human induced pluripotent stem cells (hiPSCs) to produce sympathetic neurons spheroids (SNSs); and (iv) aggregating EMOs with the SNSs to produce a heart organoid (HO).

[0224] Materials and Methods

[0225] The standard operating procedure for an innervated heart organoid is provided herein. All steps are to be performed under aseptic conditions.

[0226] Al. Myocardium and Pro-Epicardium Organoid differentiation from hiPSCs

[0227] Cell seeding on AggreWell plates and pre-differentiation culture

[0228] 1. Start the protocol with hPSCs cultured on Matrigel-coated 6 well-plates at 70% of confluence after 3 consecutive passages.

[0229] 2. Aspirate the exhausted medium and wash the cells with 1 mL / well of PBS.

[0230] 3. Add 800 pL / well of Accutase solution and incubate at 37 °C for 7 min.

[0231] 4. Flush the cells with the Accutase solution and transfer this suspension to a 50 mL conical tube with two times the volume of Accutase used. Wash the well with 1 mL / well of DMEM / F12 medium to collect the remaining cells and transfer the medium to the same conical tube. Repeat the process for all the wells.

[0232] 5. Centrifuge the 50 mL conical tube at 1000 rpm for 3 min.

[0233] 6. Discard the supernatant and resuspend the pellet in mTeSRl medium supplement with 10 pM of ROCK inhibitor for cell counting.

[0234] 7. Perform cell counting using a haemocytometer and the trypan blue dye exclusion test, under an optical microscope. Calculate the cell suspension volume necessary to inoculate the desired number of wells on the AggrewellTM800 plate (Aggrewell plate)*l.

[0235] 8. Prepare the AggreWellTM plate. Add 1 mL / well of AggreWell™ Rinsing Solution. Centrifuge at 3500 rpm for 3 minutes. Remove the solution and add 500 pL of mTeSRl supplemented with 10 pM of ROCK inhibitor. Centrifuge at 3500 rpm for 3 minutes.

[0236] 9. Transfer 1 mL of cell suspension to each well of the Aggrewell plate.

[0237] 10. Centrifuge the Aggrewell plate at 1000 rpm for 3 minutes. This step corresponds to Day -3 of differentiation.

[0238] 11. Replace exhausted medium by fresh mTeSRl after 24 and 48 hours post-seeding (Day -2 and Day -1, respectively).

[0239] 12. Register aggregate diameter at Day 0, which should be between 280-320 pm of diameter. Take BF images of 20 aggregates for analysis.

[0240] *Note 1 : The number of cells / microwell (equivalent to number of cells / aggregate) is cell line dependent and should be tested to ensure an aggregate diameter at DO within 280- 320 pm range. This value should be optimised between 3000-4500 cells / aggregate.

[0241] 3D differentiation of hiPSCs spheroids in myocardium and pro-epicardium organoids 1. On Day 0, prepare RPMI basal media supplemented with B27 minus insulin (RPMI / B27(-)) and the small molecule CHIR99021*2. Add a volume of 1.5 mL of this media to each well of the AggreWell plate and record the time. Put the AggreWell plate back into the humidified incubator at 37 °C and 5% CO2.

[0242] 2. On Day 1 of differentiation, at the same time, aspirate the medium from each well of the Aggrewell plate and replace it with 1.5 ml of RT RPMI / B27(-). Put the plate back into the CO2 incubator.

[0243] 3. On Day 3 change the media. Aspirate half of the media from each well (750 pL) and collect it in a conical tube. Mix to the previously collected exhausted RMPI / B27(-) with an equivalent volume of fresh RPMI / B27(-), and add 5 pM of the small molecule IWP4. Aspirate the remaining medium from each well of the AggreWell plate, and then add 1.5 ml per well of the combined medium containing IWP4 into each well. Put the plate back into the CO2 incubator.

[0244] 4. On Day 5 of differentiation :

[0245] 4.2. In the case of myocardium organoid differentiation - Transfer the aggregates to a 6-well ultra-low attachment plate. First remove the exhausted medium from each well of the Aggrewell plate. Then, remove the aggregates from each well by flushing with 1 mL of RPMI / B27(-), two times, and transfer to one well of a 6-well ultra-low attachment plate. Flush again the same well with ImL of RPMI / B27(-) two times and transfer the content to the same well of the 6-well ultra-low attachment plate. At the end, each well of the Aggrewell plate should have been transferred to one well of a 6-well ultra-low attachment plate, with a final volume of 2 mL / well.

[0246] 4.3. In the case of vascularised myocardium organoid differentiation - Transfer the aggregates to a 6-well ultra-low attachment plate (same procedure described in 4.2.). Culture the organoids in 2mL / well of RMPI / B27(-) supplemented with 100 ng / mL VEGF and 25ng / mL of BMP4. After 24 hours, replace with the same media.

[0247] 4.4. In the case of pro-epicardium organoid differentiation - Aspirate the medium from each well of the Aggrewell plate, and add 1.5 ml / well of Advanced DMEM / F12 + 2.5 mM Glutamax + 100 pg / mL of ascorbic acid (from now one referred as DMEM / F12) supplemented with 3 pM CHIR, 25 ng / mL BMP4 and 4 pM Retionic Acid (RA). After 24 hours, replace with the same media.

[0248] 5. On Day 7 of differentiation :

[0249] 5.2. In the case of myocardium organoid differentiation - Change culture medium to RPMI / B27 (2ml / well)

[0250] 5.3. In the case of vascularised myocardium organoid differentiation - Change culture medium to RPMI / B27 (2ml / well) + 50 ng / mL VEGF

[0251] 5.4. In the case of pro-epicardium organoids differentiation - Transfer the aggregates to a 6-well ultra-low attachment plate. First remove the exhausted medium from each well of the Aggrewell plate. Then remove the aggregates from each well by flushing with 1 mL of DMEM / F12 two times, and transfer to one well of a 6-well ultra-low attachment plate. Flush again the same well with ImL of DMEM / F12 two times and transfer the content to the same well of the 6-well ultra-low attachment plate. At the end, each well of the Aggrewell plate should have been transferred to one well of a 6-well ultra-low attachment plate, with a final volume of 2 mL / well.

[0252] 6. On Day 9 change the media, RPMI / B27 and DMEM / F12 for myocardium and proepicardium organoids, respectively. In the case of vascularised myocardium organoid differentiation Change culture medium to RPMI / B27 + 50 ng / mL VEGF.

[0253] 7. At Day 11 of differentiation collect the organoids to start the co-culture. Perform flow cytometry analysis to check differentiation yield (cTnT and CD31 for myocardium organoids and WT1 and CD31 for pro-epicardium organoids). The average yield for each tested marker should feet the values present in Table 1. The typical morphology for each organoid at this time point of differentiation can be seen in Supplementary Figure 1.

[0254] Table 1. Percentage of cTnT, CD31, and WT1 markers assessed by flow cytometry at day 11 of differentiation cTnT WT1 CD31

[0255] Myocardium Organoid 82,8±7,4% - 2,7±0,4%

[0256] Vascularised Myocardium 71,2± 1,6% - 17,3±0,9%

[0257] Organoid

[0258] Pro-epicardium Organoid - 79,7±2,2% 2,9±l,0%

[0259] *Note 2: The concentration of the small molecule CHIR99021 is cell line dependent and should be tested within the range of 7-11 pM.

[0260] A2. Production of epicardium-myocardium organoids through myocardium and pro-epicardium organoids co-culture

[0261] 1. Singularise Day 11 myocardium and Day 11 pro-epicardium organoids using 0.25% Trypsin-EDTA for 7 min at 37°C.

[0262] 2. Perform cell counting using a haemocytometer and the trypan blue dye exclusion test, under an optical microscope. Combine both cell populations in a proportion of 87% myocardium organoid cell population and 13% pro-epicardium organoid cell population in DMEM / F12 media supplemented with 10 pM of ROCK inhibitor (1.5 ml / well), and then seed on the Aggrewell plate*3.

[0263] 3. Centrifuge the Aggrewell plate at 1000 rpm for 3 minutes, twice. This corresponds to Day 0 of co-culture (Day 0 CC).

[0264] 4. At Day 1 CC, aspirate the medium from each well and transfer the organoids to a 6-well ultra-low attachment plate. The content of each well should be divided into two wells of a 6-well ultra-low attachment plate. Culture the organoids in DMEM / F12 supplemented with 5 ng / mL FGF2. Do not change media until Day 6 CC.

[0265] 5. At Day 6 CC, prepare a combined medium of 1 ml of exhausted DMEM / F12 media and 1 ml of fresh DMEM / F12 media supplemented with PDGFBB (lOng / mL) and / or VEGF (50 ng / mL). Change the media of each 6-ULA plate.

[0266] 6. At Day 8 CC, transfer the organoids to a U-shaped 96-ULA plate previously coated with anti-adherent solution. At this time, supplement again with PDGFBB (lOng / mL) and / or VEGF (50 ng / mL), combining again new and old media in 50:50 proportion.

[0267] 7. At Day 10 CC perform co-culture with SN spheroids

[0268] *Note 3: Consider a cell seeding of 12000 cells / aggregate, which corresponds to a total of 3.13 x 106 myocardium cell population and 0.47 x 106 pro-epicardium cell population per well. Use information present in Table 2 to plan the number of wells of myocardium and pro-epicardium organoids at Dll that are needed. In average, for one well of coculture, it is necessary 3 / 4 wells of myocardium organoids and 1 well of pro-epicardium organoids.

[0269] Table 2 - Average* cell number well of the 6-well ultra-low attachment at day 11 of differentiation Myocardium organoid 1.7x106 cells

[0270] Pro-epicardium organoid 5.2x106 cells information taking into to account n=7 independent experiments

[0271] 8. Singularise Day 11 myocardium and Day 11 pro-epicardium organoids using 0.25% Trypsin-EDTA for 7 min at 37°C.

[0272] 9. Perform cell counting using a haemocytometer and the trypan blue dye exclusion test, under an optical microscope. Combine both cell populations in a proportion of 87% myocardium organoid cell population and 13% pro-epicardium organoid cell population in DMEM / F12 media supplemented with 10 pM of ROCK inhibitor (100 pL / well), and then seed on the V-shaped Ultra-Low Attachment (ULA) 96-well plate*4.

[0273] 10. Centrifuge the ULA 96-well plate at 1000 rpm for 3 minutes, twice. This corresponds to Day 0 of co-culture (Day 0 CC).

[0274] 11. At Day 1 CC, aspirate the medium from each well and culture the organoids in DMEM / F12 supplemented with 5 ng / mL FGF2, 50ng / mL HGF and 50ng / mL IGFBP-2 (100 pL / well). Do not change media until Day 6 CC.

[0275] 12. At Day 6 CC, prepare a combined medium (50: 50) of exhausted DMEM / F12 media and fresh DMEM / F12 media supplemented with PDGFBB (lOng / mL) and / or VEGF (50 ng / mL). Transfer the organoids to a U-shaped ULA 96-well plate and culture the organoids in the prepared media.

[0276] 13. At Day 8 CC, aspirate 40 uL of exhausted media and add 50uL of fresh DMEM / F12 media supplemented with PDGFBB (lOng / mL) and / or VEGF (50 ng / mL).

[0277] 14. At Day 10 CC perform co-culture with SN spheroids. For quality control assessment perform immunostaining of organoid slices at this time point of co-culture. See Table 3 for antibody combinations guideline. The typical organoid struture at this time point of differentiation can be seen in Supplementary Figure 2.

[0278] *Note 4: Consider a cell seeding of 19000 cells / aggregate, which corresponds to a total of 1.65 x 104 myocardium cell population and 0.25 x 104 pro-epicardium cell population per well. Use information present in Table 2 to plan the number of wells of myocardium and pro-epicardium organoids at Dll that are needed. In average, to seed a complete 96-well plate, it is necessary only 1 well of myocardium organoids and 1 well of proepicardium organoids.

[0279] Table 3. Immunostaining analysis of epicardium-myocardium organoids

[0280] VIM + NG2 + cTnT Quantification of NG2 within the myocardium region

[0281] WT1 + cTnT + CD31 Identification of myocardium and epicardium regions Identification of vascularisation localisation Quantification of CD31 + cells within myocardium and epicardium regions Understand if vascularisation may be derived from epicardial-like cells

[0282] Laminin + Fibronectin Evaluate ECM deposition

[0283] + Collagen I

[0284] NG2 + CD31 + Laminin Deeper characterisation of the vascular plexus + DCH 1

[0285] KRT18 + WT1 + ISL1 Deeper characterisation of epicardium region cTnT + NKX2.5 + Ki-67 Quantification of myocardium growth / CMs proliferation and myocardium compaction

[0286] B. Sympathetic Neurons Spheroids Differentiation from hiPSCs

[0287] 1. Start the protocol with hPSCs cultured on Matrigel-coated plates at 70% of confluence after 3 consecutive passages.

[0288] 2. Aspirate the exhausted medium and wash the cells with 1 mL / well of PBS.

[0289] 3. Add 800 pL / well of Accutase solution and incubate at 37 °C for 7 min.

[0290] 4. Flush the cells with the Accutase solution and transfer this suspension to a 50 mL conical tube with two times the volume of Accutase used. Wash the well with 1 mL / well of DMEM / F12 medium to collect the remaining cells and transfer the medium to the same conical tube. Repeat the process for all the wells.

[0291] 5. Centrifuge the 50 mL conical tube at 1000 rpm for 3 min.

[0292] 6. Discard the supernatant and resuspend the pellet in mTeSRl medium supplement with 10 pM of ROCK inhibitor for cell counting.

[0293] 7. Perform cell counting using a haemocytometer and the trypan blue dye exclusion test, under an optical microscope. Calculate the cell suspension volume necessary to inoculate the desired number of wells on the AggrewellTM800 plate (Aggrewell plate)*5.

[0294] 8. Prepare the AggreWellTM plate. Add 1 mL / well of AggreWell™ Rinsing Solution. Centrifuge at 3500 rpm for 3 minutes. Remove the solution and add 500 pL of mTeSRl supplemented with 10 pM of ROCK inhibitor. Centrifuge at 3500 rpm for 3 minutes.

[0295] 9. Transfer 1 mL of cell suspension to each well of the Aggrewell plate.

[0296] 10. Centrifuge the Aggrewell plate at 1000 rpm for 3 minutes. This step corresponds to Day -1 of differentiation.

[0297] 11. At day 0 change the media to StemFlex® Medium supplemented with 500 nM LDN193189 (LDN) and 10 pM SB431542 (SB) (1,5 mL / well)

[0298] 12. At day 2 change the media to StemFlex® Medium supplemented with 500 nM LDN193189 (LDN) + 10 pM SB431542 (SB) + 3 pM CHIR99021 + 10 pM DAPT

[0299] 13. At Day 3 cahnge the media to StemFlex® Medium supplemented with 10 pM SB431542 (SB) + 3 pM CHIR99021 + 10 pM DAPT + 1 pM SAG

[0300] 14. At Day 4, transfer the aggregates to a ULA 6-well plate (1 well of Aggrewell to 1 well of 6-well plate) and change the media to StemFlex® Medium (3 / 4) + N2 media (1 / 4) supplemented with 3 pM CHIR99021 + 10 pM DAPT + 1 pM SAG (2 mL / well)

[0301] 15. At Day 5, replace with the same media (StemFlex® Medium (3 / 4) + N2 media (1 / 4) supplemented with 3 pM CHIR99021 + 10 pM DAPT + 1 pM SAG

[0302] 16. At Day 6, change the media to StemFlex® Medium (1 / 2) + N2 media (1 / 2) supplemented with 3 pM CHIR99021 + 10 pM DAPT + 1 pM SAG

[0303] 17. At Day 7, change the media to StemFlex® Medium (1 / 2) + N2 media (1 / 2) supplemented with 1 pM SAG

[0304] 18. At Day 8, change the media to StemFlex® Medium (1 / 4) + N2 media (3 / 4) supplemented with 1 pM SAG

[0305] 19. At Day 9, change the media to N2 media supplemented with 1 pM SAG + 10 ng / mL BMP4

[0306] 15. At Day 11, perform the re-aggregation of sympathetic neuron progenitors in V- shaped ULA 96-well plate.

[0307] 16. Singularise sympathetic neuron progenitor aggregates using Accutase for 10 min at 37OC. 17. Perform cell counting using a haemocytometer and the trypan blue dye exclusion test, under an optical microscope. Resuspend the cells in N2 maturation media (NeurobasalPlus medium + B27Plus + Glutamax + N2 + 0,2 mM Ascorbic Acid + 10 ng / mL NGF + 10 ng / mL BDNF + 10 ng / mL GDNF) supplemented with 10 pM of ROCK inhibitor (100 pL / well), and then seed on the V-shaped ULA 96-well plate*6.

[0308] 18. Centrifuge the ULA 96-well plate at 1000 rpm for 3 minutes, twice. This corresponds to Day 0 of maturation (Day 0 MT).

[0309] 19. After 24 hours, transfer the aggregates to a U-shaped ULA 96-well plate. From that point on, the medium should be changed between 3-4 days and maturation should be prologued for 30-40 days.

[0310] 20. At Day 20 MT perform a quality control assessment. Replate Day 20 MT spheroid in Matrigel-coated coverslips and perform immunostaining after 5 days. See Table 4 for antibody combinations guideline. Additionally, assess SNs activity replating Day 20 MT spheroids in Matrigel coated multielectrode array chips.

[0311] 21. At Day 30 MT, fluorescently label the spheroids using viral transfection. For this, for each well add 0,5 uL of virus in 40 pL of maturation media. After 24 hours, add 100 pl of maturation medium. In the next day change the entire medium and add 100 pl of fresh maturation medium.

[0312] 22. At Day 40 MT, perform co-culture of SN spheroids with EMOs.

[0313] *Note 5: The number of cells / microwell (equivalent to number of cells / aggregate) is cell line dependent and should be tested to ensure an aggregate diameter at DO of ±200 pm. Cell seeding densities in the range of 1000-2500 cells / aggregate should be tested.

[0314] *Note 6: The optimal seeding density is 10000 cells / aggregate. This should correspond to a sympathetic neuron progenitor size 24 hours after re-aggregation of ±300 pm.

[0315] Table 4. Immunostaining analysis of replated sympathetic neuron spheroids (antibody combinations)

[0316] TH + DBH + PRPH

[0317] GATA3 + PHOX2B + PRPH

[0318] C. Production of innervated heart organoids through assembly of epicardiummyocardium organoids and sympathetic neurons spheroids

[0319] 1. Assembly D10CC EMOs with D40 MT SNSs in a ULA 96-well plate. Transfer the each D10CC EMOs to one well of D40 MT SNSs, using P200 multichannel pipete (cut the extremity of P200 tips).

[0320] 2. Culture the assembloids in 50:50 N2 maturation medium and DMEM / F12 + 50 ng / mL of VEGF. Change the media every 3 days. 3. Follow SN projections towards the EMO region using a fluorescence microscope. After 10 days a functional innervated heart organoid should be obtained.

[0321] 4. To confirm the functional interaction between SNs and CMs, perform stimulation with 1 uM of Nicotine (Stock: Sigma: N1019-25 mL 35% (w / v) in H2O - 829 mM), and record contraction after 10 minutes.

[0322] (Prepared 5 ml of Nicotine ImM by diluting 6uL of Stock). to improve vascularisation of hPSC-derived epicardium-

[0323] Previous work developed by the inventors' group has reported a platform to produce epicardium-myocardium organoids (EMOs) from hiPSCs that present a self-organised epicardium layer that surrounds the entire surface area of a ventricle myocardium-like region, mimicking the in vivo structure of the ventricle wall (Branco et aL, 2023 Nature Communications). To progress the recreation of human heart organogenesis in vitro, the inventors followed the in vivo embryogenesis process, and designed a new model that incorporates the autonomic nervous system (ANS) of the heart in EMOs. Knowing that neurotrophic factors secreted by epicardium-derived vascular cells have been described to be involved in the extension of axons through the subepicardium and in the invasion into the myocardium, the vascularisation of EMOs was analysed. Immunostaining analysis revealed that although some endothelial-like CD31+ cells were present within and surrounding the myocardial region, the establishment of a network of vascular plexus was not evident. Therefore, the inventors incorporated an additional step into the previously reported protocol to potentiate the vascularisation of EMOs before moving to the innervation stage. Platelet-derived growth factor beta signalling has been described to be required for efficient epicardial cell migration and derivation into coronary vascular smooth muscle cell population [13,14]. In addition, the growth factor PDGFBB is a commonly used inducer of vascular smooth muscle cells specification from hPSC-derived mesoderm progenitors (Patsch et aL, 2015; Orlova et aL, 2014; Cheung et aL, 2014), specifically from hPSC-derived epicardial-like cells (Iyer et aL, 2015) in vitro. On the other hand, VEGF-A is a common inducer of endothelial-like cells specification and maintenance from hPSCs. Therefore, the combined (or stepwise) supplementation of PDGFBB and VEGFA was tested in EMOs after 6 days of co-culture. It was observed that the addition of PDGFBB induced a homogenous increase of the epicardial region, with the production of a subepicardial space composed mainly by ECM proteins (Laminin / Fibronectin and Collagen I). Additionally, the inventors observed increased staining for the pericyte marker NG2 within the epicardial region. The addition of VEGFA resulted in the production of a network of connected CD31+ tubules at the interface of epicardial and myocardial regions, with endothelial branches sprouting toward the myocardial regions. Furthermore, the inventors showed that the endothelial cells coexpress the WT1 marker, which may indicate that these cells are in fact specified from epicardial progenitors.

[0324] Example 2 - Adaptation of vascularised EMOs production to a high throughout screening platform

[0325] To improve the robustness, reproducibility, and consequent applicability of the produced platform, the inventors decided to adapt the new EMOs production protocol to a high throughput screening setting. The process of EMOs production starts with the reaggregation of both cell populations from myocardium and pro-epicardium organoids in the commercially available Aggrewell plates. After 24 hours, the re-aggregates organoids are transferred to 6-well ultra-low attachment plates (1 well of Aggrewell into 2 wells of 6-well plate), being cultured for 6 days without medium change, period in which the segregation and self-organisation of epicardial and myocardial cells occur. The translation of the re-aggregation step to a 96 well plate impaired the self-organisation process to occur in a reproducible manner. Therefore, the inventors hypothesised that the concentration of secreted factors on those 6 days of culture could be critical to induce the process. Conditioned medium after 6 days of EMOs culture was collected, and a membrane array to detect 41 targets was used to screen for possible important secreted factors. From this analysis, the inventors identified two growth factors, HGF and IGFBP2, that were considerably expressed. To test the inventors' hypothesis, the supplementation of both identified growth factors was performed in the first 6 days of EMOs production in 96-well plates. The inventors observed that this strategy proved to be sufficient to compensate for the absence of a concentrated growth factor environment, as it is present in the 6-ULA plate, and to rescue the self-organisation pattern. A scheme of the final platform can be seen in figures 1 and 2.

[0326] Example 3 - Production of Functional Sympathetic Neuron Spheroids from hiPSCs For the production of the innervated heart organoid model, the inventors first established a differentiation platform to obtain sympathetic neuron spheroids (SNS) from hiPSCs. In order to do so, the inventors performed their differentiation protocol in a three-dimensional environment. Differentiation was confirmed by RT-PCR, with a significant up-regulation of sympathetic neurons markers ASCL1 and PHOX2B at the end of the induction period (Day 10 / 12), and by immunostaining of TH, PRPH, DBH, GATA3, and PHOX2B after 30 days of differentiation (20 of maturation). The functionality of sympathetic neurons was assessed using a multielectrode array (MEA) after 50 days of differentiation. Example 4 - Assembly and functional characterisation of innervated EMOs After protocol optimisation for the production of SNS, the inventors assembled vascularised EMOs after 10 days of culture and SNS (> day 50 of differentiation) in 96- well plates. To visually follow SNs axon projections towards the EMOs organoid, SNS were fluorescently labelled with a lentiviral vector comprising a red fluorescence protein, prior to assembly. After 48 hours post-assembly, the inventors found that it was already possible to see the beginning of axon projections towards the EMOs region. After 2 weeks of co-culture, to assess the presence of a functional interaction between ventricle CMs and SN, the inventors examined the effect of SN stimulation on contraction activity of the myocardial region. In order to do so, the inventors treated assembloids with nicotine, which activates nicotinic acetylcholine receptors of post-ganglionic neurons in the ANS, and assessed beating rate of EMOs. The inventors registered a significant increase in beating rate upon nicotine stimulation, which was not observed in EMOs without innervation.

[0327] Example 5 - Use of the innervated heart organoid in a cardiotoxicitv study

[0328] Referring to Figure 3, the inventors, using the innervated heart organoids, or precursors thereof, of the invention, would be able to assess the impact of drug exposure on normal organoid function and / or structure. Thus, the inventors would be informed how exposure of said drug would impact normal in vivo heart function and / or structure.

[0329] The inventors would use day 11 MOs, day 11 EMOs, and / or day 10 iHO. The inventors would then select an appropriate range of test agent (drug) concentrations, and timings for drug exposure.

[0330] The experiment could be designed to assess the impact of acute drug exposure, i.e., up to 24 hours of exposure, by selecting timings for drug exposure of 3, 6, 12, and 24 hours, for example. Alternatively, the experiment could be designed to assess the impact of prolonged drug exposure, by selecting timings for drug exposure of 1, 2, 4, and 7 days) (see Figure 3). The inventors would use, as controls, 1) organoids exposed only to the vehicle in which the drug is diluted, for example DMSO or H2O; 2) untreated organoids; 3) organoids exposed to a cardiotoxic drug, i.e., positive controls (see Table 6 for further positive controls); and 4) organoids exposed to a non-cardiotoxic drug, such as Acetylsalicylic Acid (Aspirin), i.e., negative controls.

[0331] After drug exposure to the defined time, the inventors would wash the organoids with PBS, and culture the organoids in a drug-free culture medium for three hours. After that time, the inventors would proceed to organoid function and structure analysis (see Table 5 for readouts). All the readouts selected for analysis after drug exposure would also be acquired before drug exposure to allow for comparison.

[0332] Table 5. Readouts for function and structure analysis of heart organoids

[0333] Table 6. Cardiotoxic drugs which may be used as positive controls

[0334] Example 6 - Use of the innervated heart organoid in a developmental toxicity study, i.e., a teratogenicity study.

[0335] The inventors, using the innervated heart organoids, or precursors thereof, of the invention, would be able to assess the impact of drug exposure on normal heart development (heart organogenesis). Thus, the inventors would be informed how exposure of said drug would impact normal in vivo heart development.

[0336] The inventors would use MOs, PEOs, EMOs and / or HOs (e.g., iHOs) from any stage of development, i.e., any day of the differentiation / maturation process. For example, the inventors could use day 5-10 MOs and PEOs, day 1-10 EMOs, and / or day 1-10 HOs (e.g., iHOs). Thus, this experimental design would allow the inventors to distinguish the impact of drug exposure at different stages of heart development. The inventors would then select an appropriate range of test agent (drug) concentrations, and timings for drug exposure. The inventors would then contact the drug with MOs, PEOs, EMOs and / or HOs (e.g., iHOs), during the differentiation / maturation process.

[0337] The experiment could be designed to assess the impact of acute drug exposure, i.e., up to 24 hours of exposure, by selecting timings for drug exposure of 3, 6, 12, and 24 hours, for example. Alternatively, the experiment could be designed to assess the impact of prolonged drug exposure, by selecting timings for drug exposure of 1, 2, 4, and 7 days). The inventors would use, as controls, 1) organoids exposed only to the vehicle in which the drug is diluted, for example DMSO or H2O; 2) untreated organoids; 3) organoids exposed to a teratogenic drug, such as Thalidomide or Valproic Acid, i.e., positive controls; and 4) organoids exposed to a non-teratogenic drug, such as Folic Acid, i.e., negative controls.

[0338] The inventors would then proceed to develop the MOs, PEOs, EMOs and / or HOs (e.g., iHOs) in accordance with the methods of the invention. Once mature HOs (e.g., iHOs) are obtained, for example, at least day 20 HOs (e.g., iHOs), the inventors would perform organoid function and structure analysis (see Table 5 for readouts). The selected redouts would be translatable to clinically relevant parameters, as discussed in more detail below. Heart organoid size and morphology, as well as function (using a fluorescent calcium sensitive dye), would be assessed, through the use of bright field and live- fluorescence microscopy analysis, respectively.

[0339] Depending on whether either the innervated heart organoid, or a precursor of the organoid, is contacted with the test compound, data concerning different biologically related developmental aspects will be gathered . For example, if MOs were contacted with the test compound, the inventors would be able to see if the myocardium layer or the organoid develops normally, or whether myocardium cavity formation is abnormal. In another example, if EMOs were contacted with the test compound, using fluorescently labelled epicardial cells, the inventors would assess whether the epicardial layer develops as expected, or abnormally.

[0340] In another example, the inventors predict that differences in EMO size, which would be assessed through bright field imaging, could be indicative of compromised myocardium growth. In a further example concerning HOs (e.g., iHOs), sympathetic neurons would be fluorescently labelled to confirm the formation of an innervated network at the surface of the organoid. In addition, contraction profile of the organoids would be assessed to investigate possible functional defects.

[0341] As discussed above, the selected redouts would be translatable to clinically relevant parameters. For example, MOs represent the establishment of the myocardium layer of the heart, and the myocardium cavity of the innervated heart organoid; assessing the impact of test compound exposure on heart organoid formation would be of significant importance as, in vivo, defects at this early stage can be indicative of embryo death and abortion. In another example, PEOs and / or EMOs represent the next stage of heart development; assessing the impact of test compound exposure on epicardial layer formation and myocardium growth and maturation progression would also be of importance as, in vivo, abnormal development at this stage can be indicative of growth retardation and / or congenital anomalies and / or malformations. In a further example, assessing the impact of the test compound on SNs and / or HOs (e.g., iHOs) would be important for identifying alterations in organoid function, and thus indicative of normal in vivo heart function. This is because physiological cardiac function is regulated by the autonomic nervous system, thus suggesting that alterations in excitability, density, distribution, and / or neurotransmitter content of cardiac sympathetic fibres, each of which may be studied using the organoids of the invention, may cause arrhythmic events. Example 7 - Use of the innervated heart organoid to study heart pathologies and for disease modelling, wherein the study of a cardiomyopathy is exemplified The inventors, using the innervated heart organoids, or precursors thereof, of the invention, would be able to study heart pathologies, such as cardiomyopathies, and model heart diseases. For example, the inventors could study dilated cardiomyopathy.

[0342] The inventors would use day 11 MOs, day 10 EMOs, and / or day 10 HOs (e.g., iHOs). The inventors would use hiPSCs derived from patients with dilated cardiomyopathy, the respective isogenic controls, such as patients without dilated cardiomyopathy, i.e., negative controls, and heart tissue derived from the same patients with dilated cardiomyopathy, i.e., positive controls. The inventors would then differentiate both hiPSC cell lines, i.e., those derived from patients and the respective isogenic controls, using the same conditions, for example, using those described in the above Materials and Methods. The inventors would then assess the differences in the resulting tissues in terms of differentiation progression, normal function, and structure. Table 5 shows the applicable readouts for such function and structure analysis.

[0343] Example 8 - Epicardium-myocardium organoid (EMO) composition analysis Referring to Figure 4, there is shown an illustration of a cross-section of an epicardiummyocardium organoid (EMO), comprising, from the outermost layer to the innermost layer, 1) the epicardial region, comprising the mesothelial epicardium, the subepicardial space comprising coronary vascularisation (endothelial cells and pericytes) and adipose tissue, 2) the myocardial region, comprising the compact myocardium comprising coronary vascularisation (endothelial cells and pericytes) and fibroblasts and smooth muscle cells, and the trabecular myocardium. A depiction of the production of an EMO is also shown, comprising the re-aggregation of at least one "day 11" (Dll) proepicardium organoid (PEO) and at least one "day 11" (Dll) myocardium organoid (MO) in a 96-ULA well plate together with fibroblast growth factor 2 (FGF2) between around "day 0" of co-culture (D0CC) and around "day 6" of co-culture (D6CC) and vascular endothelial growth factor A (VEGFA) / platelet-derived growth factor-BB (dimer composed of two B subunits) (PDGFBB) between around "day 6" of co-culture (D6CC) and around "day 10" of co-culture (D10CC). Around D10CC, an EMO is produced.

[0344] Referring to Figure 5, there is shown representative images of immunofluorescent stained D10CC epicardium-myocardium organoids (EMOs) of the embodiment depicted in Figure 4 (organoid slices). The images highlight the 1) two distinct layers in the epicardium region, the mesothelium (WT1+ / NR2F2+ / DACH1+ cells) and the subepicardium space (WT1- / +Iow, NR2F2+ and DACH1+ cells), involving the NKX2.5+ myocardial area, 2) the coronary-like vasculature (CD31+ / DACH1+ cells), co-stained with the ECM laminin (LAM) and the pericyte marker NG2, and 3) two distinct subpopulations of ECs, namely CD31+ / NR2F2+ cells within the sub-epicardium space and in the myocardium surface, and CD31+ / WT1+ / NR2F2- cells within the myocardium region. Scale bars, 100 pm.

[0345] Referring to Figure 6, there is shown representative images of immunofluorescent stained D10CC epicardium-myocardium organoids (EMOs) of the embodiment depicted in Figure 4 (organoid slices), highlighting the fibroblast population (ECM Collagen I and Fibronectin, and Vimentin (VIM)), mainly within the compact myocardial region, and fatty acid deposition in the epicardium space. Scale bars, 100 pm. composition analysis

[0346] Referring to Figure 7, there is shown an illustration of a cross-section of a heart organoid (HO) (e.g., an innervated heart organoid (iHO)), comprising an autonomic nervous system spheroid (ANSS), and an EMO comprising, from the outermost layer to the innermost layer, the mesothelial epicardium comprising innervation, the subepicardial space comprising innervation, coronary vascularisation (endothelial cells and pericytes), and adipose tissue, the compact myocardium comprising coronary vascularisation (endothelial cells and pericytes) and fibroblasts and smooth muscle cells, and the trabecular myocardium. A depiction of the production of a HO (e.g., an iHO) is also shown, comprising the assembly of at least one "day 10" (D10) epicardium-myocardium organoid (EMO) and at least one "day 40 to 50" (D40-D50) autonomic nervous system spheroid (ANSS) in a 96-ULA well plate. Around "day 10 to day 15" (DIO-15) after assembly, a HO is produced.

[0347] Referring to Figure 8, there is shown bright-field / fluorescent images of heart organoids (HOs) (e.g., innervated heart organoids (iHOs)) of the embodiment depicted in Figure 8 (CD31 - endothelial cells, TUJ1 - neurons, cTnT - cardiomyocytes). The illustration of Figure 8 and the images of Figure 8 represent an embodiment of the final HO product of the invention, comprising innervation, vascularisation, and adipose tissue.

[0348] Referring to Figure 9, there is shown a strip plot for the effect of sympathetic neuron stimulation on contraction activity of the heart organoids (HOs) (e.g., innervated heart organoids (iHOs)) of the invention. The spontaneous beating rate of the control organoids, i.e., organoids which were not contacted with a stimulus, was between 5 and 30 beats per minute (BPM). The beating rate of the experimental organoids, i.e., organoids which were been contacted with a stimulus (i.e., nicotine, a stimulant), was between 20 and 50 beats per minute (BPM). Accordingly, there was a significant increase in beating rate upon contact of the organoids with nicotine, which was not observed in organoids without innervation.

[0349] Summary

[0350] The inventors have developed a novel methodology that enables the consistent and reproducible production of heart organoids that are innervated, vascularised, and / or comprise adipose tissue, and typically, the organoids are innervated. The inventors' methodology recreates the sequential stages of in vivo heart embryogenesis in a physiologically relevant manner, culminating with a final stage of ventricle myocardiumlike tissue innervation, to produce a heart organoid (HO), such as an innervated heart organoid (iHO). The inventors have demonstrated, firstly, the production of a ventricle myocardium organoid that incorporates an epicardium-like layer at the outer surface, followed by a step of coronary-like vascular plexus induction.

[0351] As such, the present invention provides a heart organoid (HO) (which is typically an innervated heart organoid (iHO), but may also be vascularised and / or comprise adipose tissue) model that incorporates the ANS, thus providing an improved platform for the evaluation of the efficacy, cardiotoxicity, and teratogenicity of pharmaceuticals, as well as for the study of heart disease and / or pathology, and in particular those that may be linked to an unbalanced, degenerate, or absence of myocardium innervation.

[0352] References

[0353] 1. Volz, K.S.; Jacobs, A.H.; Chen, H.I.; Poduri, A.; McKay, A.S.; Riordan, D.P.; Kofler, N.; Kitajewski, J.; Weissman, I.; Red-Horse, K. Pericytes Are Progenitors for Coronary Artery Smooth Muscle. eLife 2015, 4, 1- 22, doi: 10.7554 / eLife.10036.

[0354] 2. Nam, J.; Onitsuka, I.; Hatch, J.; Uchida, Y.; Ray, S.; Huang, S.; Li, W.; Zang, H.; Ruiz-Lozano, P.; Mukouyama, Y.S. Coronary Veins Determine the Pattern of Sympathetic Innervation in the Developing Heart. Development (Cambridge) 2013, 140, 1475-1485, doi: 10.1242 / dev.087601.

[0355] 3. Ge, Y.; Smits, A.M.; van Munsteren, J.C.; Gittenberger-de Groot, A.C.; Poelmann, R.E.; van Brakel, T.J.; Schalij, M .; Goumans, MJ.; DeRuiter, M.C.; Jongbloed, M.R.M . Human Epicardium-Derived Cells Reinforce Cardiac Sympathetic Innervation. Journal of Molecular and Cellular Cardiology 2020, 143, 26-37, doi:10.1016 / j.yjmcc.2020.04.006.

[0356] 4. Takeuchi, A.; Nakafutami, S.; Tani, H.; Mori, M.; Takayama, Y.; Moriguchi, H.; Kotani, K.; Miwa, K.; Lee, J.K.; Noshiro, M.; et al. Device for Co-Culture of Sympathetic Neurons and Cardiomyocytes Using Microfabrication. Lab on a Chip 2011, 11, 2268-2275, doi: 10.1039 / c0lc00327a.

[0357] 5. Oh, Y.; Cho, G.-S.; Li, Z.; Hong, I.; Zhu, R.; Kim, M.-J.; Kim, Y.J.; Tampakakis, E.; Tung, L.; Huganir, R.; et al. Functional Coupling with Cardiac Muscle Promotes Maturation of HPSC-Derived Sympathetic Neurons. Cell Stem Cell 2016, 19, 95-106, doi : 10.1016 / j . stem.2016.05.002.

[0358] 6. Oiwa, K.; Shimba, K.; Numata, T.; Takeuchi, A.; Kotani, K.; Jimbo, Y. A Device for Co-Culturing Autonomic Neurons and Cardiomyocytes Using Micro-Fabrication Techniques. Integrative Biology (United Kingdom) 2016, 8, 341-348, doi: 10.1039 / c5ib00273g. 7. Takayama, Y.; Kushige, H.; Akagi, Y.; Suzuki, Y.; Kumagai, Y.; Kida, Y.S. Selective Induction of Human Autonomic Neurons Enables Precise Control of Cardiomyocyte Beating. Scientific Reports 2020, 10, 1- 13, doi: 10.1038 / S41598-020-66303-3.

[0359] 8. Sakai, K.; Shimba, K.; Ishizuka, K.; Yang, Z.; Oiwa, K.; Takeuchi, A.; Kotani, K.; Jimbo, Y. Functional Innervation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes by Co-Culture with Sympathetic Neurons Developed Using a Microtunnel Technique. Biochemical and Biophysical Research Communications 2017, 494, 138-143, doi: 10.1016 / j.bbrc.2017.10.065.

[0360] 9. Winbo, A.; Ramanan, S.; Eugster, E.; Jovinge, S.; Skinner, J.R.; Montgomery, J.M. Functional Coculture of Sympathetic Neurons and Cardiomyocytes Derived from Human-Induced Pluripotent Stem Cells. American Journal of Physiology - Heart and Circulatory Physiology 2020, 319, H927-H937, doi: 10.1152 / AJPHEART.00546.2020.

[0361] 10. Kowalski, W.J.; Garcia-Pak, I.H.; Li, W.; Uosaki, H.; Tampakakis, E.; Zou, J.; Lin, Y.; Patterson, K.; Kwon, C.; Mukouyama, Y.S. Sympathetic Neurons Regulate Cardiomyocyte Maturation in Culture. Frontiers in Cell and Developmental Biology 2022, 10, 1-13, doi: 10.3389 / fcell.2022.850645.

[0362] 11. Li, N.; Edel, M.; Liu, K.; Denning, C.; Betts, J.; Neely, O.C.; Li, D.; Paterson, D.J. Human Induced Pluripotent Stem Cell-Derived Cardiac Myocytes and Sympathetic Neurons in Disease Modelling. Philosophical Transactions of the Royal Society B: Biological Sciences 2023, 378, doi: 10.1098 / rstb.2022.0173.

[0363] 12. Hernandez, I.; Ramirez, S.P.; Salazar, W. V.; Mendivil, S.; Guevara, A.; Patel, A.; Loyola, C.D.; Dorado, Z.N.; Joddar, B. A Semi-Three-Dimensional Bioprinted Neurocardiac System for Tissue Engineering of a Cardiac Autonomic Nervous System Model. Bioengineering 2023, 10, doi: 10.3390 / bioengineeringl0070834.

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Claims

Claims1. A heart organoid that: i) is innervated; ii) is vascularised; and / or iii) comprises adipose tissue.

2. The heart organoid according to claim 1, wherein the heart organoid comprises:(i) a model autonomic nervous system (ANS);(ii) an autonomic nervous system spheroid (ANSS); and / or(iii) at least 0.0001% of its surface area covered by sympathetic neurons (SNs) and / or parasympathetic neurons (PSNs).

3. The heart organoid according to either claim 1 or claim 2, wherein the heart organoid comprises an inner myocardial region, optionally wherein the inner myocardial region comprises compact myocardium and trabecular myocardium, and / or an outer region, optionally wherein the outer region comprises epicardium and subepicardial space.

4. The heart organoid according to any preceding claim, wherein the heart organoid comprises at least one cardiac tissue type selected from a group of cardiac tissue types consisting of: sympathetic neuron, parasympathetic neuron, epicardium, subepicardium, myocardium, endocardium, coronary vascular plexus, and adipose tissue, optionally wherein the epicardium comprises mesothelial cells, adipocytes, and / or connective tissue, and / or wherein the myocardium comprises cardiomyocytes.

5. The heart organoid according to claim 4, wherein:(i) the myocardium comprises at least one neuron;(ii) the heart organoid comprises at least one sympathetic neuron (SN) located at an interface of the myocardium and the epicardium of the organoid and / or within the myocardium and / or epicardium of the organoid;(iii) the adipose tissue is located between the myocardium and the epicardium; and / or(iv) the subepicardium comprises an extracellular matrix (ECM) protein.

6. The heart organoid according to any preceding claim, wherein the heart organoid comprises a sinoatrial (SA) node, an atrioventricular (AV) node, an AV bundle / bundle of His, and / or Purkinje fibres.

7. The heart organoid according to any preceding claim, wherein the heart organoid comprises:(i) a blood vessel, optionally wherein the blood vessel is an artery, an arteriole, a capillary, a venule, and / or a vein; and / or(ii) a vascular plexus, optionally a coronary vascular plexus disposed in a subepicardial space and / or beneath the epicardium.

8. The heart organoid according to any preceding claim, wherein the heart organoid is contractile, optionally wherein(i) the heart organoid contracts spontaneously;(ii) the heart organoid contracts at a rate of between 1 and 100 BPM; and / or(iii) the heart organoid contracts when contacted with a stimulus.

9. A method of producing a heart organoid, wherein the heart organoid is innervated, is vascularised, and / or comprises adipose tissue, the method comprising :(i) differentiating at least one induced pluripotent stem cell (iPSC) to produce at least one pro-epicardium organoid (PEO) and at least one myocardium organoid (MO);(ii) contacting the at least one PEO and the at least one MO to produce at least one epicardium-myocardium organoid (EMO);(iii) differentiating at least one induced pluripotent stem cell (iPSC) to produce at least one sympathetic neuron spheroid (SNS) and / or parasympathetic neuron spheroid (PSNS); and(iv) contacting the at least one MO and / or EMO with the at least one SNS and / or at least one PSNS to produce a heart organoid (HO) that is innervated, is vascularised, and / or comprises adipose tissue.

10. The method according to claim 9, wherein the method comprises differentiating at least one animal induced pluripotent stem cell (iPSC), at least one mammal iPSC, at least one pig iPSC, at least one mouse iPSC, and / or at least one human iPSC (hiPSC), to produce at least one pro-epicardium organoid (PEO) and at least one myocardium organoid (MO).

11. The method according to either claim 9 or claim 10, wherein the at least one EMO is derived from PEOs and MOs at a ratio of between 1 : 1 and 1:50.

12. The method according to any one of claims 9 to 11, wherein the at least one PEO and the at least one MO is contacted with at least one growth factor.

13. The method according to any one of claims 9 to 12, wherein the method comprises differentiating at least one animal induced pluripotent stem cell (iPSC), at least one mammal iPSC, at least one pig iPSC, at least one mouse iPSC, and / or at least one human iPSC (hiPSC), to produce at least one sympathetic neuron spheroid (SNS) and / or at least one parasympathetic neuron spheroid (PSNS).

14. The method according to any one of claims 9 to 13, wherein the at least one iPSC is differentiated to produce at least one SNS and / or PSNS in a three-dimensional environment.

15. The method according to any one of claims 9 to 14, wherein the at least one iPSC is contacted with at least one growth factor.

16. The method according to any one of claims 9 to 15, wherein:(i) steps (i)-(iv) are performed separately, consecutively, and / or at the same time; and / or(ii) the method of step (i) is performed prior to step (ii), the method of step (ii) is performed prior to step (iv), the method of step (iii) is performed prior to, or subsequent to steps (i) and / or (ii), the method of step (iii) is performed prior to step (iv), and / or the method of step (iv) is performed subsequent to steps (i), (ii), and / or (iii).

17. The method according to any one of claims 9 to 16, wherein the method further comprises, subsequent to the method of step (iv) :(v) maintaining the heart organoid (HO) that is innervated, is vascularised, and / or comprises adipose tissue in media, optionally wherein maintaining the heart organoid comprises culturing the heart organoid in the media for at least one hour.

18. A heart organoid that is innervated, is vascularised, and / or comprises adipose tissue obtained, or obtainable by, the method according to any one of claims 9 to 17.

19. Use of the heart organoid that is innervated, is vascularised, and / or comprises adipose tissue according to any one of claims 1-8 or claim 18, or a precursor thereof, in a drug evaluation screen.

20. The use of the heart organoid according to claim 19, wherein the precursor of the heart organoid comprises a human induced pluripotent stem cell (hiPSC), proepicardium organoid (PEO), myocardium organoid (MO), epicardium-myocardium organoid (EMO), sympathetic neuron spheroid (SNS), and / or parasympathetic neuron spheroid (PSNS).

21. A method of carrying out a drug evaluation screen, the method comprising contacting the heart organoid that is innervated, is vascularised, and / or comprises adipose tissue according to any one of claims 1-8 or claim 18, or a precursor thereof according to claim 20, with a test agent, and analysing the effects of the test agent on the heart organoid, or the precursor thereof.

22. The use of the heart organoid according to either claim 19 or claim 20, wherein:(i) the heart organoid is used for drug efficacy analysis, toxicity analysis, teratogenicity analysis, pharmacokinetics analysis, pharmacodynamics analysis, and / or functional analysis;(ii) the heart organoid is used for assessing drug mechanism of action, adverse effects, and / or drug-induced cardiac injury; and / or(iii) the heart organoid is used in an in vitro and / or ex vivo assay.

23. The method according to claim 21, wherein the test agent is a chemical compound or biological agent, optionally wherein the test agent is a drug or pharmaceutical.

24. An apparatus for carrying out a drug evaluation screen, the apparatus comprising :(i) a heart organoid according to any one of claims 1-8 or claim 18, or a precursor thereof according to claim 20; and(ii) a vessel configured to permit contacting the heart organoid, or the precursor thereof, and a test agent.

25. The apparatus according to claim 24, wherein the vessel is a test tube, a microtube, a well plate, a spot plate, a microplate, a multiwell plate, a microwell plate, a tip, a reagent container, a vial, a microchannel device, and / or a microfluidic chip.

26. Use of the heart organoid according to any one of claims 1 to 8 or claim 18, or a precursor thereof according to claim 20, to study a heart disease and / or pathology.

27. The use of the heart organoid, or precursor thereof, according to claim 26, wherein:(i) the heart organoid, or precursor thereof, is used for disease modelling;(ii) the heart organoid, or precursor thereof, is genetically modified and / or genome edited; and / or(iii) the heart organoid, or precursor thereof, is used in an in vitro and / or ex vivo assay.

28. Use of the heart organoid according to either claim 26 or claim 27, wherein the heart disease and / or pathology is a cardiomyopathy, a cardiac arrhythmia, and / or a genetic disease.

29. A method of carrying out a study of a heart disease and / or pathology, the method comprising :(i) producing a diseased heart organoid (HO), or a precursor thereof, either from a cell obtained from a patient with a heart disease and / or pathology, or from a genetically modified cell exhibiting a heart disease and / or pathology;(ii) producing a control heart organoid (HO), or a precursor thereof, either from a cell obtained from a patient without a heart disease and / or pathology, or from a genetically modified cell not exhibiting a heart disease and / or pathology; and(iii) comparing the diseased heart organoid, or the precursor thereof, and the control heart organoid, or the precursor thereof, to study heart disease and / or pathology.

30. The method according to claim 29, wherein the method comprises performing genomic, transcriptomic, proteomic, and / or metabolomic analysis on the diseased organoid and / or the control organoid.

31. The method according to either claim 29 or claim 30, wherein the method comprises comparing the at least one diseased organoid and the at least one control organoid to identify the genesis of the heart disease and / or pathology and / or to identify a therapeutic agent for use in treating the heart disease and / or pathology.

32. An apparatus for carrying out a study of a heart disease and / or pathology, the apparatus comprising :(i) a heart organoid according to any one of claims 1-8 or claim 18, or a precursor thereof according to claim 20; and(ii) a vessel configured to permit contacting the heart organoid, or the precursor thereof, and a test agent.

33. A microfluidic chip comprising the heart organoid according to any one of claims 1 to 8 or claim 18.

34. The microfluidic chip according to claim 33, wherein the microfluidic chip comprises a chamber in which the heart organoid is disposed, optionally wherein the microfluidic chip comprises a media inlet in fluid communication with the chamber and / or a media outlet in fluid communication with the chamber.

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