Plurality of cardiac tissue culture fusions

Abstract

The present invention provides a cardiac tissue model comprising cardiac tissue having at least one lumen or central cavity, wherein the cardiac tissue model comprises at least two different cardiac tissues selected from left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular canal tissue, sinoatrial node tissue, and atrioventricular node tissue, the central cavity can be shared by at least two different cardiac tissues, and / or at least two different cardiac tissues comprise the ability to propagate calcium signaling connections and / or tissue contractions, a method of generating such a tissue model, and the use of the tissue model for screening purposes.

Description

[Technical field]

[0001] The present invention relates to the field of cardiac tissue model generation. [Background technology]

[0002] Congenital heart disease (CHD) is the most common birth defect in human development and the most common cause of embryonic and fetal death. CHD occurs most often in specific compartments of the embryonic heart, such as the outflow tract (OFT), atrium, atrioventricular canal (AVC), right ventricle (RV), and most rarely in the left ventricle (LV). In about 75% of CHDs, the underlying cause remains unknown. These may result from undiscovered genetic mutations, environmental factors, or a combination of these effects. To test and study possible causes, we need models that represent all compartments of the developing human heart.

[0003] CHD often occurs early in embryonic development before pregnancy is detected, making it difficult to characterize defect etiology. These difficulties are exacerbated by the lack of control over genetic background and environmental interactions during human embryonic development. The complexity, speed, inaccessibility, and species-specific physiological differences make it difficult to understand the pathogenesis of human compartment-specific defects in animals. Thus, recent human self-organizing cardiac organoid models are complementary, offering greater accessibility, reductionist dissection of mechanisms, and high-throughput statistical significance capabilities. However, these systems do not allow for mechanistic analysis of defects that represent all interacting compartments (OFT, AVC, atrium, RV, and LV) of the human embryonic heart.

[0004] WO 2019 / 174879(A1) describes engineered cardiac tissue organoids grown into multi-layered aggregates containing large amounts of non-cardiac cells, such as foregut endoderm cells.

[0005] Hofbauer et al. (Cell 2021;184(12):3299-3317.e22) and International Publication No. 2021 / 186044 (A1) disclose the generation of left ventricular cardiac organoids (cardioids) by self-organization. Although the conventional artificial heart valve tissue is improved by self-organization after initiating cardiac differentiation using specific drift factors, the present invention has not yet shown the diversity of in vivo heart development.

[0006] Therefore, it is an object to provide different and more advanced artificial heart tissues that can model different or greater diversity of the developing heart. SUMMARY OF THE INVENTION

[0007] The present invention provides a cardiac tissue model comprising cardiac tissue having at least one inner cavity or central chamber, the cardiac tissue model comprising at least two different cardiac tissues selected from left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular canal tissue, sinoatrial node tissue, and atrioventricular node tissue, the central chamber being shareable by at least two different cardiac tissues, and / or at least two different cardiac tissues comprising the ability to propagate calcium signaling connections and / or tissue contractions. Also provided is a cardiac tissue model comprising cardiac tissue having a central chamber, wherein the central chamber is shareable by at least two different cardiac tissues selected from left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular canal tissue, sinoatrial node tissue, and atrioventricular node tissue.

[0008] The present invention further provides a method for generating a heart tissue model, comprising generating at least two different heart tissues in vitro, wherein the different heart tissues are selected from a left ventricular progenitor first heart region tissue, a right ventricle / outflow tract progenitor second heart region tissue, an atrial progenitor second heart region tissue, an atrioventricular canal progenitor second heart region tissue, a sinoatrial node progenitor second heart region tissue, and an atrioventricular node tissue; fusing at least two heart tissues; culturing the fused tissue model; and propagating calcium signal transduction connections and / or the ability to contract tissues and / or forming a central heart cavity between different heart tissues.

[0009] The present invention further provides a heart tissue model selected from a right ventricular tissue model comprising cells expressing expression markers IRX1, IRX2 and PRDX1, an atrial tissue model comprising cells expressing expression markers NR2F1, NR2F2 and HEY1; an outflow tract tissue model comprising cells expressing expression markers WNT5A, MSX1, BMP4 and RSPO3; an atrioventricular canal tissue model comprising cells expressing expression markers TBX2, MSX2 and RSPO3; a sinoatrial node tissue model comprising cells expressing expression markers SHOX2, TBX3, HCN4, ISL1 and GJC1; and an atrioventricular node tissue model comprising cells expressing expression markers TBX3, TBX5, KCNE1, HCN4 and GJC1.

[0010] The present invention further provides a method for screening or testing a candidate compound for its effect on heart development and / or function, comprising generating a heart tissue model according to the present invention while treating cells with the candidate compound, and comparing the development of the heart tissue model with the development and / or function of a heart tissue model not treated with the candidate compound.

[0011] Furthermore, a method for observing the effects of genes that are suppressed, mutated, or overexpressed during heart development, comprising generating a heart tissue model according to the present invention, wherein the cells have a suppressed or mutated candidate gene or overexpress a candidate gene, and comparing the development of the heart tissue model with the development of a heart tissue model not generated using a suppressed, mutated, or overexpressed gene.

[0012] Furthermore, a method for screening or testing a candidate compound for its effect on heart function, comprising treating any heart tissue model of the present invention with the candidate compound and comparing the function of the heart tissue model not treated with the candidate compound.

[0013] The present invention further provides a method for treating heart damage in a patient, comprising transplanting cells derived from the heart tissue model of the present invention into the damage.

[0014] The present invention further provides a cell culture medium useful for any one of the methods or specific method steps of the present invention. Such media can be combined in kits of different media or kits of one or more media and other means used in the methods of the present invention (e.g., carriers or templates used for the fusion of cultured tissues).

[0015] All embodiments of the present invention are described in conjunction with the following detailed description, and all preferred embodiments relate to all embodiments, aspects, methods, heart tissue models, organoids, uses, media, and kits, etc. For example, the medium and kit or their components can be used in the method of the present invention or can be suitable for the method of the present invention. Any component used in the described method can be part of the medium or kit. The tissue model or organoid of the present invention is the result of the method of the present invention or can be used in the method and use of the present invention. The preferred detailed description of the method of the present invention is similarly interpreted with respect to the suitability of the organoid or tissue model of the present invention obtained or used. All embodiments can be combined with each other unless otherwise specified.

Mode for Carrying Out the Invention

[0016] The present invention provides a cardiac tissue model comprising cardiac tissue having at least one lumen or central lumen, wherein the cardiac tissue model comprises at least two different cardiac tissues selected from left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular canal tissue, sinoatrial node tissue, and atrioventricular node tissue, the central lumen can be shared by at least two different cardiac tissues, and / or at least two different cardiac tissues comprise the ability to propagate calcium signaling connections and / or tissue contractions. Also provided is a cardiac tissue model comprising cardiac tissue having a central lumen, wherein the central lumen is shared by at least two different cardiac tissues selected from left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular canal tissue, sinoatrial node tissue, and atrioventricular node tissue. The tissue model of the present invention can be generated from mesodermal cells (mesodermal cells can be generated from pluripotent cells, such as induced pluripotent cells, or embryonic stem cells). The tissue model comprises a lumen in at least one of the different cardiac tissues. The lumen can extend into at least one other of the different cardiac tissues, for example, the lumen is directly surrounded by at least two of the different cardiac tissues. Here, such a lumen is referred to as the central lumen. The central lumen is formed from the tissue fusion of cardiac tissues selected from left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular canal tissue, sinoatrial node tissue, and atrioventricular node tissue, which are at least two different cardiac tissues, or their tissue precursors, left ventricular progenitor first heart field tissue, right ventricular / outflow tract progenitor anterior second heart field tissue, atrial progenitor posterior second heart field tissue, outflow tract progenitor anterior second heart field tissue, atrioventricular canal progenitor posterior second heart field tissue, and sinoatrial node progenitor posterior second heart field tissue. The atrioventricular canal progenitor second heart field tissue is the progenitor tissue of the atrioventricular canal tissue and the atrioventricular node tissue. Usually, the central lumen is formed after fusion. The central lumen can start as a lumen in one of the different tissues or precursors and then extend into one or more of the other different cardiac tissues. Precursors of the central lumen, such as small tissue vesicles or lumens, can already be present in different cardiac tissues before fusion. The different cardiac tissues or tissue precursors are also aspects of the present invention.

[0017] The tissue model of the present invention preferably includes at least two different heart tissues including electrophysiological signal transduction connections, such as calcium signal transduction connections in particular. An electrophysiological signal transduction connection means that different heart tissues are functionally connected and can communicate through cell - to - cell connections and through calcium / voltage signal transduction events. Such electrophysiological signal transduction connections can be due to gap junctions and / or ion channels between cells of different heart tissues. Furthermore, tight junctions and / or desmosomes may be present for electrophysiological signal transduction connections to maintain the structural connection of cells and to facilitate or improve signal propagation. Electrophysiological signal transduction can be traced through either calcium or voltage signal propagation. A calcium signal can be the release of calcium from the ER within the cell to the cytoplasm. Such a signal can be propagated to adjacent cells. Calcium signal transduction connections can be observed by dyes or reporter strains that trace calcium or voltage signal propagation. The dye can be an intracellular calcium - sensitive dye or a voltage - sensitive dye (e.g., FluoroVolt). The dye can enter the cell by expressing a calcium - or voltage - sensitive dye as a protein or a transgene. Alternatively, the dye can be introduced into the cell through the cell - culture medium. Further methods for measuring signal transduction events and voltage signal transduction connections between different heart tissues can be detected using multi - electrode arrays.

[0018] The tissue model of the present invention may include at least two different heart tissues having the ability to propagate contraction. The contraction is tissue contraction, and the cells of the tissue can perform contraction activities. Usually, for example, stimulation by cell action potential and / or calcium signal transduction can cause cell contraction. Contraction propagation means that the contraction of one of the heart tissues can be propagated to another one of the different heart tissues, usually the adjacent heart tissue. The contraction can be stimulated by the contraction of the adjacent heart tissue, but is usually stimulated via a calcium signal transduction connection. Therefore, these embodiments may be combined, that is, the heart tissue model may include a calcium signal transduction connection and contraction propagation between at least two different heart tissues. The contraction can start in one of the different tissues and be observed as a pulsatile behavior that propagates or projects to the adjacent heart tissue. The adjacent heart tissue can start to contract or pulsate in response to the pulsation or contraction of the initial heart tissue. The response may have a short time delay. The contraction propagation may be the contraction of the lumen or the central heart cavity. The contraction may be the contraction by cardiomyocytes. Cardiomyocytes are usually the main contractile cells. Other cell types such as fibroblasts and endothelial cells may be particularly involved in signal propagation for contraction.

[0019] The heart tissue model of the present invention is an advanced tissue model that reproduces the function of a specific heart compartment. Therefore, this is preferably a heart organoid, also called a cardioide.

[0020] "Selected from" means that the selected species can be derived from any one of the members classified as selectable species.

[0021] The present invention further provides a method for generating a heart tissue model, comprising generating at least two different heart tissues in vitro, wherein the different heart tissues are selected from a left ventricular progenitor first heart region tissue, a right ventricle / outflow tract progenitor anterior second heart region tissue, a right ventricle progenitor anterior second heart region tissue, an atrium progenitor posterior second heart region tissue, an outflow tract progenitor anterior second heart region tissue, and an atrioventricular canal progenitor posterior second heart region tissue; fusing at least two heart tissues; culturing the fused tissue model; and enabling calcium signal transduction connection, the ability to propagate contraction, and / or form a central heart cavity between different heart tissues. Fusion is the combination of tissues to form one (combined or fused) tissue. Fusion can be facilitated by placing different heart tissues in proximity to each other, preferably in contact with each other. Fusion can occur by and / or after cell growth and / or movement, whereby at least two different heart tissues are combined. Preferably, tissue fusion occurs during the progenitor stage of the tissue, as further described herein. Preferably, the connection region between the two tissues at the fusion junction is at least 5000 μm 2 and / or at least 100 μm in length. The fused tissue can be elongated. Preferably, the fused tissue can pass calcium and voltage signals from one tissue to another. The right ventricle progenitor (anterior) second heart region tissue and the outflow tract (anterior) second heart region tissue are also referred to as the right ventricle / outflow tract progenitor anterior second heart region tissue. This is one of the progenitor tissues of the (anterior) second heart region tissue that can differentiate into right ventricle tissue and outflow tract tissue.

[0022] To generate a controlled in vitro system of human heart development, in vivo principles governing the coalescence of all lineages that form the heart are used. Most heart structures are derived from three precursor populations that give rise to specific cardiomyocyte (CM) lineages. The first heart field (FHF) precursors become the developing left ventricle (LV) tissue, the anterior second heart field (aSHF) becomes the developing right ventricle (RV) tissue and most of the outflow tract tissue (OFT), and the posterior second heart field (pSHF) becomes most of the atria, atrioventricular canal tissue (AVC), atrioventricular node tissue (AVN), and part of the sinoatrial node tissue (SAN). The development of these structures is time-dependent. For example, FHF-derived CMs form the heart tube, which then grows into the LV, while aSHF and pSHF precursors differentiate and form other compartments in a delayed, progressive manner. This process is orchestrated by developmental signaling via multiple pathways (WNT, activin / nodal, BMP, RA, FGF, NOTCH, etc.) at specific stages of cardiogenesis. These signaling pathways control downstream important compartment-specific TFs (TBX1, TBX5, FOXF1, TBX3, ISL1, IRX4, HEY1 / 2, etc.) and direct precursor specification, morphogenesis, and physiological functions at specific stages. Some of these signals are generated by the developing tissues or their environment. Some signaling factors can be supplied to the developing tissues to advance development along the developmental pathway or to create artificial alterations in development.

[0023] Preferably, the heart tissue model and different heart tissues are mammalian tissues, preferably human or non-human primate tissues. Similarly, preferably, the heart tissue model is a mammalian or human or non-human primate tissue culture, e.g., a mammalian, human or non-human primate cell aggregate. Different heart tissues can be derived, for example, from culturing mammalian, human or non-human primate cells such as pluripotent cells, e.g., induced pluripotent stem cells (iPS cells) or embryonic stem cells. Human cells, tissues, and cultures are particularly preferred.

[0024] "Pluripotent" cells cannot grow into an entire organism, but can give rise to cell types derived from all three germ layers, namely the mesoderm, endoderm, and ectoderm, and may be able to give rise to all cell types of an organism. Pluripotency can be a characteristic of the cell itself, for example in embryonic stem cells, or can be artificially induced. For example, in a preferred embodiment of the present invention, pluripotent stem cells are derived from somatic cells, multipotent cells, unipotent cells or progenitor cells in which pluripotency is induced. Such cells are referred to herein as induced pluripotent stem (iPS) cells. Somatic cells, multipotent cells, unipotent cells or progenitor cells can be patient-derived, for example, and are converted into pluripotent cells for use in the methods of the present invention. Such cells or the resulting tissue cultures can be studied, for example, for abnormalities during heart tissue development according to the methods of the present invention. The patient may, for example, be suffering from a heart disorder or heart tissue deformation. The characteristics of the disorder or deformation can be reproduced and investigated in the tissue cultures of the present invention.

[0025] "Multipotent" cells can give rise to at least one cell type from each of two or more different organs or tissues of an organism, and the cell types can be derived from the same or different germ layers, but cannot give rise to all cell types of an organism. An example of a multipotent cell is a mesodermal cell.

[0026] In contrast, "unipotent" cells can differentiate into cells of only one cell lineage.

[0027] "Progenitor cells" are cells that, like stem cells, have the ability to differentiate into specific cell types, have limited options for differentiation, and usually have only one target cell. Progenitor cells are usually unipotent cells, but can also be multipotent cells.

[0028] Similar to progenitor cells, "progenitor tissue" or "precursor tissue", if left undisturbed, includes differentiated cells that determine tissue developmental fate. Examples are the left ventricular (LV) progenitor first heart field (FHF) tissue, which is destined to develop into LV tissue; the second heart field (SHF) tissue, which is destined to develop into right ventricular (RV) tissue or outflow tract (OFT) tissue, particularly the anterior second heart field (aSHF) tissue, i.e., the RV / OFT progenitor SHF tissue; the RV progenitor SHF tissue, which is the SHF tissue, particularly the aSHF tissue, destined to develop into RV tissue; the atrial progenitor SHF tissue, which is the SHF tissue, particularly the posterior second heart field (pSHF) tissue, destined to develop into atrial tissue; the outflow tract progenitor SHF tissue, which is the SHF tissue, particularly the aSHF tissue, destined to develop into OFT tissue; the atrioventricular canal (AVC) progenitor SHF tissue, which includes the SHF tissue, particularly the pSHF tissue, destined to develop into AVC tissue or atrioventricular node (AVN) tissue; and the sinoatrial node (SAN) progenitor SHF tissue, which includes the SHF tissue, particularly the pSHF tissue, destined to develop into SAN tissue.

[0029] As used herein, gene names or symbols refer to human genes and are described in databases such as GeneCards (www.genecards.org) or the HGNC database (www.genenames.org). Gene symbols are defined, for example, by the "HUGO Gene Nomenclature Committee" (HGNC). Other names, such as long names, can be found on those websites.

[0030] In the heart tissue model of the present invention or in different heart tissues, preferably, the left ventricular tissue or the left ventricular progenitor first heart region comprises at least 60% of heart cells selected from cardiomyocytes, endocardial cells, and epicardial cells. Such tissues have been described previously (Hofbauer et al., Cell 2021;184(12):3299-3317.e22; and International Publication No. WO 2021 / 186044(A1); both incorporated herein by reference) and can be used in the present invention. Preferably, at least 70% or at least 80% of the cells of the left ventricular tissue or the left ventricular progenitor first heart region are heart cells selected from cardiomyocytes, endocardial cells, and epicardial cells. Another term for endocardial cells is endocardial endothelial cells.

[0031] In a preferred embodiment, the right ventricular tissue and / or the right ventricular progenitor second heart region tissue comprises at least 60% cardiomyocytes. Preferably, the content of cardiomyocytes in these tissues is at least 70%, particularly preferably at least 80%.

[0032] In a preferred embodiment, the atrial tissue and / or the atrial progenitor second heart region tissue comprises at least 60% cardiomyocytes. Preferably, the content of cardiomyocytes in these tissues is at least 70%, particularly preferably at least 80%.

[0033] In a preferred embodiment, the outflow tract tissue and / or the outflow tract progenitor second heart region tissue comprises at least 60% cardiomyocytes. Preferably, the content of cardiomyocytes in these tissues is at least 70%, particularly preferably at least 80%.

[0034] In a preferred embodiment, the right ventricular tissue and / or the right ventricular / outflow tract progenitor second heart region tissue comprises at least 60% cardiomyocytes. Preferably, the content of cardiomyocytes in these tissues is at least 70%, particularly preferably at least 80%.

[0035] In a preferred embodiment, the atrioventricular canal tissue and / or the atrioventricular canal progenitor second heart field tissue contains at least 60% cardiomyocytes. Preferably, the content of cardiomyocytes in these tissues is at least 70%, particularly preferably at least 80%.

[0036] In a preferred embodiment, the atrioventricular node tissue contains at least 60% cardiomyocytes. Preferably, the content of cardiomyocytes in these tissues is at least 70%, particularly preferably at least 80%.

[0037] In a preferred embodiment, the sinoatrial node tissue and / or the sinoatrial node progenitor second heart field tissue contains at least 60% cardiomyocytes. Preferably, the content of cardiomyocytes in these tissues is at least 70%, particularly preferably at least 80%. In the subsequent development of the heart tissue model of the present invention, in addition to cardiomyocytes, the number of other heart cells such as endocardial cells and epicardial cells can also increase in these tissues. Therefore, the present invention also contemplates that any of the right ventricular tissue, atrial tissue, outflow tract tissue, and atrioventricular canal tissue contains at least 60% heart cells selected from cardiomyocytes, endocardial cells, and epicardial cells. Preferably, at least 70% or at least 80% of the cells of these tissues are heart cells selected from cardiomyocytes, endocardial cells, and epicardial cells.

[0038] Of course, in the heart tissue model of the present invention, any of these cell numbers, if present, can be combined for each tissue. The heart tissue model itself can contain at least 60% heart cells selected from cardiomyocytes, endocardial cells, and epicardial cells, particularly when left ventricular tissue is present. The left ventricular tissue, if present, is usually a large part of the heart tissue model. Preferably, at least 70% or at least 80% of the cells of the heart tissue model are heart cells selected from cardiomyocytes, endocardial cells, and epicardial cells. In embodiments combinable with these numbers, at least 40%, preferably at least 50%, particularly preferably at least 60% or at least 70% of the cells of the heart tissue model are cardiomyocytes.

[0039] The heart tissue model of the present invention includes a central heart cavity that is at least one lumen or one large heart chamber, which simulates the heart chambers of the natural heart. When functional, for example, in a healthy state without interfering with mutations or chemicals, a pulsatile rhythm with fluctuations in the volume distribution of the lumen or central heart chamber can be observed. The heart tissue model is still artificial and is not connected to the blood circulation system as a pump therein, so usually lacks any large blood vessels that enter (and exit from) the lumen or central heart chamber. Preferably, the lumen or central heart chamber is completely surrounded by tissue selected from different heart tissues, in particular, left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, or atrioventricular canal tissue. Alternatively, or in combination therewith, the volume of the lumen or central heart chamber does not communicate with major blood vessels and / or does not develop blood vessels. It is possible to artificially graft blood vessels into the heart tissue model, for example surgically, but such blood vessels are not naturally formed from the fusion products of the present invention without artificial intervention. A "major blood vessel" is a blood vessel (vena cava and pulmonary vein; aorta and pulmonary artery) similar to one of the large blood vessels of the mammalian heart. Such a major blood vessel may have a diameter of 10% or more of the diameter of one of the different heart tissues forming the heart tissue model. Such a major blood vessel usually refers to one of the different heart tissues because it enters the lumen or central heart chamber in one of the tissues. Preferably, the volume of the lumen or central heart chamber does not communicate with the blood vessels.

[0040] The tissue model of the present invention is artificial and is grown in culture using the natural principles of development, but is not an in vivo growing heart in any of the in vivo heart developments. The culture process usually limits the size. In a preferred embodiment of the present invention, the heart tissue model has a size with a maximum dimension of 0.3 mm to 50 mm, more preferably 1 mm to 40 mm, or particularly preferably 2 mm to 30 mm. Due to the fusion of different heart tissues, the heart tissue model can have an elongated shape. For reference, the size of the heart tissue model is determined using the maximum dimension of its shape.

[0041] The inner cavity or central cavity is a cavity within the heart tissue model, which is very large (compared to previous heart tissue models) and occupies a majority of the volume of the tissue model. Preferably, the size at the maximum dimension of the inner cavity or central cavity is at least 30% of the size at the maximum dimension of the heart tissue model. If there are more cavities other than the inner cavity or central cavity, this only applies to the largest inner cavity or central cavity (and the surrounding tissue that does not extend up to the tissue layer surrounding another cavity). In a preferred embodiment, the size at the maximum dimension of the inner cavity or central cavity is at least 40%, more preferably at least 50%, even more preferably at least 60% of the size at the maximum dimension of the heart tissue model. Both the central cavity and the heart tissue model can be in an elongated shape by the fusion of different heart tissues and the expansion of the central cavity into different heart tissues. Similarly to the above, the maximum dimension within the shape is used.

[0042] In some embodiments (e.g., multi-chamber cardioids), at least two, such as three or more, of the different heart tissues have a cavity. The cavities of the different heart tissues may be as described above, for example, may have a size of at least 30% of the size of the heart tissue model. Any of the cavities may be smaller, especially when limited to a single different heart tissue. The cavity can have a size of at least 20% of the size at the maximum dimension of the heart tissue model at its maximum dimension. Also, the cavity may have a size at the maximum dimension of at least 30%, preferably at least 40% of the size of the heart tissue at its maximum dimension. Here, the heart tissue only considers the part of the tissue model belonging to one specific heart tissue that limits the cavity.

[0043] Cardiomyocytes are heart muscle cells and are mainly involved in the beating activity or tissue contraction of the heart tissue model or different heart tissues. These are the main components of the tissue model of the present invention and form layers in the tissue model surrounding the lumen or central heart cavity. In early developmental tissues, they may directly face the lumen or central heart cavity, but in later and more developed tissues, the inner layer is formed by endocardial cells. Thus, in a preferred embodiment, cardiomyocytes or endocardial cells directly face the lumen or central heart cavity of a heart tissue selected from a heart tissue model and / or different heart tissues, particularly preferably left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular canal tissue, sinoatrial node tissue, and atrioventricular node tissue.

[0044] Not only the heart tissue model and its different heart tissues, but also the different heart tissues themselves (before fusion) forming a further aspect of the present invention, as well as precursor tissues such as FHF, aSHF, pSHF, or pluripotent or multipotent stem cells, can be characterized by specific expression patterns or expression markers. These expression patterns are the expression patterns of the cells constituting the heart tissue / tissue model / precursor. Gene names or symbols (see above) are used to characterize the expression markers. Such patterns are shown in the figures. The expression of cell markers can be determined by any suitable technique such as immunocytochemistry, immunofluorescence, RT-PCR, immunoblotting, fluorescence-activated cell sorting (FACS), RNA sequencing, single-cell RNA sequencing, and enzyme analysis. Some characteristic expression markers are shown below. Some markers can be overexpressed or underexpressed, and in some cases, are below the detection limit. Preferably, overexpression means an increase in expression compared to the expression in human embryonic stem cells. Preferably, underexpression means a decrease in expression compared to the expression in human embryonic stem cells. Underexpression may also not constitute detectable expression.

[0045] The term "expressing an expression marker" means that the expression of the mRNA encoding the marker is detectable above background levels using RT-PCR or RNA sequencing or single-cell RNA sequencing, preferably RT-PCR. The expression level of the expression marker can be compared to the expression level obtained from a negative control (i.e., cells known to be marker-free) or an isotype control (i.e., a control antibody that lacks the relevant specificity and binds non-specifically only to cellular proteins, lipids, or carbohydrates). Thus, a cell that "expresses" a marker has a detectable expression level that exceeds the expression level determined for the negative control for that marker. Alternatively, a cell surface marker can be detectable above background levels on the cell using a flow cytometry method such as immunofluorescence microscopy or fluorescence-activated cell sorting (FACS).

[0046] The terms "lacking expression", "not expressing", and "absence of a marker" mean that the expression of the mRNA for a given expression marker cannot be detected above background levels when using RT-PCR or RNA sequencing or single-cell RNA sequencing, preferably RT-PCR. The expression level of the expression marker can be compared to the expression level obtained from a negative control (i.e., cells known to be marker-free) or an isotype control (i.e., a control antibody that lacks the relevant specificity and binds non-specifically only to cellular proteins, lipids, or carbohydrates). Thus, a cell that "lacks expression" of a marker is considered to be similar to the negative control with respect to that marker. Alternatively, a cell surface marker may not be detected above background levels on the cell using a flow cytometry method such as immunofluorescence microscopy or fluorescence-activated cell sorting (FACS).

[0047] Preferably, the left ventricular tissue cells express one or more expression markers selected from NPPA, IRX4, and HEY2; and / or the left ventricular tissue cells lack the expression of one or more expression markers selected from NR2F2, TBX2, and TBX3. NR2F2, TBX2, and TBX3 may be absent or underexpressed after the maturation of the left ventricular tissue.

[0048] Preferably, the right ventricular tissue cells express one or more expression markers selected from NPPA, IRX1, IRX2, and PRDX1; and / or the right ventricular tissue cells lack the expression of one or more expression markers selected from NR2F2, TBX2, and WNT5A. NR2F2, TBX2, and WNT5A may be absent or underexpressed after the maturation of the right ventricular tissue.

[0049] Preferably, the atrial tissue cells express one or more expression markers selected from NPPA, NR2F1, NR2F2, and HEY1; and / or the atrial tissue cells lack the expression of one or more expression markers selected from IRX1, IRX4, and HEY2. IRX1, IRX4, and HEY2 may be absent or underexpressed after the maturation of the atrial tissue.

[0050] Preferably, the outflow tract tissue cells express one or more expression markers selected from WNT5A, MSX1, BMP4, WNT11, and RSPO3; and / or the outflow tract tissue cells lack the expression of one or more expression markers selected from TBX3, NR2F1, and NPPA. TBX3, NR2F1, and NPPA may be absent or underexpressed after the maturation of the outflow tract tissue.

[0051] Preferably, the atrioventricular canal tissue cells express one or more expression markers selected from TBX2, MSX2, and RSPO3; and / or the atrioventricular canal tissue cells lack the expression of one or more expression markers selected from IRX1, IRX4, and NPPA. IRX1, IRX4, and NPPA may be absent or underexpressed after the maturation of the atrioventricular canal tissue.

[0052] Preferably, the sinoatrial node tissue cells express one or more expression markers selected from SHOX2, TBX3, HCN4, ISL1, and GJC1; and / or the sinoatrial node tissue cells lack the expression of one or more expression markers selected from NKX2.5, IRX1, IRX4, and NPPA. NKX2.5, IRX1, IRX4, and NPPA may not be present or may be underexpressed after the maturation of the sinoatrial node tissue.

[0053] Preferably, the atrioventricular node tissue cells express one or more expression markers selected from TBX3, TBX5, KCNE1, HCN4, and GJC1; and / or the atrioventricular node tissue cells lack the expression of one or more expression markers selected from RSPO3, MSX2, IRX4, and NPPA. RSPO3, MSX2, IRX4, and NPPA may not be present or may be underexpressed after the maturation of the atrioventricular node tissue.

[0054] In a further aspect, the present invention provides a method for generating a heart tissue model of the present invention. The method comprises generating at least two different heart tissues in vitro, wherein the different heart tissues are selected from left ventricular progenitor first heart field tissue, right ventricle / outflow tract progenitor second heart field tissue, atrial progenitor second heart field tissue, outflow tract progenitor second heart field tissue, atrioventricular canal progenitor second heart field tissue, sinoatrial node progenitor second heart field tissue, and atrioventricular node tissue. These at least two different heart tissues may be provided as pre-grown tissues, for example, in a frozen state. Then, the method uses the thawed viable heart tissue. The method further comprises fusing at least two heart tissues, culturing the fused tissue model, and forming a central heart cavity between calcium signal transduction connections, tissue contraction propagation behavior, and / or different heart tissues.

[0055] The present invention also provides a heart tissue model obtainable by any method of the present invention. The obtained heart tissue model may have any of the structural elements as described above.

[0056] Fusing at least two heart tissues may include placing different heart tissues in contact with each other and fusing the tissues. Fusion can include cell growth and / or cell migration. Fusion can be achieved by placing two or more heart tissues, preferably left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrial tissue, a progenitor stage of outflow tract tissue, or a progenitor stage of pacemaker cells (sinoatrial node tissue and atrioventricular node tissue), in proximity to each other, preferably on a plate. Typically, within one day after fusion, the tissues form a structural connection with each other, such as the connection region described above. After several days, the structural connection results in a functional interaction. Preferably, when three or more heart tissues are fused, an elongated fusion pattern is used to control the arrangement of the heart tissues. Fusion of tissues by live cells of different heart tissues facilitates the formation of a heart tissue model. Further growth results in the formation of a central heart cavity once fused. For fusion, at least two different heart tissues preferably do not include a major central heart cavity or lumen here. This is because it may impair fusion efficiency and later the formation of a central heart cavity spanning two or more of the at least two different heart tissues. However, the left ventricular progenitor first heart region tissue is an exception, which may include a lumen or central heart cavity at the time of fusion and may allow for efficient fusion and the formation of a lumen or central heart cavity extending into the other of the two heart tissues. One or more of the different heart tissues may include a lumen when placed for fusion or during fusion.

[0057] Calcium signaling connections and / or contraction propagation can develop between different heart tissues after fusion.

[0058] Since the developmental stages can be better controlled, it is preferable to grow at least two different heart tissues in situ before fusion. Some of the developmental stages are described for each culture day. These culture days refer to culturing at the optimal temperature (for example, 37°C for human cells for a given cell type) and continuous growth in a growth medium. Freezing the cells from which the tissue or tissue trunk is derived interrupts the count of the culture days. Thus, "culture day" refers to the time corresponding to culturing under optimal growth conditions for a given cell type.

[0059] In a preferred embodiment, different heart tissues are cultured and differentiated from pluripotent cells. That is, the pluripotent cells are grown and differentiated to yield different heart tissues. Fusion starts from culturing the pluripotent cells and is preferably carried out from the pluripotent stage on the 1st to 7th day of culture.

[0060] In a preferred embodiment, the right ventricle / outflow tract precursor second heart field tissue, the atrial precursor second heart field tissue, and the atrioventricular precursor second heart field tube tissue are also fused here on the second to fifth days of culture, counted from the pluripotent stage. Preferably, the sinoatrial node precursor second heart field tissue and / or the atrioventricular node tissue are fused on the sixth to tenth days of culture, and particularly preferably, they are fused to tissues of approximately the same age. Alternatively, or in combination, the optimal fusion time is determined by specific expression markers of the tissue. Preferably, the left ventricle precursor first heart field tissue is fused when expressing the expression markers TBX5 and / or HAND1. Preferably, the right ventricle / outflow tract precursor second heart field tissue is fused when expressing the expression markers TBX1, FOXC1, and / or FOXC2. Preferably, the atrial tissue is fused when expressing the expression markers HOXB1, TBX5, and / or OSR1. Preferably, the atrioventricular canal precursor second heart field tissue is fused when expressing the expression markers TBX3, FOXF1, and / or HOXB1. Preferably, the sinoatrial node precursor second heart field tissue is fused when expressing the expression markers SHOX2, TBX3, HCN4, ISL1, and / or GJC1. Preferably, the atrioventricular node tissue is fused when expressing the expression markers TBX3, TBX5, KCNE1, HCN4, and / or GJC1. These markers indicate the development towards conditions suitable for efficient fusion.

[0061] Generating different tissues can be done using a medium and / or growth factors and / or differentiation factors. Examples are shown in the Examples section of this specification, particularly Examples 1 and 2. Any of the differentiation factors described therein can be used in any combination, alone, or in combination with other differentiation markers that achieve the same differentiation development for the method of the present invention. Some preferred components for culturing are described below for specific tissues.

[0062] In a particularly preferred embodiment, at least one of the at least two different cardiac tissues is a left ventricular progenitor first cardiac region tissue. Generating the left ventricular progenitor first cardiac region tissue includes bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, a Wnt inhibitor, preferably Wnt-C59 or IWP2, and retinoic acid, and may include differentiating mesodermal cells into left ventricular progenitor cells in a medium having a concentration of retinoic acid in the medium of 5 nM to 100 nM. Culturing with this medium is preferably performed on about the second day of culture, for example, between 1.5 days and 3 days of culture.

[0063] Particularly preferably, at least one of the at least two different cardiac tissues is a right ventricular progenitor anterior second cardiac region tissue or a right ventricular / outflow tract progenitor anterior second cardiac region tissue. Generating the right ventricular progenitor anterior second cardiac region tissue or the right ventricular / outflow tract progenitor anterior second cardiac region tissue may include differentiating mesodermal cells into right ventricular progenitor cells in a medium containing a TGF-beta inhibitor, preferably SB431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939. Culturing with this medium is preferably performed on about the second day of culture, for example, between 1.5 days and 3 days of culture. Generating the right ventricular progenitor anterior second cardiac region tissue, particularly further developing it into right ventricular tissue, may further include a Wnt inhibitor, preferably Wnt-C59 or XAV-939, bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, and preferably further includes growing it in a medium having a concentration of retinoic acid in the medium of 50 nM to 500 nM.

[0064] Preferably, at least one of the at least two different heart tissues is the second heart region tissue in front of the outflow tract precursor. Generating the second heart region tissue in front of the outflow tract precursor may include differentiating mesoderm cells into outflow tract tissue precursor cells in a medium containing a TGF-beta inhibitor, preferably SB431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939. Culturing with this medium is preferably carried out on about the second day of culture, for example, between 1.5 days and 3 days of culture. Generating the second heart region tissue in front of the outflow tract precursor or further generating it into outflow tract tissue may further include growing in a medium containing a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and lacking retinoic acid.

[0065] Preferably, at least one of the at least two different heart tissues is the second heart region tissue behind the atrial precursor. Generating the second heart region tissue behind the atrial precursor may include differentiating mesoderm cells into atrial tissue precursor cells in a medium containing a TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, preferably at a concentration of 300 nM to 800 nM. Culturing with this medium is preferably carried out on about the second day of culture, for example, between 1.5 days and 3 days of culture. Generating the second heart region tissue behind the atrial precursor or further differentiating it into atrial precursor tissue may further include growing in a medium containing a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably at a concentration of 300 nM to 800 nM.

[0066] Preferably, at least one of the at least two different heart tissues is the second heart field tissue posterior to the atrioventricular canal precursor. Generating the second heart field tissue posterior to the atrioventricular canal precursor may include differentiating mesodermal cells into atrioventricular canal tissue precursor cells in a medium containing a TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a concentration of 300 nM to 800 nM of retinoic acid. Culturing with this medium is preferably performed on about the second day of culture, for example, between 1.5 days and 3 days of culture. Generating the second heart field tissue posterior to the atrioventricular canal precursor or further differentiating into atrioventricular canal tissue may include further growing in a medium containing a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably at a concentration of 300 nM to 800 nM of retinoic acid.

[0067] Preferably, at least one of the at least two different heart tissues is the second heart field tissue posterior to the sinoatrial node precursor, and generating the second heart field tissue posterior to the sinoatrial node precursor includes differentiating mesodermal cells into sinoatrial node tissue precursor cells in a medium containing a TGF-beta inhibitor, preferably SB431542, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a concentration of 300 nM to 800 nM of retinoic acid.

[0068] Preferably, one of at least two different heart tissues is atrioventricular node tissue, and generating atrioventricular node tissue involves TGF-beta inhibitor, preferably SB431542, Wnt inhibitor, preferably Wnt-C59 or XAV-939, bone morphogenetic protein, preferably BMP4 and retinoic acid, and preferably differentiating mesoderm cells into atrioventricular canal tissue precursor cells in a medium where the concentration of retinoic acid is 300 nM to 800 nM. Generating atrioventricular node tissue may further include differentiating atrioventricular canal posterior second heart field tissue into atrioventricular node tissue by further growing it in a medium containing an activator or inhibitor of sonic hedgehog signaling and / or a bone morphogenetic protein.

[0069] Fusion of heart tissues is performed under culture conditions that allow fusion and do not prevent the differentiation of those precursor tissues into their target tissues during or after fusion. Preferably, fusing at least two heart tissues involves culturing in a medium containing a Wnt inhibitor, preferably Wnt-C59, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, and preferably where the concentration of retinoic acid is 300 nM to 800 nM, particularly preferably 500 nM. These are excellent culture conditions for the heart tissues referred to herein.

[0070] The above are mesoderm cells as precursors of tissue differentiation of the present invention. Mesoderm cells can be derived from pluripotent cells. Preferably, generating at least two different heart tissues in vitro includes generating mesoderm cells. Mesoderm cells can be generated by differentiating pluripotent cells in a medium containing activin and a Wnt activator, preferably CHIR99021, preferably until the differentiated cells express an expression marker selected from BRA, EOM, MIXL1, FOXA2, preferably in the absence of SOX2. Preferably, the medium for generating mesoderm cells further contains a PI3 kinase inhibitor such as LY294002. Using a PI3 kinase inhibitor is particularly preferred because it results in the most contamination-free and most homogeneous tissue model. The PI3 kinase inhibitor has the effect that many pluripotent cells exit pluripotency and differentiate into the mesoderm lineage, which determines the subsequent highly homologous heart tissue model. LY294002 is preferably used at a concentration of 3 μM to 15 μM.

[0071] Already at the mesoderm cell stage, it is promising for differentiation into specific tissues. For example, mesoderm cells can be precursors of the first heart field (FHF) or the second heart field (SHF), such as aSHF and pSHF.

[0072] For mesodermal cells that further differentiate into left ventricular tissue (FHF), activin is preferably at a concentration of 1 ng / mL to 8 ng / mL, more preferably about 5 ng / mL, and / or CHIR99021 is preferably at a concentration of 1 μM to 6 μM, more preferably about 3 μM. For mesodermal cells that further differentiate into right ventricular tissue, atrial tissue, or outflow tract tissue (a / pSHF), activin is preferably at a concentration of 30 ng / mL to 100 ng / mL, more preferably about 50 ng / mL, and / or CHIR99021 is preferably at a concentration of 2 μM to 8 μM, more preferably about 4 μM. For mesodermal cells that further differentiate into atrioventricular canal tissue (partially pSHF) or atrioventricular node tissue, activin is preferably at a concentration of 6 ng / mL to 30 ng / mL, more preferably about 10 ng / mL, and / or CHIR99021 is preferably at a concentration of 0.4 μM to 4 μM, more preferably about 2 μM. In mesodermal cells that further differentiate into sinoatrial node tissue, preferably, activin is at a concentration of 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or CHIR99021 is at a concentration of 0.1 μM to 4 μM, preferably about 1 μM.

[0073] Preferably, the pluripotent stem cells are grown in a medium containing at least 1.5% (w / v) (at least 1.5 g / l) albumin, preferably BSA, and / or at least 100 ng / ml fibroblast growth factor, preferably FGF2.

[0074] Preferably, the growth of pluripotent stem cells and their differentiation into mesodermal cells are performed in 2D culture. Subsequent steps, such as the growth of mesodermal cells and their differentiation into progenitor cells and the generation of different heart tissues and tissue models, are performed in 3D culture.

[0075] 2D culture may include growing or maintaining cells on a surface such as a tissue culture plate. Such a surface may be coated with a suitable culture substrate such as vitronectin.

[0076] In 3D culture, the cells or aggregates are not attached to the surface, but the cells can aggregate with each other or remain freely suspended, so that expansion in all 3D directions is uniformly possible. Such cultures are carried out in low-attachment cultures so that the cells or aggregates do not attach to the walls of the culture vessel. Preferably, the low-attachment culture involves culturing the cells in a vessel having a low-attachment surface that inhibits cell attachment to the surface. The low-attachment surface is preferably hydrophilic, neutrally charged or non-ionic. They may have a hydrogel layer or coating that repels cell attachment and keeps the cells in suspension. Such low-attachment culture vessel is known in the art and is described, for example, in International Publication No. WO 2019 / 014635 or International Publication No. WO 2019 / 014636. Preferably, the bottom of the culture vessel is circular, particularly concave. Alternatively, it may be flat or have a V-bottom shape.

[0077] Next, preferably, the pluripotent cells can be dissociated cells to initiate the culture. Then, they can form aggregates. The mesodermal cells are preferably cultures that are already in an aggregated state, such as obtained by culturing and differentiating pluripotent cells, for example. Then, the aggregates form different tissues and tissue models as an expansion of the aggregates.

[0078] Preferably, a cell culture medium such as E8 medium is used. The cell culture medium used in the process of the present invention preferably contains amino acids and carbohydrates necessary for cell growth, particularly preferably an energy source such as glucose. It further contains salts and ions necessary for cell growth, such as Ca, Fe, Mg, K, Na, Zn, Cl, SO4, NO3, PO4 ions. Further preferred components of the medium are vitamins such as vitamin B-12, biotin, choline, folic acid, inositol, niacinamide, pantothenic acid, pyridoxine, riboflavin, thiamine.

[0079] Preferred amino acids include essential amino acids, preferably any one of the following: alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.

[0080] In a preferred embodiment, the cells are cultured or treated for at least 1 day, preferably at least 2 days, for example 1 - 30 days, for example 2 - 20 days, while maintaining the pluripotency process, in such a medium, particularly a medium containing albumin and / or fibroblast growth factor, or using such a medium.

[0081] A preferred bone morphogenetic protein (BMP) is BMP4, particularly human BMP4. Preferably, the medium containing BMP contains at least 8 ng / ml of bone morphogenetic protein, preferably BMP4.

[0082] Some media or cultures contain FGF (fibroblast growth factor), preferably FGF2, FGF4, FGF8, FGF17, FGF18. Particularly preferred is FGF2. FGF2 is preferably used at a concentration of at least 6 ng / ml, unless otherwise specified.

[0083] WNT inhibitors are known in the art. WNT inhibitors are disclosed, for example, in Nusse and Clevers, Cell 169, 2017: 985 - 999. Further WNT activators are disclosed on the website web.stanford.edu / group / nusselab / cgi-bin / wnt / smallmolecules. WNT inhibitors are preferably selected from Wnt - C59, IWR - 1, XAV939, IWP - 2, IWP - 4, DKK1 or combinations thereof. Particularly preferred WNT inhibitors are IWP - 2 and XAV.

[0084] Any medium containing a Wnt inhibitor or activator preferably contains albumin. The albumin may be bovine serum albumin (BSA). Albumin is recommended to protect cells from the toxicity of small molecules such as WNT activators / inhibitors.

[0085] Since TGF-beta inhibitors inhibit TGF-beta function, they can be TGF-beta signaling pathway inhibitors. This can be an inhibitor of the TGF-beta superfamily pathway. Preferably one, more preferably two, or more TGF-beta inhibitors are used. In some preferred embodiments, this consists of or comprises one or more inhibitors of the TGF-beta receptor or the TGF-beta pathway. In a preferred embodiment, at least one TGF-beta (pathway) inhibitor is at least one SMAD inhibitor, preferably DMH1 (dorsomorphin homolog 1)) and / or SB431542 (4-[4-(2H-1,3-benzodioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl]benzamide). Further preferred TGF-beta inhibitors are alternatives to or combinable with those described above and are noggin (a protein that binds to and inactivates BMP proteins belonging to the TGF-beta superfamily), A83-01 (a small molecule), LDN193189 (a small molecule) and / or dorsomorphin. Further TGF-beta inhibitors are known in the art, for example as disclosed at www.medchemexpress.com / Targets / TGF-(beta)%20 Receptor.html.

[0086] Activin, such as activin A, may be used to specify cardiac tissue precursors for LV, a / pSHF and SAN, or AVC fates. Generally, high activin (e.g., more than 10 ng / ml) directs the tissue model towards a / pSHF and SAN fates, medium activin (e.g., about 10 ng / mL) directs the tissue model towards an atrioventricular fate, and low activin (e.g., less than 10 ng / ml) directs the tissue model towards a left ventricular fate. These exemplary concentrations may vary depending on the cell type used.

[0087] Wnt activators are known in the art and are described, for example, in Nusse and Clevers, Cell 169, 2017:985-999. Nusse and Clevers discuss the Wnt / b-catenin signaling pathway and its manipulation in stem cells. The Wnt activator may be a GSK3 inhibitor. Further WNT activators are disclosed on the website web.stanford.edu / group / nusselab / cgi-bin / wnt / smallmolecules. In a preferred embodiment, the Wnt activator is a WNT ligand such as WNT-3a, or CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile). Even more preferred Wnt activators are WAY-316606, ABC99, IQ1, QS11, SB-216763.

[0088] Activators of Sonic hedgehog (SHH) signaling are preferably smoothened agonists, particularly preferably SAG (N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane), cyclopamine or purmorphamine (PMA), or combinations thereof. Inhibitors of Sonic hedgehog (SHH) signaling are preferably cyclopamine.

[0089] The present invention further provides the above different heart tissues and their precursors as tissue models.

[0090] Accordingly, in a further aspect, a right ventricular tissue model is provided. The right ventricular tissue model may include cells expressing the expression markers IRX1, IRX2, and PRDX1. The entire disclosure herein regarding right ventricular tissue also applies to the right ventricular tissue model.

[0091] An atrial tissue model is further provided. The atrial tissue model may include cells expressing the expression markers NR2F1, NR2F2, and HEY1. The entire disclosure herein regarding atrial tissue also applies to the atrial tissue model.

[0092] An outflow tract tissue model is further provided. The outflow tract tissue model may include cells expressing the expression markers WNT5A, WNT11, MSX1, BMP4, and RSPO3. The entire disclosure herein regarding outflow tract tissue also applies to the outflow tract tissue model.

[0093] An atrioventricular canal tissue model is further provided. The atrioventricular canal tissue model may include cells expressing the expression markers TBX2, MSX2, and RSPO3. The entire disclosure herein regarding atrioventricular canal tissue also applies to the atrioventricular canal tissue model.

[0094] A sinoatrial node tissue model is further provided. The sinoatrial node tissue model may include cells expressing the expression markers SHOX2, TBX3, HCN4, ISL1, and GJC1. The entire disclosure herein regarding sinoatrial node tissue also applies to the sinoatrial node tissue model.

[0095] An atrioventricular node tissue model is further provided. The atrioventricular node tissue model may include cells expressing the expression markers TBX3, TBX5, KCNE1, HCN4, and GJC1. The entire disclosure herein regarding atrioventricular node tissue also applies to the atrioventricular node tissue model.

[0096] The right ventricular tissue model, atrial tissue model, outflow tract tissue model, atrioventricular canal tissue model, sinoatrial node tissue model, and atrioventricular node tissue model, collectively also referred to as tissue models, may not be fused, or they may be fused to another tissue. The tissue model reproduces a specific part of the heart and, together with the left ventricular tissue as in International Publication No. WO 2021 / 186044 (A1), can also be considered a heart organoid or cardioid.

[0097] Any tissue model may be subject to cavitation and may then, similar to the fused tissue model as described above, have an inner cavity or a central heart cavity by itself. Preferably, the size at the maximum dimension of the inner cavity or central heart cavity is at least 30% of the size at the maximum dimension of the tissue model. If there are more cavities in addition to the central heart cavity or the largest inner cavity, this only applies to the largest inner cavity or central heart cavity (and does not extend to the surrounding tissue, the tissue layer surrounding another inner cavity). In a preferred embodiment, the size at the maximum dimension of the inner cavity or central heart cavity is at least 40% of the size at the maximum dimension of the tissue model, more preferably at least 50%, and even more preferably at least 60%.

[0098] The tissue model may include calcium signaling or tissue contraction behavior or the ability of tissue contraction. Calcium signaling and contraction can be observed as described above for the fused heart tissue model. Of course, a single-tissue heart model may not show calcium signaling connections or contraction propagation to another tissue. However, similar connections and propagation can be observed between the cells of the tissue model.

[0099] Preferably, the tissue model includes at least 60% of heart cells selected from cardiomyocytes, endocardial cells, and epicardial cells. Preferably, at least 70% or at least 80% of the cells of the tissue model are heart cells selected from cardiomyocytes, endocardial cells, and epicardial cells. Particularly preferably, the tissue model includes at least 60% of cardiomyocytes.

[0100] By the culturing process, the size of these tissue models is usually limited. In a preferred embodiment of the present invention, the tissue model has a size with a maximum dimension of 0.2 mm to 30 mm, more preferably 0.5 mm to 25 mm, or particularly preferably 1 mm to 20 mm.

[0101] The heart tissue model can be obtained or produced by the methods described above for different heart tissues without fusing with another one of the different heart tissues. In particular, the heart tissue model can be obtained by the culturing method described above for different heart tissues.

[0102] The present invention, namely the heart tissue model, method and different tissue models, can be used for screening the effects of candidate compounds and / or the effects of gene modifications or environmental factors. Such a method may include generating a heart tissue model according to the present invention while treating cells with a candidate compound (at any stage during development). Also, the candidate compound may be tested using the final tissue model.

[0103] For example, the present invention provides a method for screening or testing a candidate compound for its effect on heart development and / or function, comprising generating a heart tissue model according to the method of the present invention while treating cells with the candidate compound, and comparing the development of the heart tissue model with the development and / or function of a heart tissue model not treated with the candidate compound.

[0104] Furthermore, a method for observing the effect of a gene suppressed, mutated, or overexpressed during heart development is provided, comprising generating a heart tissue model according to the present invention, wherein the cells have a suppressed or mutated candidate gene or overexpress a candidate gene, and comparing the development of the heart tissue model with the development of a heart tissue model not generated using the suppressed, mutated, or overexpressed gene.

[0105] Of course, these methods for screening / testing candidate compounds and genetic changes can be combined. For example, in combination with observing the effects of genes suppressed or overexpressed during heart development according to the present invention, a method for screening or testing a candidate compound for the effect of the candidate compound on heart development and / or function according to the present invention is provided.

[0106] Also provided is a method for screening or testing a candidate compound for its effect on heart function, comprising treating a heart tissue model according to the present invention with the candidate compound and comparing it with the function of a heart tissue model not treated with the candidate compound.

[0107] The method may include comparing the development or functionality of the heart tissue model with that of a heart tissue model not treated with the candidate compound. For comparison, all treatment steps should, of course, be the same except for treatment with the candidate compound. Examples of functions are, for example, beating behavior (e.g., intensity and / or rhythm, e.g., arrhythmia), or any cardiac function such as morphological changes or metabolic turnover or gene expression of a gene of interest that can be affected by the candidate compound. In the lumen or central heart cavity, fluid flow and / or electrical flow within the tissue surrounding the lumen or central heart cavity can also be observed. The development of the function can be the occurrence or function of toxicity that can be caused by the candidate compound. Thus, the method or tissue model of the present invention can be used for toxicity testing or screening.

[0108] Gene changes can be tested. For example, the present invention is a method for observing the effects of genes that are mutated (e.g., disease-related), suppressed, mutated, or overexpressed during heart development, which comprises generating a heart tissue model according to the present invention, wherein the cells have a suppressed or mutated candidate gene or overexpress a candidate gene; comparing the development of the heart tissue model with the development of a heart tissue model not generated using the suppressed, mutated, or overexpressed gene. Here, the suppression or overexpression is related to a normal state without modification of the expression or mutation of the candidate gene. The mutation, overexpression, or suppression of expression can be performed using any method known in the art, such as suppression by gene knockout, siRNA inhibition, or CRISPR / Cas-based inactivation. Overexpression can be performed by introducing a transgene or applying a gene activator that results in upregulation of gene expression. Also, using such mutations in mutant tissues or cells, the method for generating the heart tissue of the present invention using these cells can be used for screening or testing candidate compounds as described above. Thus, the present invention provides for the use of the heart tissue or method of the present invention as a disease model, e.g., a heart disease model, particularly for studying heart function. Accordingly, a method for screening or testing a candidate compound can also be combined with observing the effects of a suppressed or overexpressed gene. In the combined method, the comparison can be made between variants of tissues or methods with (''druged'') or without (''not-druged'') a candidate treatment, both in a druged configuration, between variants with or without mutations, or between all four states (mutated + druged, mutated + not-druged, non-mutated + druged, non-mutated + not-druged).

[0109] The heart tissue of the present invention can be used to reproduce a specific normal or abnormal state of the heart (e.g., a disease, disorder or injury), as well as recovery from such a state. The disease can be, for example, a congenital defect of the heart or a myopathy or hypertrophy. For example, factors investigated by creating cells or aggregates during the method, or by making the tissue model sensitive to or exposing it to such factors, are, for example, genetic defects, environmental conditions, maternal diabetes, toxins, such as plastic particles like microplastics. The heart tissue model of the present invention can include damage or healed damage. The method of the present invention may include causing damage to the tissue model and optionally further causing recovery from such damage. An agent such as a test compound can be tested to cause such a state, or to test the effect of the compound during damage and during the recovery and / or healing process. Examples of damage-related effects studied are fibrosis after damage and, for example, the healing of such fibrosis by treatment with a candidate compound.

[0110] The tissue model of the present invention can be used to study cardiac chamber dilation, fibrillation, or the transmission between the RV tissue and the LV tissue, particularly when it includes LV tissue and / or RV tissue. When it includes atrial tissue or sinoatrial node tissue, it can be used to study atrial fibrillation. Usually, the effect of a drug or an environmental effect on cardiac function has a compartment-specific effect. Such effects can be studied in the tissues of the present invention including LV, RV, OFT, atria, AVC, SAN and / or AVN tissue. In particular, in a fused tissue model having different tissues, the effects on these tissues can be studied in comparison with the effects on other tissues within the tissue model.

[0111] The present invention further provides a method for treating cardiac injury in a patient, which includes transplanting cells derived from the cardiac tissue model of the present invention into the injury. The cells may preferably be cardiomyocytes, fibroblasts, endothelial cells, smooth muscle cells or pacemaker cells, or combinations or mixtures thereof. Cardiomyocytes are particularly preferred. Pacemaker cells are also preferred. Pacemaker cells are preferably obtained from sinoatrial node tissue or a sinoatrial node tissue model. It is possible to provide the tissue model of the present invention, isolate the cells necessary for the regeneration of the injury, such as cardiomyocytes or progenitor cells, and provide these cells to the injury site. Incorporating the cells of the tissue model into the wound usually includes a regeneration process improved by the presence of the cells of the tissue model of the present invention. These cells can connect to the damaged heart of the patient and improve its regeneration. An example of cardiac injury is an infarcted heart.

[0112] The present invention further includes a cell culture medium for carrying out the method of the present invention. This medium can be used for the above-mentioned steps. Any specific combination of the above-mentioned compounds can be provided in the medium. In particular, a cell culture medium is provided, which includes the following. a) Bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, and retinoic acid has a concentration of less than 100 nM; b) A TGF-beta inhibitor, preferably SB431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939; c) A Wnt inhibitor, preferably Wnt-C59 or XAV-939, bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably, retinoic acid has a concentration of 50 nM to 500 nM in the medium; d) A TGF-beta inhibitor, preferably SB431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939; e) A medium lacking a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid; f) A TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, preferably, the retinoic acid is at a concentration of 300 nM to 800 nM; g) A Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably, the retinoic acid is at a concentration of 300 nM to 800 nM; h) A TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4 and retinoic acid, preferably, the retinoic acid is at a concentration of 300 nM to 800 nM; i) Activin and CHIR99021; preferably, Activin is at a concentration of 1 ng / mL to 8 ng / mL, preferably about 5 ng / mL, and / or CHIR99021 is at a concentration of 1 μM to 6 μM, preferably about 3 μM; or preferably, Activin is at a concentration of 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or CHIR99021 is at a concentration of 2 μM to 8 μM, preferably about 4 μM; or preferably, Activin is at a concentration of 6 ng / mL to 30 ng / mL, preferably about 10 ng / mL, and / or CHIR99021 is at a concentration of 0.4 μM to 4 μM, preferably about 2 μM; or preferably, Activin is at a concentration of 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or CHIR99021 is at a concentration of 0.1 μM to 4 μM, preferably about 1 μM; or j) A Wnt inhibitor, preferably Wnt-C59, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably, the retinoic acid is at a concentration of 300 nM to 800 nM, particularly preferably 500 nM.

[0113] Any of the media may contain amino acids and an energy source necessary for cell growth, such as carbohydrates, particularly preferably glucose. These may further contain salts and ions necessary for cell growth, such as Ca, Fe, Mg, K, Na, Zn, Cl, SO4, NO3, PO4 ions. More preferred components of the medium are vitamins such as vitamin B12, biotin, choline, folic acid, inositol, niacinamide, pantothenic acid, pyridoxine, riboflavin, thiamine, etc.

[0114] Preferred amino acids include essential amino acids, preferably any one of the following: alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.

[0115] Any of these media can be combined in a kit of different media or a kit of one or more media and other means (e.g., carriers or templates used for the fusion of cultured tissues) used in the method of the present invention. The medium can be provided in a kit that further includes instructions for carrying out the method of the present invention. Such instructions may be in a form printed on a suitable data carrier or in a computer-readable format. The carrier or template can be a plate having recesses in a shape for fusing the different tissues described above. One example is an elongated recess for arranging a linear array of different heart tissues in contact with each other.

[0116] According to the present invention, the following numbered embodiments are preferred. Any of the numbered embodiments can, of course, be combined with the above-described embodiments and preferred options, or with the corresponding elements of the Examples section.

[0117] Embodiment 1 A cardiac tissue model comprising cardiac tissue having at least one lumen or central lumen, wherein the cardiac tissue model comprises at least two different cardiac tissues selected from left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular canal tissue, sinoatrial node tissue, and atrioventricular node tissue, the central lumen can be shared by at least two different cardiac tissues, and / or at least two different cardiac tissues include an electrophysiological signal transmission connection or a calcium signal transmission connection or the ability to propagate tissue contraction.

[0118] Embodiment 2 The left ventricular tissue comprises at least 60% cardiac cells selected from cardiomyocytes, endocardial cells, and epicardial cells. The right ventricular tissue comprises at least 60% cardiomyocytes. The atrial tissue comprises at least 60% cardiomyocytes. The outflow tract tissue comprises at least 60% cardiomyocytes. The atrioventricular canal tissue comprises at least 60% cardiomyocytes. The sinoatrial node tissue comprises at least 60% cardiomyocytes and / or The atrioventricular node tissue comprises at least 60% cardiomyocytes. The cardiac tissue model according to Embodiment 1.

[0119] Embodiment 3 The cardiac tissue model according to Embodiment 1 or 2, wherein the lumen or central lumen is completely surrounded by tissue selected from left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular canal tissue, sinoatrial node tissue, or atrioventricular node tissue, and / or the volume of the lumen or central lumen does not communicate with the major blood vessels.

[0120] Embodiment 4 The cardiac tissue model according to any one of Embodiments 1 to 3, having a size with a maximum dimension of 0.3 mm to 50 mm.

[0121] Embodiment 5 Left ventricular tissue cells express one or more expression markers selected from NPPA, IRX4, and HEY2; and / or left ventricular tissue cells lack the expression of one or more expression markers selected from NR2F2, TBX2, and TBX3, Right ventricular tissue cells express one or more expression markers selected from NPPA, IRX1, IRX2, and PRDX1; and / or right ventricular tissue cells lack the expression of one or more expression markers selected from NR2F2, TBX2, and WNT5A, Atrial tissue cells express one or more expression markers selected from NPPA, NR2F1, NR2F2, and HEY1; and / or atrial tissue cells lack the expression of one or more expression markers selected from IRX1, IRX4, and HEY2, Outflow tract tissue cells express one or more expression markers selected from WNT5A, MSX1, BMP4, WNT11, and RSPO3; and / or outflow tract tissue cells lack the expression of one or more expression markers selected from TBX3, NR2F1, and NPPA, Atrioventricular canal tissue cells express one or more expression markers selected from TBX2, MSX2, and RSPO3; and / or atrioventricular canal tissue cells lack the expression of one or more expression markers selected from IRX1, IRX4, and NPPA, Sinu-atrial node tissue cells express one or more expression markers selected from SHOX2, TBX3, HCN4, ISL1, and GJC1; and / or sino-atrial node tissue cells lack the expression of one or more expression markers selected from NKX2.5, IRX1, IRX4, and NPPA, and / or Atrioventricular node tissue cells express one or more expression markers selected from TBX3, TBX5, KCNE1, HCN4, and GJC1; and / or atrioventricular node tissue cells lack the expression of one or more expression markers selected from RSPO3, MSX2, IRX4, and NPPA, the cardiac tissue model according to any one of Embodiments 1 to 4.

[0122] Embodiment 6. The heart tissue model according to any one of Embodiments 1 to 5, wherein the size at the maximum dimension of the inner cavity and / or the central cavity is at least 30% of the size at the maximum dimension of the heart tissue model.

[0123] Embodiment 7. The heart tissue model according to any one of Embodiments 1 to 6, wherein cardiomyocytes or endocardial cells directly face the inner cavity and / or the central cavity.

[0124] Embodiment 8. A method for generating the heart tissue model according to any one of Embodiments 1 to 7, comprising generating at least two different heart tissues in vitro, wherein the different heart tissues are selected from left ventricular progenitor first heart region tissue, right ventricular / outflow tract progenitor second heart region tissue, atrial progenitor second heart region tissue, outflow tract progenitor second heart region tissue, atrioventricular canal progenitor second heart region tissue, sinoatrial node tissue, and atrioventricular node tissue; fusing at least two heart tissues; culturing the fused tissue model; and forming a central cavity between different heart tissues by calcium signal transduction connection, the ability to propagate contraction, and / or the like.

[0125] Embodiment 9. The method according to Embodiment 8, wherein fusing at least two heart tissues comprises arranging different heart tissues in contact with each other and fusing the tissues, preferably by cell growth.

[0126] Embodiment 10 Different cardiac tissues are cultured, differentiated from pluripotent cells, and fusion is performed from the pluripotent stage to day 1 to day 7 of culture. Preferably, the right ventricle / outflow tract progenitor second heart field tissue, atrial progenitor second heart field tissue, atrioventricular progenitor second heart field tube tissue, sinoatrial node progenitor second heart field tissue, and / or atrioventricular node tissue are fused on day 2 to day 5 of culture, or preferably, when expressing the expression markers IRX4, TBX5, and / or HEY2, the left ventricle progenitor first heart field tissue is fused; preferably, when expressing the expression markers TBX1, FOXC1, and / or FOXC2, the right ventricle progenitor second heart field tissue is fused, preferably, when expressing the expression markers HOXB1, TBX5, and / or OSR1, the atrial progenitor second heart field tissue is fused, preferably, when expressing the expression markers TBX3, FOXF1, and / or HOXB1, the atrioventricular canal progenitor second heart field tissue is fused, preferably, when expressing the expression markers SHOX2, TBX3, HCN4, ISL1, and / or GJC1, the sinoatrial node progenitor second heart field tissue is fused, and / or, preferably, when expressing the expression markers TBX3, TBX5, KCNE1, HCN4, and / or GJC1, the atrioventricular node tissue is fused, the method according to Embodiment 8 or 9.

[0127] Embodiment 11 One of at least two different cardiac tissues is the left ventricle progenitor first heart field tissue, and generating the left ventricle progenitor first heart field tissue includes differentiating mesoderm cells into left ventricle progenitor cells in a medium containing bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, a Wnt inhibitor, preferably Wnt-C59 or IWP2, and retinoic acid, and the retinoic acid has a concentration of 5 nM to 100 nM, the method according to any one of Embodiments 8 to 10.

[0128] Embodiment 12 One of at least two different cardiac tissues is the right ventricle / outflow tract progenitor second cardiac region tissue, and generating the right ventricle / outflow tract progenitor second cardiac region tissue involves differentiating mesodermal cells into right ventricle progenitor cells in a medium containing a TGF-beta inhibitor, preferably SB 431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and optionally, thereafter, growing in a medium containing a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably with the retinoic acid having a concentration of 50 nM to 500 nM in the medium, according to any one of Embodiments 8 to 11.

[0129] Embodiment 13 One of at least two different cardiac tissues is the outflow tract progenitor second cardiac region tissue, and generating the outflow tract progenitor second cardiac region tissue involves differentiating mesodermal cells into outflow tract tissue progenitor cells in a medium containing a TGF-beta inhibitor, preferably SB 431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and optionally, thereafter, growing in a medium containing a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and lacking retinoic acid, according to any one of Embodiments 8 to 12.

[0130] Embodiment 14 One of at least two different cardiac tissues is atrial progenitor second heart field tissue, and generating the atrial progenitor second heart field tissue involves differentiating mesodermal cells into atrial tissue progenitor cells in a medium containing a TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, preferably at a concentration of 300 nM to 800 nM, and optionally, thereafter, growing in a medium containing a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably at a concentration of 300 nM to 800 nM, the method according to any one of Embodiments 8 to 13.

[0131] Embodiment 15 One of at least two different cardiac tissues is atrioventricular canal progenitor second heart field tissue, and generating the atrioventricular canal progenitor second heart field tissue involves differentiating mesodermal cells into atrioventricular canal tissue progenitor cells in a medium containing a TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4 and retinoic acid, preferably at a concentration of 300 nM to 800 nM, and optionally, thereafter, growing in a medium containing a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably at a concentration of 300 nM to 800 nM, the method according to any one of Embodiments 8 to 14.

[0132] Embodiment 16: One of at least two different cardiac tissues is the sinoatrial node precursor second cardiac region tissue, and generating the sinoatrial node precursor second cardiac region tissue includes differentiating mesodermal cells into sinoatrial node tissue precursor cells in a medium containing a TGF-beta inhibitor, preferably SB431542, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a concentration of 300 nM to 800 nM of retinoic acid, according to any one of Embodiments 8 to 15.

[0133] Embodiment 17: One of at least two different cardiac tissues is the atrioventricular node tissue, and generating the atrioventricular node tissue includes differentiating mesodermal cells into atrioventricular canal tissue precursor cells in a medium containing a TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a concentration of 300 nM to 800 nM of retinoic acid, and further differentiating the atrioventricular canal precursor second cardiac region tissue into the atrioventricular node tissue by further maturing in a medium containing an activator and / or inhibitor of sonic hedgehog signaling and / or a BMP, according to any one of Embodiments 8 to 16.

[0134] Embodiment 18: Fusing at least two cardiac tissues includes culturing in a medium containing a Wnt inhibitor, preferably Wnt-C59, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably at a concentration of 300 nM to 800 nM, particularly preferably 500 nM of retinoic acid, according to any one of Embodiments 8 to 17.

[0135] Embodiment 19 Generating at least two different cardiac tissues in vitro involves generating mesodermal cells by differentiating pluripotent cells in a medium containing activin and a Wnt activator, preferably CHIR99021, and preferably until the differentiated cells express an expression marker selected from BRA, EOM, MIXL1, and FOXA2, preferably in the absence of SOX2; and / or For mesodermal cells that further differentiate into left ventricular tissue, activin is at a concentration of 1 ng / mL to 8 ng / mL, preferably about 5 ng / mL, and / or CHIR99021 is at a concentration of 1 μM to 6 μM, preferably about 3 μM; For mesodermal cells that further differentiate into right ventricular tissue, atrial tissue, or outflow tract tissue, activin is at a concentration of 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or CHIR99021 is at a concentration of 2 μM to 8 μM, preferably about 4 μM, and / or For mesodermal cells that further differentiate into atrioventricular canal tissue, activin is at a concentration of 6 ng / mL to 30 ng / mL, preferably about 10 ng / mL, and / or CHIR99021 is at a concentration of 0.4 μM to 4 μM, preferably about 2 μM, and / or For mesodermal cells that further differentiate into sinoatrial node tissue, preferably, activin is at a concentration of 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or CHIR99021 is at a concentration of 0.1 μM to 4 μM, preferably about 1 μM, the method according to any one of Embodiments 8 to 18.

[0136] Embodiment 20 A right ventricular tissue model comprising cells expressing the expression markers IRX1, IRX2, and PRDX1.

[0137] Embodiment 21 An atrial tissue model comprising cells expressing the expression markers NR2F1, NR2F2, and HEY1.

[0138] Embodiment 22 An outflow tract tissue model comprising cells expressing the expression markers WNT5A, MSX1, BMP4, and RSPO3.

[0139] Embodiment 23 An atrioventricular canal tissue model comprising cells expressing the expression markers TBX2, MSX2, and RSPO3.

[0140] Embodiment 24 A sinoatrial node tissue model comprising cells expressing the expression markers SHOX2, TBX3, HCN4, ISL1, and GJC1.

[0141] Embodiment 25 An atrioventricular node tissue model comprising cells expressing the expression markers TBX3, TBX5, KCNE1, HCN4, and GJC1.

[0142] Embodiment 26 The tissue model according to any one of Embodiments 20 to 25, comprising at least 60% of cardiac cells selected from cardiomyocytes, endocardial cells, and epicardial cells.

[0143] Embodiment 27 The tissue model according to any one of Embodiments 20 to 26, having a size of 0.2 mm to 30 mm in maximum dimension.

[0144] Embodiment 28 The tissue model according to any one of Embodiments 20 to 27, comprising a lumen.

[0145] Embodiment 29 A method according to any one of Embodiments 8 to 19 for screening or testing a candidate compound for its effect on the development and / or function of the heart, comprising generating a heart tissue model according to any one of Embodiments 8 to 19 while treating the cells with the candidate compound, and comparing the development of the heart tissue model with the development and / or function of a heart tissue model not treated with the candidate compound.

[0146] A method for observing the effects of genes that have mutated, been suppressed, or overexpressed during heart development, the method comprising generating a heart tissue model according to any one of Embodiments 8 to 19, wherein the cells have a suppressed or mutated candidate gene or overexpress a candidate gene, and comparing the development of the heart tissue model with the development of a heart tissue model that was not generated using a suppressed, mutated, or overexpressed gene.

[0147] Embodiment 31 A method for screening or testing a candidate compound for its effect on heart development and / or function according to Embodiment 29 in combination with observing the effects of genes that have been suppressed, mutated, or overexpressed during heart development according to Embodiment 30.

[0148] Embodiment 32 A method for screening or testing a candidate compound for its effect on heart function, the method comprising treating a heart tissue model according to any one of Embodiments 1 to 7 or 20 to 28 with the candidate compound and comparing the function of the heart tissue model not treated with the candidate compound.

[0149] Embodiment 33 A method for treating heart damage in a patient, the method comprising transplanting one or more cells, preferably cardiomyocytes, derived from a heart tissue model according to any one of Embodiments 1 to 7 or 20 to 28 into the damage.

[0150] Embodiment 34 A cell culture medium comprising: a) Bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, wherein the retinoic acid has a concentration of less than 100 nM; b) A TGF-beta inhibitor, preferably SB431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939; c) A Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably, the retinoic acid has a concentration of 50 nM to 500 nM in the medium; d) A TGF-beta inhibitor, preferably SB431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939; e) A medium lacking a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid; f) A TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, preferably, the retinoic acid is at a concentration of 300 nM to 800 nM; g) A Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably, the retinoic acid is at a concentration of 300 nM to 800 nM; h) A TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4 and retinoic acid, preferably, the retinoic acid is at a concentration of 300 nM to 800 nM; i) Activin and CHIR99021; preferably, Activin is at a concentration of 1 ng / mL to 8 ng / mL, preferably about 5 ng / mL, and / or CHIR99021 is at a concentration of 1 μM to 6 μM, preferably about 3 μM; or preferably, Activin is at a concentration of 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or CHIR99021 is at a concentration of 2 μM to 8 μM, preferably about 4 μM; and / or preferably, Activin is at a concentration of 6 ng / mL to 30 ng / mL, preferably about 10 ng / mL, and / or CHIR99021 is at a concentration of 0.4 μM to 4 μM, preferably about 2 μM; and / or preferably, Activin is at a concentration of 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or CHIR99021 is at a concentration of 0.1 μM to 4 μM, preferably about 1 μM; j) A Wnt inhibitor, preferably Wnt-C59, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably retinoic acid at a concentration of 300 nM to 800 nM, particularly preferably 500 nM.

[0151] Throughout this disclosure, the articles "a", "an", and "the" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the articles.

[0152] As used herein, without limitation, approximate terms such as "about", "substantially", or "substantially" are understood not to be absolute or complete when so modified, but to refer to conditions that are considered to be close enough to a condition to ensure that there are conditions present. The degree to which the description can vary depends on the magnitude of the change that can be made, and those skilled in the art will recognize that the modified feature still has the necessary characteristics and capabilities of the unmodified feature. Generally, however, in accordance with the previous discussion, numerical values in this specification modified by approximate terms such as "about" can vary, for example, by ±10% from the stated value.

[0153] As used herein, the terms "comprising" (and any form of "comprising" such as "comprise" and "comprises"), "having" (and any form of "having" such as "have" and "has"), "including" (and any form of "including" such as "includes" and "include"), or "containing" (and any form of "containing" such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. When the expression "comprising" is used in combination with a numerical range of specific values for an element, it means that the element is limited to that range, but "comprising" still relates to the possible presence of other elements in some cases. For example, an element having a range can be subject to an implied condition excluding the presence of that element in amounts outside that range. As used herein, the phrase "consisting essentially of" requires a particular integer(s) or step, and those that do not substantially affect the characteristics or functions of the invention claimed by the claims. As used herein, the closed term "consisting of" is used to indicate the presence of only the recited elements.

[0154] The present invention will be further described by the following drawings and examples, but is not limited to these embodiments of the present invention.

Brief Description of the Drawings

[0155]

Figure 1-1

[0156] A. Protocol for differentiation into three major cardiac lineages: the first heart field (FHF), anterior second heart field (aSHF), and posterior second heart field (pSHF).

[0157] B. Real-time qPCR of TBX1 and TBX5 levels in FHF and aSHF precursors at day 3.5 in 2D, 3D, and 2D->3D protocols. Fold changes were normalized to a housekeeping gene and pluripotency.

[0158] C. RNAscope staining of TBX1 and TBX5 in aSHF cardioid cryosections at day 3.5 shows increased expression of TBX1 and absence of TBX5 expression in the 2D->3D approach compared to 3D differentiation.

Figure 1-2

[0159] D. Volcano plots of differentially expressed genes in FHF vs. aSHF precursors (top) and aSHF vs. pSHF precursors (bottom) at day 3.5.

[0160] E. Heatmap reveals upregulation of aSHF and pSHF genes in the corresponding differentiation protocols at day 3.5. General cardiac genes are more highly expressed in FHF compared to aSHF and pSHF.

[0161] F. RNAscope staining (TBX1, TBX5, and HOXB1) of FHF, aSHF, and FHF precursors at day 3.5.

Figure 1-3

[0162] G. Immunostaining (G’) and quantification of staining (G’’) of aSHF marker (FOXC2) and pSHF marker (FOXF1) in organoids at day 3.5 (N = 3, n = 3 - 4). Mean ± SD.

[0163] is a Venn diagram showing the degree of intersection among aSHF, pSHF, and FHF of the upregulated genes compared with pluripotency.

Figure 1-4

[0164] I. Three biological replicates on day 9.5 show highly robust and efficient differentiation into FHF, aSHF, and pSHF cardioids.

[0165] J. Frozen sections of FHF, aSHF, and pSHF cardioids on day 9.5 showing the expression of the CM-specific marker MYL7.

[0166] K. Quantification of TNNI1-GFP+ cells in cardioids on day 9.5 by flow cytometry. Mean ± SD (N = 3, n = 8) L. Representative flow cytometry plots of FHF-, aSHF-, and pSHF-derived CMs using reporter strains and WT strains.

[0167] All scale bars in this figure have a length of 200 mm. Cell lines used in this figure: H9 and WTC. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2-1

[0168] A. In the time course of three major lineages of cardioids from day 2.5 to day 9.5, delayed cavity formation and delayed expression of TNNI-GFP in SHF are revealed compared with the FHF lineage.

[0169] B.Quantification of the change in cardiomyoid area during differentiation in all three protocols is shown. (N = 3, n = 32)

[0170] C. & D. Cryosections of cardiomyoids from day 2 to day 5.5 showing delayed cavity initiation (white arrows) and cavity formation (yellow arrows) in the SHF lineage. Immunostaining (C) and mRNA expression quantification (D) of the proliferation marker Ki67 over time show that more aSHF is proliferating on day 4.5.

Figure 2-2

[0171] E. Principal component analysis (PCA) plot of vst using the top 1000 variable genes is shown. VST: variance-stabilizing transformation count.

[0172] F. Expression of lineage-specific genes over time is shown. aSHF cardiomyoids express RV-specific genes, while pSHF cardiomyoids express atrial-specific genes.

Figure 2-3

[0173] G. Volcano plots show differentially expressed genes in FHF vs. aSHF (top) and FHF vs. pSHF (bottom) cardiomyoids at day 9.5.

[0174] H. Lineage-specific staining of IRX1 (RV marker) and NR2F2 (atrial marker) and quantification of staining (H’) at day 14 are shown. Each data point represents one organoid. (N = 3, n = 3 - 4) All scale bars in this figure have a length of 200 mm.

[0175]

Figure 3-1

[0176] A. Schematic showing the differentiation protocol of aSHF precursors into RV and OFT cardioids depending on the addition or absence of RA during patterning stage 2.

[0177] B. RNAseq over time of developing RV and OFT cardioids reveals the expression of lineage-specific genes.

[0178] C. Shows the overall gene expression differences between RV and OFT cardioids at E9.5.

[0179] D. and E. OFT cardioids highly express the OFT markers ISL1 (D) and WNT5A (E) at E14 compared to RV cardioids.

[0180] F. Whole-mount images of RV, OFT, atrial, and AVC cardioids, which are three biological replicates at E9.5.

Figure 3-2

[0181] G. Schematic showing the differentiation protocol of atrial and AVC cardioids using different induction conditions and addition of BMP during patterning stage 2 of AVC.

[0182] H. Shows genes differentially expressed at E1.5 in AVC, FHF, and SHF precursors.

[0183] I. Volcano plot showing the genes most differentially expressed in AVC compared to atrial precursors at E3.5.

[0184] J. Shows the expression of AVC organoids over time.

[0185] K. AVC cardioids upregulate TBX2 compared to atrial cardioids but still express TBX3.

[0186] Addition of RA and FGF, and inhibition of NOTCH and BMP in atrial cardioids after day 7.5 results in upregulation of chamber markers.

[0187] RV and LV cardioids maintained in the maturation medium published after 7.5 days showed an increase in chamber-specific markers. Atrial cardioids maintained in the maturation medium published after RA, FGF, NOTCHi, and BMPi downregulated AVC-specific markers compared to cardioids in CDMI.

[0188] All scale bars in this figure have a length of 200 mm.

Figure 4-1

[0189] A. Bar graph showing the rate at which organoids contract within 1 minute, from recordings on both day 6 and day 9.

[0190] B. Shows quantification of the number of beats per minute (BPM) of different types of organoids on both day 6 and day 9.

[0191] C. Shows quantification of the number of pixels that move during contraction, divided by the area of the organoid (degree of contraction). This is a proxy for how much the edge of the cardioid moves during a single contraction. Organoids that are not beating are not included.

[0192] A - C: All were performed with N = 2 - 7, and for each biological replicate, there were 16 technical replicates, and 80, 65, 48, 48, and 33 organoids were obtained for LV, RV, OFT, atrium, and AVC respectively.

[0193] D. Shows RNA expression of HCN4 throughout differentiation quantified by bulk RNA-seq. Each dot represents the mean (N = 3), and the error bars represent the standard deviation. The dots are connected by lines to facilitate the following trends.

[0194] E. Bulk RNA-seq showing both L-type and T-type calcium channels on day 9.5 of differentiation.

Figure 4-2

[0195] F. Representative calcium signal propagation in the whole LV, RV, and atrial cardiomyoids for one beat. The map is colored by the time when each pixel reached 50% of the peak intensity.

[0196] G. Scaled heatmap (G) of RNA-seq on day 9.5, showing the expression of key ion channels involved in contraction.

[0197] H. Patch-clamp analysis performed on cells dissociated from different RV and atrial cardiomyoids. Representative AP curves for each cell type are shown. Top: RV Bottom: Atrium.

[0198] I. Action potential duration (APD90) measured from peak to 90% repolarization is shown.

[0199] J. Amplitude of the AP measured from the peak is shown.

[0200] K. Resting membrane potential of each cell type is shown.

[0201] K - M: Each point represents the mean from one cell. The bar represents the mean of the cells, and the error bar represents the standard deviation.

[0202] * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5-1

[0203] On day 3.5, it is shown that 2D heart precursors are dissociated and mixed to form cardioids of the same or different precursors.

[0204] B. Different types of heart precursors labeled with H2B-GFP or LMNB1-RFP are sorted on day 7.5, and precursors of the same type are mixed.

[0205] C. The cardioids maintain their characteristics upon mixing. The RV precursors also differentiate into RV CMs here, as shown by IRX1 staining.

[0206] D. CDH1 staining of cardioids on day 4.5, 24 hours after mixing different or the same precursors, is shown.

[0207] E. Cardioids can be fused together on day 3.5 in different combinations by fusing any of two or three different compartments together. By doing this with fluorescent green and red lines, it is possible to accurately track how different compartments interact with each other.

[0208] F. Representative bright-field images of fused cardioids of the atrium (A), LV, and RV cardioids on day 6.5 are shown.

[0209] G. Representative calcium signal propagation through a three-chambered cardioids for one heartbeat is shown. The map is colored by the time when each pixel reaches 50% of the peak intensity.

Figure 5-2

[0210] H. Cardioids maintain their characteristics upon fusion, and all compartments uniformly express the CM marker TNNT2.

[0211] I. Percentage of pulsating organoids for each fusion type for day 6 and day 9 are shown.

[0212] J. Beats per minute (BPM) for different fusion types on both day 6 and day 9 are shown.

[0213] K. Cardioids in which pulsation has started are shown as a percentage of all recorded cardioids (shown by color). Mix indicates that pulsation was initiated by different organoids for each beat, and None indicates either that the fused cardioids are not pulsating organoids at all or that there is no interaction between the fused cardioids. These are shown for both day 6 and day 9.

Figure 5-3

[0214] L. Next, the timing of heart progenitor fusion was optimized to promote the formation of shared cavities between cardioids by aggregating FHF progenitors at day 1.5, aggregating a / pSHF progenitors at day 3.5, and fusing the aggregates 4 hours after aggregation.

[0215] M. Representative image of a cardioids with three compartments using the protocol shown in M. Atria are shown in red, LV in gray, and RV in green.

[0216] N. Representative frozen sections of three fused cardioids using the protocol shown in M, stained with the pan - cardiac marker TNNT2. Arrows indicate cavities shared between heart chambers.

[0217] O. Frozen sections of fused cardioids of two different compartments using the protocol shown in M. The fused cardioids share several cavities indicated by blue arrows and express TNNT2.

[0218] All scale bars in this figure have a length of 200 mm.* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6-1

[0219] A. On day 9.5, ISL1 KO cardioids show a dramatic decrease in size when using the atrial and OFT protocols, and a slight decrease in size when using the LV and RV protocols.

[0220] B. Cross-sections of cardioids show a decrease in TNNT2 expression in the RV, atrium, and OFT in ISL1 KO compared to WT.

[0221] C. RNAseq analysis showing misregulated genes in ISL1 KO cardioids compared to WT on day 9.5.

[0222] D. OFT ISL1 KO cardioids show a characteristic switch and express the atrial marker NR2F2. Atrial and OFT cardioids have less TNNT2 expression in the ISL1 KO strain compared to WT.

[0223] E. Contraction analysis on day 9 reveals that atrial WT cardioids have more beats per minute (BPM) compared to atrial ISL1 KO. On day 14, atrial ISL1 KO cardioids have a higher contraction rate compared to WT. On day 14, OFT ISL1 KO cardioids begin to contract, while OFT WT cardioids do not contract. (N = 1, n = 24)

[0224] F. Shows the overall gene expression differences in OFT cardioids using the ISL1 KO strain compared to the WT strain on day 9.5.

Figure 6-2

[0225] G. Compared with WT, the atrial and AVC precursors do not express HOXB1, a pSHF marker, in TBX5 KO.

[0226] H. Representative whole-mount images of TBX5 KO and WT cardioids on day 9.5 and quantification of the cardioids area (H’) (N = 3, n = 8) are shown.

[0227] I. TBX5 KO cardioids downregulate NPPA and TNNT2 in the LV and RV protocols compared with WT. Atrial and AVC TBX5 KO cardioids cannot differentiate into CMs.

[0228] J. RNAseq analysis showing differentially expressed TBX5 KO cardiac chamber-specific genes (NPPA, NPPB) and characteristic genes compared with WT cardioids on day 9.5 is shown.

Figure 6-3

[0229] K. Global gene analysis of cardiac precursors on day 3.5 shows misregulated genes in FOXF1 KO compared with WT.

[0230] L. The time course of atrial and AVC cardioids in FOXF1 KO and WT is shown.

[0231] M. Representative real-time qPCR on day 3.5 is shown, comparing atrial and AVC precursors using the OXF1 KO strain and the WT strain. Fold changes were normalized to housekeeping genes and pluripotency.

[0232] On day 9.5, a decrease in cardioid size of FOXF1 KO cardioids using the LV and AVC protocols is shown compared to WT cardioids. RV and atrial cardioids have the same size in FOXF1 KO and WT strains on day 9.5.

[0233] In the contraction analysis of LV, atrial, and AVC cardioids on days 6.5 and 9.5, a decrease in BMP is shown in FOXF1 KO compared to WT on day 6.5. (N = 1, n = 16) All scale bars in this figure have a length of 200 mm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 7-1

[0234] A. Whole - mount images of cardioids treated with different concentrations of thalidomide starting from mesoderm induction are shown compared to control cardioids (day 9.5). A’ shows quantification of the cardioid area of cardioids treated with thalidomide on day 9.5. (N = 1, n = 8)

[0235] B. Representative real - time qPCR from 9.5 - day - old cardioids treated with thalidomide showing misregulation of lineage - specific genes. Fold changes were normalized to housekeeping genes and pluripotency.

Figure 7-2

[0236] C. Comparison of the size and TNNI - GFP expression of 4.5 - day - old cardioids treated with different concentrations of acitretin.

[0237] D. Shows inefficient CM differentiation and morphological changes of cardioids treated with acitretin.

[0238] E. Shows representative real-time qPCR of cardioids treated with actitretin on days 3.5 and 9.5. Fold changes were normalized to housekeeping genes and pluripotency.

Figure 7-3

[0239] F. Shows whole-mount images of cardioids treated with BPA (B), PFO (P), and nanoplastics (NP) compared to control cardioids (day 9.5).

[0240] All cardioids were treated with teratogens from mesoderm induction (day 0) to day 9.5. All scale bars in this figure have a length of 200 mm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 8-1

[0241] B. Shows SOX2 and EOMES staining after mesoderm induction for aSHF (day 1.5) using the 3D vs 2D->3D protocol.

[0242] C. Shows a heatmap of head mesoderm markers for all precursor populations on day 3.5.

[0243] D. Shows real-time qPCR of TBX1 and TBX5 levels testing factors important for aSHF development alone and in combination.

[0244] E. Optimization of the aSHF protocol by testing different BMP and RA concentrations during the two-step patterning is shown.

Figure 8-2

[0245] G. RNAscope staining of TBX1 and TBX5 of aSHF precursors using different activin concentrations during mesoderm induction is shown.

[0246] H. TBX5 staining of all three precursors on day 3.5 is shown.

[0247] I. A figure showing the absence of homogeneous NKX2-5 expression and SOX2+ cells in all precursors on day 3.5.

[0248] J. Due to low cell density during mesoderm induction, homogeneous TNNI expression is brought about in aSHF-derived cardioids on day 9.5. J’. SOX1 / 2+ cores of aSHF cardioids when using high cell density during mesoderm induction are shown.

[0249] K. Cardioids of all lines showing very few cells expressing PECAM1 and FOXA2 and the absence of COL1A1 and SOX2 are shown. L. Endothelial cell differentiation of all three precursor populations in 2D 24-well plates is shown.

[0250] All scale bars in this figure have a length of 200 mm.

Figure 9

[0251] B. RV (IRX1) and atrial (NR2F2) specific staining of cardioids derived from the hESC line H9 are shown.

[0252] Figure showing a Venn diagram indicating the degree of LV, RV, and atrial crossover of upregulated genes compared to pluripotency on day 9.5.

[0253] Figure showing a volcano plot of differentially expressed genes in the RV vs. atrium on day 9.5.

[0254] All scale bars in this figure have a length of 200 mm.

Figure 10-1

Figure 10-2

[0255] Figure showing higher protein expression of HAND1 and HAND2 in OFT cardioids compared to RV cardioids.

[0256] Figure showing quantification of changes in cardioids area during differentiation in all five protocols. (N = 3, n = 32) All scale bars in this figure have a length of 200 mm.

Figure 11-1

[0257] Figure showing a representative image indicating the degree of contraction for different organoid types. Red pixels represent changes in pixels during contraction.

[0258] Figure showing 30 seconds of f / f0 with different beating patterns for each cardioid in the calcium trace.

[0259] Shows the FluoroVolt-AP curves recorded from 3D cardioids for D.FHF, RV, atrium, and AVC. Each column represents a different cardioids, and different positions of the cardioids are overlaid in each quadrant.

Figure 11-2

[0260] H. Shows the cAPD90 of the patch clamp data in Figure 4K. Corrected using the Fridericia correction method.

[0261] I. Shows the beat intervals of the cells recorded in Figure 4K.

Figure 12-1

[0262] B. Shows sectioned cardioids of each mixing condition stained for TNNT2.

[0263] C. Shows the differences in cadherin expression over time in control LV, RV, and atrial cardioids.

[0264] D. Shows control conditions of LV, RV, and atrial precursor cardioids stained for CDH2 and CDH1 on day 3.5 of differentiation.

[0265] E. Shows cardioids fused together at different time points during differentiation. Cardioids were brought together either on day 3.5 or day 5.5. Then, images were taken on day 9.5 (R = biological replicates). Cardioids fused on day 3.5 are more connected to each other compared to day 5.5.

[0266] F. Shows the cardioids one day after fusion (day 4.5). The right side shows the RV labeled with LMN1-RFP and the FHF labeled with H2B-GFP, and the left side is the reverse.

Figure 12-2

[0267] H. Shows the time course of contraction of the two-chambered cardioids.

[0268] I. Shows the ICC staining of the two-chambered cardioids. Here, the LV cardioids are colored in gray, the RV cardioids are colored in green, and the atrial cardioids are colored in red. Staining was performed for NR2F2, IRX1, and TNNT2.

[0269] All scale bars in this figure have a length of 200 mm.

Figure 13-1

[0270] B. Shows the RNAseq analysis indicating genes misregulated in all protocols in ISL1 KO vs WT on day 3.5.

[0271] C. Shows strong downregulation of WNT5A (OFT marker) in OFT ISL1 KO cardioids compared to WT on day 14.5.

[0272] D. Analysis of the degree of contraction reveals that on day 9.5, atrial ISL1 KO cardioids have less contraction compared to WT, but on day 14.5, the contractions are similar.

[0273] E. Shows the time course of RV, atrial, and OFT cardiogenesis using the ISL1 KO strain and the WT strain.

[0274] Quantification of the cardiomyoid area of ISL1 KO and WT cardiomyoids on day 3.5 (N = 4, n = 8 - 24) and day 9.5 (N = 2, n = 8 - 24) is shown.

Figure 13-2

[0275] H. Verification of the TBX5 KO strain of LV, RV, atrium, and AVC cardiomyoids on day 3.5 is shown.

[0276] I. Representative RT-qPCR of atrial and AVC TBX5 KO cardiomyoids compared to WT on days 3.5 and 9.5 is shown. Fold change was normalized to the housekeeping gene and pluripotency.

[0277] J. Verification of the FOXF1 KO strain of atrial and AVC cardiomyoids on day 3.5 is shown.

[0278] K. Area analysis of WT and FOXF1 KO organoids of the full protocol on day 9.5 is shown. (N = 3 - 4 (LV, RV, atrium, AVC) and N = 1 (OFT), n = 8 - 16).

[0279] L. Expression of TNNT2 in FOXF1 WT vs KO cardiomyoids on day 9.5 is shown.

Figure 13-3

[0280] N. Representative RT-qPCR of atrial and AVC FOXF1 KO cardiomyoids compared to WT is shown. Fold change was normalized to the housekeeping gene and pluripotency.

[0281] All scale bars in this figure have a length of 200 mm. * p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001.

Figure 14-1

[0282] B. Real-time qPCR of cardioids treated with aspirin, showing no obvious gene expression difference compared to control cardioids.

Figure 14-2

[0283] D. Quantification of the TNNI-GFP signal in the OFT revealed a higher GFP signal in OFT cardioids treated with 5 nM and 10 nM acitretin on days 4.5 and 9.5 compared to untreated cardioids. (N = 1, n = 8)

Figure 14-3

[0284] F. Real-time qPCR showing downregulation of the OFT and upregulation of ventricular genes in OFT cardioids treated with all-trans retinol.

[0285] G. Holmount images showing low efficiency of CM differentiation in RV and atrial cardioids when treated with different combinations of plastic residues.

[0286] From mesoderm induction (day 0) to day 9.5, all cardioids were treated with teratogens. ** p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 15

[0287] B. Verification of the sinoatrial node protocol showing protein expression of SHOX2 (an important SAN marker), NKX2.5 (not present in part of the SAN), and HCN4 (an important SAN marker).

[0288] C. The heatmap shows bulk RNA expression at day 3.5 of SAN differentiation, indicating having a gene profile similar to that of the atrium at this time point.

[0289] D. Bulk RNA expression at day 9.5 of SAN differentiation, and a heatmap showing that it upregulates SAN markers compared to the atrial protocol.

[0290] E. Analysis of contraction speed shows that the SAN beats faster than other cardioids and approaches the speed of the paced heart.

Figure 16

[0291] B. And B’ Optimization of the aggregation density of FHF, aSHF, and pSHF precursors (day 3.5) results in different cardioide formation efficiencies later.

[0292] C.178 / 5 hPSC strain shows the beating rates of LV, RV, and atrial cardioids.

[0293] D. OFT precursors form α-SMA-, SM22-, and calponin-positive smooth muscle cell precursors.

Figure 17

[0294] B. Shows specific NR2F2 immunostaining in atrial cardioids.

[0295] C.178 / 5 hiPSC cell line shows the formation of LV, RV, and atrial cardioids.

[0296] D. Shows an overview of the cardioids maturation conditions.

[0297] E. Shows the expression of MYL2, NPPA, and NPPB cardiac chamber markers during RV and LV cardioids maturation.

[0298] F. Shows the expression of markers during atrial cardioids specification and maturation.

[0299] G. Shows immunostaining for the MYL2 cardiac maturation marker in mature LV cardioids and mature RV cardioids.

Figure 18

[0300] B. Shows that the ratio of MYH7 / MYH6 increases in mature cardioids.

[0301] C. Shows the sarcomeric structure in mature cardioids visualized by α-actinin immunostaining.

[0302] D. Shows the contraction analysis of mature cardioids.

[0303]

Figure 19A

[0304]

Figure 19B

[0305]

Figure 19C

Example

[0306] Example 1: Generation of Cardioids Cell Lines WiCell Institute (USA) provided the human H9 (female) ES cell line. The WTC iPS cell line (male, derived from skin fibroblasts) was purchased from the Coriell Institute for Medical Research (USA). The reporter cell line from the Allen Institute for Cell Science was derived from the WTC cell line and received from the Coriell Institute for Medical Research (USA).

[0307] All human pluripotent stem cell lines were grown in customized in-house media using the E8 culture system (Chen et al. (2011) Nat. Methods 8, 424 - 429). To the original E8 mix (R&D #RD - 240 - B - 010), 0.5% BSA (Europa Biosciences, #EQBAH70), in-house manufactured FGF2, and 1.8 ng / ml TGFb1 were added. Cells were cultured on Eppendorf (Eppendorf SE, #0030 721.110) or TPP (TPP Techno Plastic Products AG, #92012) tissue culture treated plates coated with Vitronectin XF (Stem Cell Technologies, #7180) and passaged every 2 - 4 days at approximately 70% confluence using Try-pLE Express Enzyme (GIBCO, #12605010). Cells were regularly tested for mycoplasma contamination.

[0308] Generation of ISL1, TBX5, and FOXF1 knockout cell lines ISL1, TBX5, and FOXF1 were knocked out in WTC cells using CRISPR / Cas9 multi-guide sgRNAs (Synthego) targeting sites on exon 3 of ISL1, exon 5 of TBX5, and exon 1 of FOXF1 (Figure M1I - K). Cells were transfected using the P3 Primary Cell 4D-Nucleofector X Kit S (Lonza - BioResearch, #:V4XP - 3032) and Amaxa 4D-Nucleofector (Lonza - BioResearch). After nucleofection, cells were incubated in E8 supplemented with 5 μM Y - 27632 (Tocris, #72302) on 6-well plates pre-coated with Vitronectin XF (StemCell Technologies, #7180). After 2 days, the medium was changed to Y - 27632-free E8 every other day.

[0309] When the cells reached approximately 70% confluence, single-cell seeding was performed and the remaining cells were harvested for gDNA extraction. The success of the editing was first evaluated at the pool level using agarose gel and Sanger sequencing. Subsequently, single colonies were picked, genotyped, and the knockout was confirmed. Colonies were collected using a microscope (EVOS) and transferred to a pre-coated 96-well plate (Corning, catalog number CLS3370) containing 150 μl / well of E8 supplemented with 5 μM Y-27632 and antibiotics-antifungals. Genome editing at the pool and clone levels was analyzed using the Synthego online tool ICE (https: / / ice.synthego.com / # / ).

[0310] Cardioid generation hPSCs (WTC or H9 line) are seeded at 30 - 40 k cells / well in a 24-well plate (TPP, #92024) in E8 medium + ROCKi (5 μM Y-27632, Tocris #1254). All differentiation media are based on CDM containing 5 mg / ml bovine serum albumin (Europa Biosciences, #EQBAH70) in 50% IMDM (Gibco, #21980065) + 50% F12 NUT-MIX (Gibco, #31765068) supplemented with 1% concentrated lipids (Gibco, #11905031), 0.004% monothioglycerol (Sigma, #M6145-100ML), and 15 μg / ml transferrin (Roche, #10652202001) (Mendjan et al., Cell Stem Cell 2014, 15:310-325; and Hofbauer et al., Cell 2021, 184(12):3299-3317.e22). The medium contains BSA, which is important for efficient cardioid generation. Twenty-four hours after seeding in the 24-well plate, the cells are induced with mesoderm induction medium. The mesoderm induction medium consists of CDM containing FGF2 (30 ng / ml, QKine, Cambridge / UK), LY294002 (5 μM, Tocris, #1130), activin A (specific concentrations for different cardioid subtypes - see below, QKine, Cambridge / UK), BMP4 (10 ng / ml, R&D Systems RD-314-BP-050), and CHIR99021 (specific concentrations for different cardioid subtypes - see below, R&D Systems RD-4423 / 50). After 36 - 40 hours, the cells are dissociated with TrypleE (Gibco, #12605010) and seeded at 15 - 20 k cells / well in a Corning ultra-low attachment 96-well plate (Corning, #7007) in cardiac mesoderm patterning medium 1 composed of CDM containing ROCK inhibitor, and for all protocols except LV, seeded with 1 μg / ml insulin (Roche, #11376497001) with specific factors added according to the cardioid subtype (see below). After seeding, the cells are spin-down in a centrifuge at 200 g for 4 minutes.This protocol is called the 2D-3D standard protocol. Alternatively, hPSCs were seeded at a density of 5000 cells / well in a Corning ultra-low attachment 96-well plate. The cells were seeded in a volume of 200 ml containing E8+ROCKi and collected by centrifugation at 200 g for 5 minutes. As another option, 2500 cells / well were seeded directly into the induction medium + ROCKi and collected by centrifugation at 200 g for 5 minutes. For both protocols, the cells were induced in mesoderm induction medium as described for the 2D->3D protocol. These were named 3D protocols. For both protocols, after 24 hours (or on day 2.5), the cells were fed with cardiac mesoderm patterning medium 1. For the next two days, the medium was exchanged daily with cardiac mesoderm patterning medium 2 composed of CDM containing specific factors depending on the cardioid subtype (see below). For the subsequent two days, the medium was exchanged daily with cardiomyocyte differentiation medium CDM medium containing BMP4 (10 ng / ml), FGF2 (8 ng / ml), and insulin (10 μg / ml). On subsequent culture days, the medium was exchanged every other day with CDM containing insulin (10 μg / ml).

[0311] Alternatively, the entire protocol can be performed completely in 2D by seeding 80,000 - 170,000 cells / well in a 24-well plate coated with vitronectin and adding the medium along the same time axis as for the cardioids. This was named 2D differentiation.

[0312] Mesoderm induction medium (days 0 - 1.5) For the differentiation of left ventricular (LV-FHF precursor-derived) cardioids, Activin 5 ng / mL and CHIR99021 3 μM are used. For the differentiation of right ventricle (RV), atrium, and outflow tract (OFT), Activin 50 ng / mL and CHIR99021 4 μM are used. For AVC, Activin 10 ng / mL is used and CHIR99021 2 μM is used. For SAN, Activin 50 ng / ml is used and CHIR99021 1 μM is used. These mesoderm induction conditions result in high (>70%) efficacy, homogeneity, and reproducibility of different cardioids subtypes. This stage is characterized by the expression of BRA, EOM, MIXL1, FOXA2, and other early mesoderm markers, and the absence of SOX2, a pluripotency and early neural marker. This medium functions best for the WTC strain.

[0313] Alternative mesoderm induction medium (days 0 - 1.5) For the differentiation of LV (FHF-derived) cardioids, Activin 5 ng / mL and CHIR99021 1 μM are used. For the differentiation of RV (aSHF-derived) and atrium (pSHF-derived), Activin 50 ng / mL and CHIR99021 1.5 μM are used. This medium functions best for the H9 strain.

[0314] Cardiac mesoderm patterning medium 1 (days 1.5 - 3.5) For LV (FHF precursor): BMP4 (10 ng / ml), FGF2 (8 ng / ml), insulin (10 μg / ml), C59 (2 μM, Tocris, #5148 / 10), and retinoic acid (50 nM, Sigma Aldrich, #R2625). The retinoic acid concentration needs to be low because high concentration (>100 nM) leads to atrial fate from FHF instead of ventricular fate. The early specific markers of LV are IRX4 and HEY2, which are characterized in Hofbauer et al., Cell 2021, 184(12):3299 - 3317.e22.

[0315] RV (aSHF precursor): Either TGF-beta inhibitor SB431542 (10 μM, Tocris, #1614 / 10) and C59 (2 μM) or XAV-939 (5 μM, SelleckChem, #S1180). By using SB431542, the SHF lineage is obtained and thus the RV, OFT, SHF-derived atrium, and AVC fates. The earliest aSHF markers on day 3.5 are TBX1 and FOXC1 / 2, which are shared by the RV and OFT.

[0316] OFT (aSHF precursor): SB431542 (10 μM) and XAV-939 (5 μM). It is the same as the RV at this stage.

[0317] Atrium (pSHF precursor): SB431542 (10 μM), XAV-939 (5 μM), and retinoic acid (500 nM). The combination of SB and high concentrations of retinoic acid results in the characteristics of pSHF precursors characterized by high levels of HOXB1, TBX5, and FOXF1 and the absence of TBX1 or IRX4 expression on day 3.5.

[0318] AVC and AVN (pSHF / AVC precursor): SB431542 (10 μM), XAV-939 (5 μM), retinoic acid (500 nM), and BMP4 (10 ng / ml). The combination of SB, high retinoic acid, and additional BMP4 at this stage results in the characteristics of pSHF / AVC precursors characterized by high expression of MSX2 and TBX2 in addition to standard pSHF markers.

[0319] Sinoatrial node (SAN) - Similar to the AVC, but during the induction process, high activation of activin and low activation of WNT (activin 50 ng / ml and CHIR99021 1 μM) are used. Optionally, WNT inhibition is not included during patterning 1. Different from the AVC.

[0320] Cardiac mesoderm patterning medium 2 (days 3.5 - 5.5) LV: BMP4 (10 ng / ml), FGF2 (8 ng / ml), insulin (10 μg / ml), C59 (2 μM), and retinoic acid (50 nM) (see Hofbauer et al. above).

[0321] RV: Either C59 (2 μM) or XAV-939 (5 μM), BMP4 (10 ng / ml), FGF2 (8 ng / ml), insulin (10 μg / ml), and retinoic acid (in the range of 50 - 500 nM). Here, retinoic acid is added to assist in driving RV characteristics.

[0322] OFT: XAV-939 (5 μM), BMP4 (10 ng / ml), FGF2 (8 ng / ml), and insulin (10 μg / ml). The absence of retinoic acid at this stage results in specific OFT characteristics characterized by high expression of WNT5A, MSX1, BMP4, and RSPO3.

[0323] Atrium, AVC, SAN, and AVN: XAV-939 (5 μM), BMP4 (10 ng / ml), FGF2 (8 ng / ml), insulin (10 μg / ml), and retinoic acid (500 nM). For the SAN, it is the same, but there is no WNT inhibition (without XAV-939) or WNT activation (CHIR99021). For the AVN, similar to the AVC, it is further advanced in the AVN direction, for example, by BMP and sonic hedgehog signaling (Figure 15). The atrium begins to express NR2F1 / 2 and HEY1 at this stage, and in the AVC, high levels of TBX2, MSX2, and RSPO3 expression start.

[0324] Example 2: Cardioid Example 2.1: Heart Chamber Specification Protocol For the atrial specification protocol, on day 7, atrial cardioids were transferred to CDM medium containing retinoic acid (500 nM, Sigma Aldrich, #R2625), FGF2 (15 ng / mL, Cambridge University), LDN-193189 (200 nM, Stemgent, #04-0074) and LY-411575 (3 μM, MedChemExpess, #HY-50752) until day 10. From day 10 to day 21, the atrial cardioids were transferred into DMEM containing low glucose (1 g / L, Sigma Aldrich, #G8644) and containing dexamethasone (250 nM, Sigma Aldrich, #D4902), indomethacin (50 μM, Sigma Aldrich, #I7378), T3 hormone (4 nM, Sigma Aldrich, #T6397) and chemically defined lipid concentrate (1X, Invitrogen, #11905031).

[0325] For the ventricular specific protocol, on day 7, LV and RV cardioids were transferred to DMEM containing high glucose (4.5 g / L, Sigma Aldrich, #G8644) and containing IGF2 (25 ng / mL), CHIR99021 (1 μM, R&D Systems RD-4423 / 50) and human insulin (10 ng / ml, Sigma) until day 14. From day 14 to day 16, the LV and RV cardioids were maintained in DMEM containing high glucose (4.5 g / L) and containing XAV-939 (4 μM, SelleckChem, #S1180). Finally, from day 14 to day 16, the LV and RV cardioids were transferred into DMEM containing low glucose (1 g / L, Sigma Aldrich, #G8644) and containing dexamethasone (250 nM, Sigma Aldrich, #D4902), indomethacin (50 μM, Sigma Aldrich, #I7378), T3 hormone (4 nM, Sigma Aldrich, #T6397) and chemically defined lipid concentrate (1X, Invitrogen, #11905031).

[0326] Example 2.2: 2D Differentiation of Endothelial Cells hPSCs were seeded at 100,000 cells / well in 24-well plates coated with vitronectin in E8 medium supplemented with 5 mM ROCK-i. The next day, for H9 cells, cells were induced with FLyAB and 1 - 3 mM CHIR99021 and incubated for 36 - 40 hours. For the next two days, the medium was changed to the respective cardiac mesoderm patterning medium 1 for FHF, aSHF, and pSHF. Thereafter, CDM containing 200 ng / ml VEGF (200 ng / ml, Peprotech, #AF - 100 - 20) and 2 mM forskolin (Sigma - Aldrich, #F3917) was given for two days, and then the cells were cultured for one day in CDM containing 100 ng / ml VEGF.

[0327] Example 2.3: Mixing of Precursors Cardiac differentiation of different precursor cell populations (FHF, aSHF, and pSHF) was carried out until day 3.5 in 24-well plates coated with vitronectin (2D differentiation). The cell populations were labeled using different colored cell lines (WTC:H2B - GFP, WTC:LMNB1 - RFP). On day 3.5, the precursor cells were dissociated by adding 200 μl of Try-pLE Express Enzyme (GIBCO, #12605010) at room temperature for 3 - 4 minutes. Dissociation was stopped by adding 1 ml of CDM containing ROCKi (5 mM). After centrifugation at 130 g for 4 minutes, the cells were resuspended in CDM containing ROCKi (5 mM). Then, two precursor populations were mixed by seeding 15,000 - 20,000 cells per precursor population into ultra-low attachment (corning) in co-development patterning medium containing C59 (2 μM), BMP4 (10 ng / ml), FGF2 (8 ng / ml), insulin (10 μg / ml), and retinoic acid (500 nM), as well as ROCKi (5 mM). On day 5.5, and then for two days, the medium was changed to cardiomyocyte specification medium.

[0328] Example 2.4: Preparation of Multicavitary Cardioids Cardioids are generated as described above until day 3.5 of differentiation. On day 3.5, the developing cardioids are combined in two different manners (see the following description) depending on whether double fusion, triple fusion, or multiple fusion is desired. The medium used for the fusion conditions is CDM-based cardiac mesoderm patterning medium 2 for LV, RV, and atria, containing BMP4 (10 ng / ml), FGF2 (8 ng / ml), insulin (10 μg / ml), C59 (2 μM), and retinoic acid (500 nM). The fused cardioids are then given cardiomyocyte differentiation medium daily for the next 2 days. The remaining time is treated every other day with CDM containing insulin (10 μg / ml).

[0329] Example 2.5: Generation of multi-chambered cardioids Double fusion On day 3.5, the developing organoids are transferred using a wide-orifice pipette from individual wells of a 96-well Corning ultra-low attachment plate to a shared well having one other desired organoid subtype. This can be achieved using any combination of LV, RV, or atrial cardioids. For this type of fusion, the cardioids are placed together in the co-development patterning medium. Alternatively, on day 1.5, LV differentiation can be combined with RV or atrial progenitor differentiation on day 3.5 in the co-development patterning medium or cardiac mesoderm patterning medium 1 to obtain a multi-chambered cardioid having at least one shared cavity. Importantly, two-chamber / multi-chamber cardioids co-develop and share cavities when fused at these early stages. Subsequent fusion (e.g., after day 5.5) impairs the formation of shared cavities.

[0330] Triple fusion A mold (made of silicone) having a shape for arranging initial cardioids that are in contact with and fused to each other in the order expected in a natural heart (e.g., a linear order) was fabricated. The mold was covered with 70% ethanol for at least 1 hour and sterilized by turning on laminar flow UV. Then, the mold was coated with an anti-adhesion rinse solution (STEMCELL Technologies, #07010) for several seconds and immediately washed once with PBS. After washing, the mold was stored in the refrigerator until the day of fusion or used immediately. On day 3.5 of cardiac differentiation, the cardioids were transferred to the mold using a wide-opening tip. By using the mold, the cardioids can be arranged in the desired orientation (e.g., the first atrium, then the LV and RV, as in vivo). While the cardioids are fusing in the mold from day 3.5 to 5.5, the medium is not changed. Earlier timing improves the fusion efficiency and results in a better morphology. Fusing with more developed cardioids may not be complete depending on the developmental stage. On day 5.5, the fused cardioids were returned to a 96-well plate and the medium exchange was continued as described above.

[0331] To track the cell populations from which the cardioids in the fusion product originated, a colored cell line (WTC:H2B-GFP, WTC:LMNB1-RFP) or a dye was used. For this purpose, the cells were stained for 1 hour before induction using SP-DiIC18(3) (Invitrogen, #D7777) to fluoresce at 564 nm or DiIC18(5) (Invitrogen, #D12730) to fluoresce at 668 nm.

[0332] Example 2.6: Mold for Multicavity Cardioid The embedded type was designed in Tinkercad, and the diameter and length were adjusted based on the cardioids size on the fusion day. The file was exported as an.stl file and loaded into the slicer software XYZ print 1.4.0. The negative was printed using transparent PLA with a filling density of 100%, a layer height of 0.1 mm, and a nozzle temperature of 215 °C. After printing, the negative was treated at 550 °C using a heat gun (Bosch Hot Air Blower 1800W) to carefully melt the surface of the negative, create a smooth finish, and remove the typical rough surface of 3D printing.

[0333] Next, the positive was cast using polydimethylsiloxane (PDMS). Briefly, 5 ml of curing agent and 45 ml of monomer (both from Sylgard® 184 Elastomer Kit, VWR) were vigorously mixed. The mixture was then spin - down to remove air bubbles and used directly.

[0334] To reduce the degree of air bubbles formed during curing, the mold was cast at a low temperature (40 °C). For this, the negative was placed in a 10 cm dish and slowly covered with 30 ml of liquid PDMS mixture. The negative was then carefully removed from the polymerized PDMS, and the remaining PDMS was cut off using a surgical scalpel. The mold was then attached to the bottom of a clean 10 cm dish using approximately 5 ml of PDMS and cured at 40 °C. To sterilize the mold, it was washed in 70% ethanol in a UV - lit draft chamber for approximately 30 minutes. To position the cardioids inside the mold, the mold was rinsed once with PBS and then coated with an anti - adhesion rinse solution (StemCell Technologies, #07010) to further increase the non - sticky behavior of the PDMS. After coating, the mold was rinsed once with PBS and then ready for use.

[0335] Example 2.7: Analytical methods Freeze - sectioning The cardioids were fixed with PBS containing 4% PFA and cryoprotected with PBS containing 30% sucrose before embedding. Embedding was performed using O.C.T. cryoembedding medium (Scigen, #4586K1). The embedded tissues were frozen using a metal surface submerged in liquid nitrogen and stored in an -80°C freezer until sectioning with a Leica cryostat. The sections were collected on SuperFrost Plus slides (Thermo Fisher Scientific, #10149870) and maintained at -20°C or -80°C until immunostaining.

[0336] Immunostaining To remove O.C.T., the fixed specimens were washed in 1×PBS for 15 minutes. Optionally, the tissues were placed in a permeabilization solution of 0.5% Triton-X100 (Sigma-Aldrich, #T8787) for 5 minutes to increase antibody penetration. The tissues were then incubated in a blocking solution (PBS (GIBCO, #14190094) containing 4% donkey serum (Bio-Rad Laboratories, #C06SB) and 0.2% Triton X-100) for at least 30 minutes. Subsequently, the specimens were incubated in a blocking solution containing the primary antibody at room temperature for 3 hours or at 4°C overnight. They were then washed in PBS containing 0.1% Tween 20 (Sigma-Aldrich, #P1379) for 20 minutes and then incubated in a blocking solution containing the secondary antibody at room temperature for 1 hour. Finally, the tissues were washed in PBS containing 0.1% Tween 20. The slides were mounted using a fluorescent mounting medium (Dako Agilent Pathology Solutions, #S3023) and covered with a coverslip (Menzel-Glaser, #631-0853 VWR).

[0337] RNAscope and in situ hybridization chain reaction (HCR) RNA-scope was performed using the ACDBio (https: / / acdbio.com) manual assay kit, following the manufacturer's instructions, with RNAscope Probe-hs-TBX1-C2 (target region: 100 - 769) and RNAscope Probe-hs-HOXB1-C2 (target region: 528 - 2015). RNAscope Probe-hs-PPIB-C1 was used as a positive control. The probes were designed and manufactured by ACDBio.

[0338] HCR fluorescence in situ was performed using the HCR kit (v.3) purchased from Molecular Instruments (molecularinstruments.org), following the manufacturer's instructions, with a slight modification of adding 100 μg / ml salmon sperm DNA to the pre-amplification solution and the amplification solution containing hairpins to reduce non-specific binding. The HCR probe WNT5A (B3) was designed and manufactured by Molecular Instruments.

[0339] Image acquisition and analysis Fixed tissue sections were imaged using a spinning disk confocal microscope (Olympus spinning disk system based on the IX3 series (IX83) inverted microscope equipped with a Yokogawa W1 spinning disk). Live imaging was performed using an inverted wide-field microscope for bright field and fluorescence (Axioobserver Z1 equipped with an sCMOS camera (Hamamatsu Orca Flash 4)). Cardioids in 96-well plates were also imaged using a Celigo Imaging Cytometer microscope (Nexcelom Biosciences, LLC).

[0340] Flow cytometry 1.5 mL of CM dissociation medium (Stem Cell Technologies, #05025) was used at 37 °C for 7 - 10 minutes to dissociate cardioids (8 cardioids per condition). CM dissociation was stopped by adding 7.5 ml of support medium. After centrifugation at 130 g for 4 minutes, the cells were resuspended in 600 μl of PBS containing 0.5 mM EDTA (Biological Industries, #01 - 862 - 1B) and 10% FBS (PAA Laboratories, #A15 - 108). Cells were acquired on a FACS LSR Fortessa II (BD) and analyzed with FlowJo V10 (FlowJo, LLC) software. FACS sorting was performed using a Sony SH800 Cell Sorter (Sony Biotechnology).

[0341] RNA extraction and bulk RNA-seq preparation and analysis RNA was isolated using the company's semi-automated RNA bead isolation kit with the KingFisher device (KingFisher Duo Prime). Bulk RNA-seq libraries (N = 3, n = 8) were generated using the QuantSeq 30mRNA-Seq Library Prep Kit FWD (Lexogen GmbH, #015) according to the manufacturer's instructions. After library preparation, the Fragment Analyzer (Advanced Analytical Technologies, Inc) was used to confirm the appropriate size distribution of the samples. Subsequently, the RNA-seq libraries were submitted to the Next-Generation-Sequencing (NGS) facility of the Vienna Biocenter Core Facilities (VBCF) for sequencing. To add UMI sequences to the read identifiers, reads were preprocessed using umi2index (Lexogen) and trimmed using BBDuk v38.06 (ref=polyA.fa.gz,truseq.fa.gz k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20). Reads mapping to abundant sequences included in the iGenomes NCBI GRCh38 reference were removed using bowtie2 v2.3.4.1 alignment. The remaining reads were analyzed using the genome and gene annotations for the GRCh38 assembly obtained from Homo sapiens Ensembl release 94. Reads were aligned to the genome using star v2.6.0c, and gene reads were counted using featureCounts (subread v1.6.2) with strand-specific read counts (-s 1). Differential gene expression analysis for raw counts and principal component analysis for variance-stabilized transformed count data were performed using DESeq2 v1.18.1.

[0342] Real-time quantitative polymerase chain reaction The isolated RNA was reverse transcribed into cDNA using a Reverse Transcription Kit (Invitrogen, #18080044) with a C100 Touch Bio-Rad Thermal Cycler. Quantitative PCR was performed using GoTaq qPCR Master Mix 2x (Promega, #A6001) with a Bio-Rad CFX384 Real-Time Thermal Cycler. Gene expression values for each sample were obtained in triplicate. Log fold changes were used from samples of PBGD as a housekeeping gene and pluripotent stem cell samples for normalization.

[0343] Analysis of contraction One to two hours before recording, fresh CDMI medium was given to the cardioids. A 96-well plate was placed in an environmental control stage incubator (37 °C, 5% CO2, water-saturated air atmosphere, Okolab Inc, Burlingame, CA, USA). Each well was imaged at 100 frames / second for 30 - 60 seconds using a wide-field phase contrast microscope (Axioobserver Z1 (inverted) with sCMOS camera, Zeis). Next, the videos were analyzed using MUSCLEMOTION. The data was loaded into custom-ordered software for the reported calculations. The percentage of beats was defined by whether the cardioids beat once during the entire recording. The total number of beats in the video was counted, divided by the length of the video in seconds, and multiplied by 60 to calculate beats per minute. The degree of contraction was the amplitude obtained from MUSCLEMOTION divided by the size of the cardioids.

[0344] Calcium transient To generate the WTC strain expressing the GCaMP6f gene, an AAVS1 integration construct with a CAG promoter followed by the GCaMP6f sequence was selected (Mandegar et al. (2016) J. Cell Stem Cell 18, 541 - 553) and introduced as previously described (Hofbauer, et al. (2021b) Cell 184, 3299 - 3317.e22).

[0345] Using the above protocol, cardiospheres were differentiated into LV, RV, atria, OFT, and AVC or multi-chamber cardiospheres. The cardiospheres were given fresh CDM-I medium 1 - 2 hours prior to recording. The 96-well plates were placed in an environmental control stage incubator (37 °C, 5% CO2, water-saturated air atmosphere, Okolab Inc, Burlingame, CA, USA). Each well was imaged at 50 - 100 (optimally 50) frames / second for 30 - 60 seconds using a wide-field microscope (Axioobserver Z1 (inverted) with sCMOS camera, Zeis). The cardiospheres were excited at 470 ± 10 nm using a light-emitting diode (LED). Analysis of signal propagation: Peaks were identified using the full cardiospheres analysis pipeline. Only pixels with a maximum intensity higher than the organoid-specific threshold were considered. The intensity was calculated for each pixel, normalized to 1, and each trace was smoothed using a rolling average over 3 frames. The frame at which the pixel reached 50% of the peak intensity was recorded. The first frame at which more than 30 pixels reached 50% of the maximum intensity was defined as the first frame. The last frame at which all pixels except for a maximum of 30 pixels reached 50% of the maximum intensity was defined as the last frame. The average position of the largest connected component of the pixels that first reached 50% of the peak intensity was considered the origin of signal propagation. Next, for all other pixels, the signal propagation speed was calculated by dividing the distance between the pixel and the origin by the frame difference between the frame at which the pixel reached 50% of the peak intensity and the origin frame. For all pixels and for all beats, the speeds were averaged to determine the signal propagation speed in the organoid. Images of signal propagation were made using the same technique and each pixel was color-coded based on the frame difference. Cardiospheres were excluded from this analysis for four reasons: (1) the cardiospheres did not beat, (2) there was no clear directionality, (3) there were two origins, and (4) less than 10% of the cardiospheres expressed the reporter protein.

[0346] Multi-electrode array (MEA): Electrophysiological recordings of extracellular electric field potentials were performed using MEA. BioCAM Duplex (3 Brain) was used together with a single-well Accura MEA chip (3 Brain). The MEA chip consists of 4096 gold-coated electrodes with a 60-μm pitch covering an area of 3.8 × 3.8 mm.

[0347] The MEA chip reservoir was rinsed with 70% ethanol, sterilized, and then washed 4 times with Mili-Q water. Then, PBS was added and the chip was left overnight with PBS to improve connectivity. Next, without allowing it to dry completely, the PBS was removed, and on day 9.6 of differentiation for single cardioids and on days 12 - 15 of differentiation for multi-chamber cardioids, a 200-μl wide-bore pipette tip (Thermofisher #2069G) was used to carefully place the cardioids in the center of the MEA chip. Their positions were fixed, and a membrane and a handmade anchor were placed on the cardioids to maximize the contact area between the cardioids and the chip. Finally, 1.5 mL of CDM-I was added to the reservoir, and the MEA chip was stored overnight at 37 °C in a 5% CO2 incubator to further improve connectivity.

[0348] Recordings were performed using BrainWave4 software with a pre-set of the cardiac organoids. The recordings were made at 37 °C, and the entire chip was covered with a black lid to prevent light exposure. The electric field potential signal from the beating cardioids was acquired through a 5-Hz high-pass filter, and electrode 1.1 was used as the reference electrode. The waveform stability was confirmed for 5 - 10 minutes to ensure consistency before recording for 5 minutes.

[0349] Patch-clamp recordings of single cardiomyocytes The STEMdiff Cardiomyocyte Dissociation Kit (Stem Cell Technologies, #05025) was used to dissociate the cardioids according to the manufacturer's protocol (incubated at 37 °C for 10 - 20 minutes to completely dissociate the organoids), and then laminin-511 E8 fragment (AMSBIO, #AMS.892 011, 0.5 μg / cm2 ) Coated 35mm tissue culture treated dishes (Corning, #430165) with low density of 15 - 40k cells were seeded.

[0350] Cells were maintained at 37°C in a humidified incubator with 5% CO2. Whole - cell patch - clamp experiments were performed on single - beating cardiomyocytes 4 - 13 days after plating. Using a Sutter P - 1000 micropipette puller (Sutter Instrument), glass micropipettes with a resistance of 1.5 - 4 MΩ were pulled from glass capillaries (Harvard Apparatus, #BS4 64 - 0792). The extracellular solution consisted of 140 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES (in mM), and the pH was adjusted to 7.4 using NaOH. The intracellular pipette solution contained 150 KCl, 5 NaCl, 2 CaCl2, 5 EGTA, 10 HEPES, and 5 MgATP (in mM), and the pH was adjusted to 7.2 using KOH. Data were acquired at 10 kHz using a HEKA EPC 10 USB Quadro (HEKA Elektronik GmbH) with PATCHMASTER NEXT software (HEKA Elektronik GmbH) and low - pass filtered at 2.9 kHz. Spontaneous electrical activity was recorded in the current - clamp mode and analyzed using custom - made MATLAB (MathWorks) software. The action potential amplitude was measured from the peak to the maximum diastolic potential, and the APD values were calculated from the action potential peak to the respective percentage of repolarization of the amplitude. Parameters were calculated individually for 15 - 20 consecutive action potentials per cell and then averaged.

[0351] Optical action potential Cardioids were dissociated in the same way as the patch - clamp experiments (see previous section), and laminin - 511 E8 fragment (AMSBIO, #AMS.892 011, 0.5 μg / cm 2) The wells of a 96-well plate (Greiner Bio-One, #655182) pre-coated with 2 were seeded with 40k cells per well and maintained at 37 °C for 7 - 11 days in a humidified incubator with 5% CO

[0352] For the CM monolayer, after repeating the washing step three times with Hank's balanced salt solution (HBSS, Gibco, #14175-053), the voltage-sensitive dye FluoVolt (FluoVolt™ Membrane Potential Kit, Thermo Fisher Scientific, #F10488) at 0.7 times the manufacturer's recommended amount was loaded. Loading was performed at room temperature for 30 minutes, after which the cells were washed three more times with HBSS. Subsequently, the 96-well plate was placed in an environmental control stage incubator (37 °C, water-saturated air atmosphere, Okolab Inc, Burlingame, CA, USA), and the fluorescence signal was recorded at an excitation wavelength of 470 ± 10 nm using a light-emitting diode (LED), and the emitted light was collected by a photomultiplier tube (PMT, Cairn Research Ltd, Kent, UK). The fluorescence signal was digitized at 10 kHz. Then, the 20 s recording was analyzed offline using custom MATLAB (MathWorks) software. APDs were measured at 30%, 50%, and 90% repolarization. The APD values were calculated from the action potential peak relative to each percentage of amplitude repolarization. For all action potentials recorded per well, the parameters were calculated individually and then averaged. The number of action potentials analyzed per well typically ranged from 5 - 20.

[0353] Example 3: Induction of Cardiomyoids from aSHF and pSHF Precursors The inventors hypothesized that aSHF is exposed to a signaling environment similar to other anterior and dorsal embryonic regions (neuroectoderm and head mesoderm), including inhibition of WNT and TGFb signaling. By combining these signaling conditions with a 3D cardioid approach (Hofbauer et al., Cell 2021, 184(12):3299-3317.e22), the inventors aimed to develop a method for inducing cardioids from the aSHF lineage. The first stage of differentiation consisted of mesoderm induction followed by an aSHF patterning stage using dual WNT and TGFb inhibition or TGF-beta signaling inhibition (Figure 1A). 3.5 days after 3D differentiation, a heterogeneous population of progenitor cells with one subpopulation expressing the aSHF markers TBX1 or FOXC2 was observed, while another subpopulation expressed the FHF and pSHF marker TBX5 (Figures 1B, 1C, and 8A). To determine the origin of this heterogeneity, the initial mesoderm introduction stage (day 1.5) was analyzed, and it was found that only the outside of the organoid expressed the mesoderm marker EOMES. In contrast, the organoid core still expressed the pluripotency and neural marker SOX2 (Figure 8B), indicating that mesoderm is not induced homogeneously in 3D. The inventors hypothesized that cells in 2D receive more equal mesoderm induction signals, leading to a homogeneous escape from pluripotency and differentiation. When mesoderm was induced in 2D and 3D differentiation was initiated only at the patterning stage (day 1.5), the cells efficiently exited pluripotency (Figure 8B), homogeneously expressed high levels of TBX1 and FOXC2, and did not express TBX5 (Figures 1C, 8A, and 8D). In addition, no expression of head mesoderm markers was observed in aSHF progenitors at day 3.5 (Figure 8C). Thus, the 2D->3D differentiation approach produces a homogeneous population of progenitor cells for both FHF and aSHF progenitor cells.

[0354] In contrast to aSHF, pSHF is exposed to retinoic acid (RA) signaling in vivo, activates pSHF regulatory factors (HOXB1+, HOXA1+, TBX5+), and inhibits the aSHF expression signature (TBX1+, FOXC2+, SIX1+). Consistent with this, it was observed that adding RA during the aSHF patterning stage resulted in a switch to pSHF characteristics (Figures 1F and 8E). Manipulation of other signaling pathways (SHH, WNT, FGF, and BMP) did not affect the major aSHF or pSHF markers (Figures 8E and 8F). Consistent with recent in vivo observations, different signaling levels during mesoderm induction promoted aSHF and pSHF more than the FHF lineage (Figure 8E). Furthermore, analyzing the three precursor subtypes by RNA-seq, the primary markers of FHF, aSHF, and pSHF were significantly those of the most differentially expressed genes (Figures 1D and 1E). Compared to pluripotency, 532 genes were specifically upregulated in aSHF cells, 408 in pSHF cells, and 1046 in FHF cells, and 1417 genes were shared among the three protocols (Figure 1H). Notably, the major markers of aSHF (TBX1, SIX1, FOXC2) had low or absent expression from FHF and pSHF, and the pSHF markers (HOXB1, HOXA1, FOXF1) were barely detectable or lost in aSHF and FHF precursors (Figures 1D - 1G). The specificity and homogeneity of the precursor populations were further emphasized by the mutually exclusive expression of the lineage - specific markers TBX1 and HOXB1 seen by RNA hybridization (Figure 1F) and the immunostaining of FOXF1, TBX5, and FOXC2 seen by immunostaining (Figures 1G and 8H). Nevertheless, all populations were positive for the cardiac precursor marker NKX2 - 5 (Figure 8I) and mostly negative for the pluripotency and neuroectodermal marker SOX2 (Figure 16A). Overall, by day 3.5 of differentiation using the cardioids system, all three major cardiac precursors can be generated efficiently and homogeneously.

[0355] FHF, aSHF, and pSHF precursors give rise to several different cardiac cell types such as CMs and endocardial cells in the embryo. The inventors have previously shown that FHF precursors generate a ventricular-like contractile cardioids containing CMs and endocardial-like cells (Hofbauer et al., supra). Following this method, a / pSHF precursors were treated with BMP, FGF, insulin, and RA and WNT signaling was inhibited (cardiac patterning 2) (Figure 1A), resulting in the formation of reproducible contractile cardioids containing cavities (Figures 1I and 1J). In contrast to FHF-derived cardioids, a / pSHF-derived cardioids required a higher dose of RA at this stage. Due to inefficient CM differentiation by the organoid core expressing neural markers at high density (Figure 8J’), efficient differentiation of a / pSHF was dependent on a lower seeding density (Figure 8J). A balanced level of WNT signaling activation during mesoderm induction and the number of seeded cells at cardiac patterning stage 1 helped optimize and fine-tune cardioids formation (Figure 8J, Figure 16B, B’). More than 85% of the cardioids cells expressed the important CM marker TNNI1 (Figures 1K and 1L), and there was no expression of endoderm marker (FOXA2), ectoderm marker (SOX2), and fibroblast marker (COLA1) (Figure 8K). Finally, aSHF cells and pSHF cells also efficiently differentiated into PECAM1+ endothelial cells in 2D when exposed to VEGF and Forskollin after the SHF patterning stage (Figure 8L). In summary, in vivo-like signaling identifies a / pSHF precursors that differentiate into CMs and the endothelial lineage, enabling the formation of cardioids.

[0356] Example 4: Formation of RV and atrial cardioids The FHF precursors differentiate early into CMs that form the cardiac tube, while the aSHF precursors first proliferate and then differentiate together with the pSHF precursors at a later time point. Therefore, the inventors hypothesized that SHF-derived cardioids would exhibit a similarly high proliferation rate, delayed morphogenesis, and differentiation as FHF-derived cardioids. Detailed time-course analysis revealed a delay in the formation of SHF cardioids (Figures 2A - 2C), a higher aSHF precursor proliferation rate and Ki67 expression (Figures 2C and 2D). The a / pSHF cardioids were smaller than the FHF cardioids and initiated the expression of the CM marker TNNI1 one day later than the FHF-derived cardioids (Figures 2A and 2B). Global gene expression confirmed that SHF-derived cardioids exhibited a delay in the expression of sarcomere and structural CM genes (Figures 2E and 2F) and a delay in the onset and formation of cavitation over time (Figures 2C, white and yellow arrows). Different cardioids subtypes also showed an increase in cell number (Figure 17A) and cell size (Figure 17C) over time. In summary, the developmental timing of SHF- and FHF-derived cardioids is consistent with the in vivo timing of SHF and FHF differentiation and morphogenesis.

[0357] Next, we also examined whether cardioids derived from a / pSHF also follow a developmental trajectory with respect to chamber characteristics. In vivo, FHF, aSHF, and pSHF give rise to the left ventricle (LV), right ventricle (RV), and atria, respectively. We tested the specification ability of a / pSHF compared to FHF precursors by modulating the concentration of RA. We observed that aSHF precursors gave rise to CMs with initial RV characteristics (IRX1+, IRX2+, IRX3+, NPPA+), while pSHF precursors differentiated into initial atrial CMs (HEY1, NR2F1, NR2F2) (Figure 2F). Global gene expression comparisons at day 9.5 of FHF-cardioids, aSHF-cardioids, and pSHF-cardioids revealed that the top differentially expressed genes included ISL1+, IRX1+, and RFTN1+ (Figures 2G and 9D), all of which are involved in ventricular characteristics and physiological functions. Furthermore, comparison of FHF cardioids with pSHF cardioids showed upregulation of TBX5, NR2F2, and NR2F1, which are consistent with initial atrial characteristics (Figures 2G and 9D, Figure 17B). These results were further confirmed at the protein level for IRX1, NR2F2, and HEY2 (Figures 2H, 2H’ and 9A). When compared to the pluripotent state, 376 genes were specifically upregulated in the RV, 645 in the atria, 449 in LV cells, and 3508 genes were shared among the three protocols (Figure 9C). Specification of CM sub-lineages was also achieved using hESC H9 (Figure 9B, Figure 17B) and other iPSC lines (Figure 16C, Figure 17C). Interestingly, it was observed that the final size of pSHF-derived cardioids was small (Figures 2A and 2B), which is consistent with the smaller size of the atria compared to the ventricles. In summary, aSHF precursors were specified into RV-like cardioids, pSHF precursors formed atrial-like cardioids, indicating that initial priming of precursors is important for obtaining different chamber characteristics in the developing heart.

[0358] Example 5: Specification to OFT, AVC, and chamber cardioids. In addition to the right ventricle, the aSHF precursors differentiate into the outflow tract (OFT), which gives rise to the aortic and pulmonary valves as well as the vascular structures. Abnormalities in these are the most frequent congenital heart defects. The inventors next investigated at which stage signaling instructs the separation of the RV and OFT and hypothesized that this occurs after aSHF specification. High doses of RA promoted aSHF specification to RV chamber identity (Figure 10B), and in the absence of RA signaling, it was observed that OFT markers (WNT11, WNT5A, ISL1, BMP4, RSPO3) were promoted in the absence of chamber markers such as NPPA (Figures 3A–3C and 10B). These observations were confirmed at the levels of WNT5A, ISL1, HAND1, and HAND2 (Figures 3D, 3E, and 10C). The differences between the RV and OFT lineages were already apparent at day 4.5 of differentiation, with an acceleration of differentiation into the RV chamber program shown compared to the OFT (Figure 10A). The gene expression profile of the OFT showed that the marker WNT5A was already upregulated at day 4.5, and OFT cardioids were generally more mesenchymal, showed a delay in differentiation, and were smaller in size compared to RV cardioids (Figures 3F and 10D). OFT cardioids also had the ability to differentiate into smooth muscle cells (Figure 16D). Thus, the aSHF precursors can then be directed to form either RV or OFT cardioids, depending on the presence or absence of RA, respectively.

[0359] In vivo, the CM derived from pSHF constitutes most of the atrium and contributes to the AVC, an important region where valves and pacemaker elements develop. The precursors of pSHF are located in different regions of the primitive streak and move at different times. The precursors of pSHF that will become the AVC move earlier, while the atrial pSHF precursors move later. Therefore, the inventors hypothesized that the mesoderm induction conditions of these two different pSHF populations are different. Consistent with this, intermediate levels of activin and WNT activation led to more highly expressed AVC-specific genes at later time points while maintaining the pSHF signature. Furthermore, after the mesoderm induction stage, three different cell populations (FHF, SHF, and AVC) show a gene expression pattern from front to back (Figure 3H). Another significant difference between AVC and atrial development in vivo is the greater exposure of the AVC region to BMP ligands. Consistent with this, adding BMP during the patterning stage upregulates early AVC markers. Finally, the combination of optimized induction and patterning stages (Figure 3G) drove the specification of pSHF towards AVC identity, as seen by RNA-seq (Figures 3I and 3J) and protein expression of TBX2 (Figure 3K). Furthermore, the AVC cardioids were smaller than the atrial cardioids over the entire time course (Figures 3F and 10D). Overall, the sub-specification of pSHF precursor cells into atrial or AVC cardioids occurs early during the mesoderm induction period, indicating the plasticity of pSHF precursors.

[0360] During the early stages of cardiogenesis, the atria and AVC have similar gene expression profiles. Subsequently, the atria, LV, and RV initiate upregulation of the chamber gene expression program, but not in the AVC and OFT. To achieve chamber specification, two previously published CM chamber specification and maturation signaling protocols were combined (Figure 17D). Importantly, in the LV and RV cardioids, the chamber markers MYL2, MYL7, NPPB, and NPPA were upregulated (Figure 3M, Figure 17E, Figure 17G). We also observed an overall increase in TNNI expression (Figure 18A), an improvement in the sarcomere (fishbone) structure (Figure 18B), an increase in the MYH7 / 6 ratio (Figure 18C), and surprisingly, an improvement in the contraction behavior / amplitude (Figure 18D). However, this approach did not stimulate the atrial chamber program and atrial characteristics were suppressed. Therefore, we screened for signaling factors that specifically promote atrial chamber differentiation (Figure 17D). We found that activation of FGF and RA and inhibition of the NOTCH and BMP pathways promote the atrial chamber gene program while downregulating AVC-specific genes (Figure 3L, Figure 17F). This treatment was then combined with a low-glucose medium similar to the ventricular chamber metabolic maturation treatment, and further chamber differentiation was observed (Figure 3M, Figure 17F). Overall, we have shown that we can specify cardioids and differentiate them into the five major compartmental characteristics found in the embryonic heart.

[0361] Finally, to confirm specification to all five lineages (LV, RV, OFT, atria, and AVC), single-cell RNAseq analysis was performed on biological duplicates (Figure 19). Using unsupervised UMAP clustering, it was found that the five cardioids / cardiomyocyte subtypes segregated into distinct clusters (Figure 19A). When well-characterized heart compartment markers were assigned to these clusters, they overlapped with the expected characteristics (Figure 19B, Figure 19C). These results confirmed that we have developed a cardioids platform that includes all the major compartmental lineage characteristics found in the human heart.

[0362] Example 6: Functional characterization of five cardioïde subtypes The heart needs to function during development. Therefore, it is also essential to understand the initial heart function during the formation of different embryonic heart compartments. Animal experiments have suggested significant differences in the spontaneous contractions (automaticity) and beating frequencies between heart compartments. The heart tube and the initial LV region derived from the FHF initiate contractions first and lose automaticity as they mature. In contrast, the atrial region (which develops the atrium and AVC) has a late onset of beating, maintains spontaneous contractions longer, and loses automaticity only after the formation of the heart pacemaker element. The inventors hypothesized that the use of fused cardioïdes would allow them to investigate these early functional developmental differences before the formation of the pacemaker and before human in vivo data could be obtained.

[0363] The fusion cardioids are particularly advantageous for investigating the contraction dynamics using a wide-field microscope. The contraction behavior of the compartment-specific cardioids on day 6.5 (Figures 11A and 11B) showed that 90 - 100% of the LV, atrium, and AVC exhibited automaticity and a greater degree of contraction. In contrast, only 18% of the RV cardioids contracted spontaneously, and similarly, only 8% of the OFT cardioids contracted with a low degree of contraction (Figures 4A and 4C). On day 9.5, the atrium and AVC retained automaticity, but the automaticity and contraction rate decreased in the LV, RV, and OFT (Figures 4A - 4C). The loss of automaticity correlated with the downregulation of HCN4, which encodes the potassium / sodium channel, in vivo (Figure 4D). Interestingly, each cardioids subtype has its distinct beating pattern. The atrium and AVC beat very regularly, while the LV and RV beat in regular bursts (Figures 11A and 11C). Importantly, these observations were reproducible in both technical and biological replicates. To gain further insight into signal propagation in the cardioids, a GcAMP reporter strain was induced to track calcium transients (Figure 4F). Within one cardioids, the origin of signal propagation changed between beats. The inventors also observed differences between cardioids subtypes, which were supported by the differences in expression seen in both T-type and L-type calcium channels (Figure 4E). Overall, each cardioids subtype has different contraction and signal propagation profiles.

[0364] Ion channel expression during early heart development is relatively uniform and later develops into chamber-specific expression profiles and action potential (AP) shapes for a particular species. Different cardioids subtypes first showed similar ion channel profiles at day 3.5 and later showed differences according to their chamber specificity at day 9.5 (Figure 4G). We characterized how APs of early human CM subtypes derived from cardioids were compared using voltage-sensitive dye (FluoroVolt) imaging and patch clamping. We verified that APs measured using FluoroVolt were consistent across different regions of the cardioids and across cardioids subtypes (Figure 11D), with slight variation in the AVC. This can be explained by the functional heterogeneity of the AVC, which has the potential to develop into different structures. By comparing AP lengths, the LV, RV, and atria were observed to have similar shapes, while the AVC had a significantly different shape (Figures 11E–11G). Next, RV and atrial cardioids were dissociated and patch clamp analysis was performed (Figure 4H). As expected from in vivo, we observed that the AP duration in atrial CMs was shorter than that in RV CMs (Figures 4H–4I and 11H–11I). Furthermore, the diastolic potential was approximately -70 mV (Figures 4J and 4K), which was close to that of in vivo CMs. Additionally, cardiac regional potentials can be measured on a multi-electrode array (MEA). In summary, the electrochemical signaling of cardioids subtypes is diverse and still immature, making it a system for investigating the developmental electrophysiology of early human heart development.

[0365] Example 7: Multi-chamber integration of cardioids subtypes The embryonic heart precursors are specified in adjacent but distinct regions. When RV and atrial precursors from the SHF migrate into the cardiac tube, they self-sort and remain in separate compartments important for heart function. Studying the molecular basis of this sorting is difficult in the embryo. The inventors hypothesized that in vitro-derived a / pSHF and FHF precursors have the ability to self-sort as they do in vivo. Indeed, when different subtypes of developing cardioids were dissociated on day 3.5 and then mixed (Figure 5A), sorting in the cardioids was observed within 24 hours (Figure 12A). In contrast, precursors of the same subtype did not sort upon mixing (Figure 5B). The highest degree of sorting was observed on day 7.5 for FHF-derived LV and aSHF-derived RV precursors (Figure 5B and Figure 5C). Sorting and patterning were consistent with differential cadherin expression signatures in different precursors that imply in vivo (Figure 5D, Figure 12C, and Figure 12D). Notably, the sorted precursors developed into TNNT2+CMs and retained appropriate chamber characteristics (Figure 5C and Figure 12B), and it was confirmed that lineage characteristics are determined by the first two stages of differentiation. This also implied that co-differentiation of different precursors was possible from day 3.5. In summary, the multi-chamber cardioids platform can be used to further study the sorting mechanism that separates the major compartments of the heart.

[0366] During development, the cardiac chambers of the LV, RV, and atria develop simultaneously. However, there is no multi-chamber model to study this important stage and process of heart morphogenesis. Since the cardiac precursors are already specified and sorted by day 3.5, although the co-developing cardioids are still separated at this stage, they were assumed to undergo morphogenesis together. When different cardioids subtypes were placed together on day 3.5 (Figure 5E), they interacted (fused) efficiently after 24 hours (Figure 12F), but maintained distinct identities and compartments (Figure 5G and Figure 5I). In contrast, the co-development of cardioids on day 5.5 did not result in efficient fusion (Figure 12E). Only the fused cardioids from day 3.5 contracted in synchrony (Figure 5G), demonstrating that different cardioids subtypes interact functionally. Hereafter, these structures are referred to as multi-chambered cardioids. Multi-chambered cardioids can be formed in all combinations, enabling the study of the interactions of two-chambered cardioids (Figure 12I) or three-chambered cardioids (atria, LV, RV fusion) in the same order as in the developing embryonic heart (Figure 5F–Figure 5H).

[0367] Directional electrochemical signal propagation occurs early in heart development and has not been traced in human embryos. The directionality of electrochemical and fluidic propagation is gradually established initially without a pacemaker, valves, or septa. The first electrochemical signal appears in the differentiating FHF / LV. As the atrial region develops, it paces other regions, thereby ensuring unidirectional signal movement and flow from the atria across the LV to the RV and OFT. The inventors first measured calcium signal propagation and tracked signal propagation on the MEA in a multi-chambered cardioids system to investigate whether this process was reproduced. Each beat originated from only one location and then propagated throughout the multi-chambered cardioids (Figs. 5G and 12G - 12H), and it was found that signal propagation was reliably a unidirectional flow. On day 6.5, most beats originated from the LV (Fig. 5K), which included pacing of the RV that did not beat independently (Fig. 5I). These observations in different multi-chambered cardioids were verified by showing that a multi-chambered cardioids paced by the LV maintained the same beat frequency as the LV cardioids (Fig. 5J). Interestingly, on day 6.5, the pacing potential was thought to be restricted. This was because the LV region could not pace the three-chambered cardioids and the atria could not pace the multi-chambered cardioids (Figs. 5I and 5L). However, since the multi-chambered cardioids had developed by day 9.5, signals originated almost exclusively from the atrial region in all combinations (Fig. 5K). Thus, it was demonstrated that multi-chambered cardioids represent a unique means for deciphering the ontogeny of electrochemical signal propagation across the concurrently developing heart chambers.

[0368] When cardiac chambers develop simultaneously, they first share a lumen before septation and valve formation occur. Therefore, the inventors further optimized the simultaneous development of shared lumens in multi-chambered cardioids. Surprisingly, immediately prior to cavity formation (FHF / LV at day 1.5, aSHF / RV and pSHF / atria at day 3.5), when developing FHF, aSHF, and pSHF cardioids were combined (Figure 5L), the shared lumens co-developed and still retained their characteristics (Figures 5L - 5O). In summary, the inventors developed a human compartment-specific multi-chamber platform to comprehensively analyze the earliest aspects of human heart development.

[0369] Example 8: Mutations cause compartment-specific defects in cardioids. Mutations in cardiac transcription factors (TFs) often cause compartment-specific congenital defects in heart development. To genetically validate the cardioids compartment platform, knockout (KO) hPSC lines were generated for important cardiac TFs (ISL1, TBX5) known to cause compartment-specific defects in vivo. Additionally, the inventors' system was used to investigate the KO effect of TF FOXF1.

[0370] ISL1 is a prominent TF, and disruption of this results in severe cardiac malformations in the OFT and RV, partial defects in the atria, and lethality in mice at E10.5. To investigate the phenotypes in the human cardioide compartment platform, the inventors developed ISL1-KO hPSCs (Figure 13A) and differentiated them into LV, RV, atrial, and OFT subtypes (Figure 6A). Misregulation of gene expression was already seen at day 3.5 in ISL1 KO, as seen by lower levels of the important cardiac TFs MEF2C, NKX2-5, and MYOCD, suggesting a slower progression of differentiation (Figure 13B). The most dramatic gene expression changes were confirmed in OFT cardioids, with HAND2 and BMP4 downregulated and TBX5 upregulated. In atrial cardioids, HOXB1, an important pSHF marker, was downregulated (Figure 13B). Time-course analysis from day 2.5 to day 9.5 revealed that ISL1 KO cardioids showed the most severe morphogenetic changes starting at day 4.5 in the OFT and atrial cardioids, while the effects on RV and LV cardioids were less apparent (Figure 13E). At day 9.5, there were significant size differences in all cardioids subtypes (Figure 6A and Figure 13F). The morphological defects at day 9.5 in KO were also reflected in the misregulation of NPPA, NPPB, NR2F2, HEY1, RSPO3, WNT5A, and MYL7 in different cardioids subtypes (Figure 6C). The efficiency of CM differentiation was low in all lineages except the LV (Figure 6B). Specifically, OFT and atrial KO cardioids showed regions without TNNT2 expression, and while atrial cardioids had delayed differentiation and contraction, they still maintained their properties (Figure 6B - Figure 6D and Figure 13D). In contrast, the major regulators in the OFT were misregulated, causing an overall gene expression shift from OFT to atrial properties (Figure 6D, Figure 6F, Figure 13C). This was also confirmed by the contraction analysis of ISL-KO OFT cardioids that acquired atrial-like beating behavior at day 14.5 (Figure 6E).

[0371] Another prominent cardiac TF is TBX5, which is an important regulator in FHF and pSHF precursors and is involved in promoting the cardiac chamber gene expression program. Disruption of TBX5 results in atrial and ventricular septal defects, conduction defects, and mutations that cause Holt-Oram syndrome in humans. In TBX5 KO (Figure 13H), it was observed that the expression of HOXB1 decreased in the atria and AVC cardioids on day 3.5 (Figures 6G and 13G). Global gene expression analysis revealed that on day 3.5, a range of aSHF markers (TBX1, FOXC2, FGF10) were upregulated in TBX5 KO atrial cardioids (Figure 13G), and TBX2 / 3 was downregulated in AVC cardioids (Figure 13I). Furthermore, TBX5 KO differentiated into the FHF lineage showed upregulation of HAND2, GATA4, and ISL1 (Figure 13G). In contrast, no major defects were detected in aSHF-derived cardioids, consistent with in vivo findings (Figure 13G). Consistent with these results, on day 9.5, the most severe morphogenetic phenotypes were observed in the LV, atria, and AVC cardioids, while the RV cardioids showed only a slight decrease in size (Figures 6H and 6H’). The atria and AVC cardioids were unable to differentiate into CMs, as confirmed by the absence of TNNT2 (Figure 6I). The LV and RV cardioids showed inefficient CM differentiation, and the cardiac chamber-specific marker NPPA was downregulated (Figure 6I). All TBX5 KO cardioid subtypes showed significant defects in the expression of ventricular chamber markers such as NPPA / B, IRX3 / 4 / 1, HEY2, and upregulation of the non-cardiac chamber marker TBX2 in the RV and LV cardioids (Figure 6J). Overall, TBX5 KO showed specific phenotypes at different stages. The LV, atria, and AVC cardioids were already affected in the progenitor stage and were unable to form CMs, while the LV and RV cardioids showed mild phenotypes in the CM specification stage when cardiac chamber-specific genes were downregulated.

[0372] Forkhead box transcription factor (FOXF1) is a specific regulatory factor of the pSHF lineage. Disruption of FOXF1 mainly results in atrial septal formation defects, but mutant mice die already by E8.0 due to extraembryonic mesoderm defects. The widespread expression of this TF in the early mesoderm is consistent with its proposed role in the establishment of cardiovascular precursor properties in the early lateral mesoderm. In all subtypes (Figure 6N), FOXF1 KO cardioids (Figure 13J) were analyzed and a severe morphological phenotype starting from day 3.5 was observed in atrial and AVC cardioids (Figure 6L). The major pSHF markers (HOXB1, OSR1) and AVC markers (TBX2, TBX3) were downregulated (Figure 6K and 6M), which was consistent with a failure of pSHF specification. LV cardioids KO showed downregulation of TBX5, while RV cardioids showed upregulation of TBX1 (Figure 6K). At day 9.5, morphological phenotypes were observed in LV and AVC KO cardioids (Figure 6N and Figure 13K). Interestingly, atrial cardioids shifted more towards ventricular characteristics (IRX1+, IRX4+, HEY2+, NRF2F2-) (Figure 13M and 13N), while AVC cardioids were unable to differentiate efficiently (Figure 13L). As expected, no phenotype was observed in the RV except for the downregulation of NPPA seen in all subtypes (Figure 13L and Figure 13M). Genes involved in cardiac contraction (ENO1, ENO2) were downregulated (Figure 13M) and a less severe phenotype appeared in LV cardioids, which was confirmed by functional assays, showing that KO LV cardioids had a lower beating rate (Figure 6O). KO atrial cardioids also showed a lower beating rate, while AVC cardioids did not contract (Figure 6O). In summary, the cardioids platform can be used to analyze stage- and compartment-specific genetic heart defects.

[0373] Example 9: Multicavity cardioids platform for screening teratogen-induced heart defects In addition to genetic causes, congenital heart defects are often induced by teratogens (e.g., drugs, toxins, metabolites). However, screening for compartment-specific teratogenic effects in a human system that is efficient, high-throughput, and easily quantifiable is not sufficient. To test the applicability of the fused cardioids teratogenicity, first, a non-teratogenic factor (aspirin, 30 μM at a high dose) was identified as a negative control. In the cardioids system, no morphological differences or significant differences in gene expression were observed (Figure 14A and Figure 14B). Next, thalidomide, a teratogen well-known in humans but not in rodents, was tested. Thalidomide binds to TBX5 and causes severe atrial septal defects, downregulation of ventricular markers, and limb malformations. Using a high-throughput platform, the effects of thalidomide at various concentrations (0.1 μM, 1 μM, 10 μM compared to the human therapeutic plasma concentration of approximately 2.4 μM) were analyzed. Dose-specific effects were observed in different compartments, with the most prominent effect on AVC formation that had not been detected previously (Figure 7A and Figure 7A’). At the same time, the RV and OFT cardioids were less affected (Figure 7A and Figure 7A’). The gene expression profiles of these treatments revealed downregulation of the cardiac chamber-specific marker NPPA and dose-specific misregulation of compartment-specific markers (NR2F2, IRX1 / 4) in all lineages except RV and OFT (Figure 7B). These results demonstrate and validate that the multi-compartment platform can recognize the initial compartment-specific effects of teratogenic drugs in a human system and associate them with the defects seen in patients.

[0374] Another class of compounds known to induce congenital anomalies are retinoid derivatives used in leukemia, psoriasis, acne creams, etc., which cause AVC defects and OFT-derived defects. Since RA plays an important role in vivo and in our cardioid system, we hypothesized that the cardioid compartment platform would enable a step-specific analysis of the underlying mechanisms. Tests with acitretin and isotretinoin were performed, and severe compartment-specific and step-specific effects were observed at significantly lower doses (e.g., 5 nM acitretin in the atrium; 1 nM isotretinoin in the LV). The OFT, atrium, and AVC cardioids had defects in specification, patterning, and morphogenesis (Figures 7C, 7D, 14C, 14C’). Surprisingly, when using all-trans retinol, only severe morphological effects on the OFT cardioids were observed, while all other cardioid subtypes were unaffected (Figures 14E and 14E’). In OFT cardioids, retinoids caused downregulation of OFT genes (WNT5A, MSX1, ISL1), as well as upregulation of ventricular and chamber genes (IRX1, IRX4, NPPA), but not atrial genes (Figures 7E and 14F). Furthermore, OFT cardioids treated with retinoids differentiated into CMs earlier (Figures 7C and 14D). In summary, the cardioid compartment platform was further validated and used to sensitively analyze the drug-specific effects on different compartments.

[0375] Different plastic residues have been ignored, but these are ubiquitous classes of compounds in the environment of the inventors and may have teratogenic effects. It is very difficult to demonstrate the in vivo effects of plastic residues, and there has been no validated system to date to elucidate the specificity and morphogenetic defects of the human heart. Here, the inventors use their cardioidal compartment platform to investigate the effects of different combinations of plastic residues (BPA, PFO, and nanoplastics). When treated with plastic residues, severe delays in the specification of RV cardioids and inefficient differentiation of RV and atrial cardioids were observed, while LV and AVC cardioids were unaffected (Figures 7F and 14G). This emphasizes that the inventors have a system for comprehensively testing the teratogenic effects of environmentally ubiquitous compounds on human heart development.

[0376] Discussion The inventors developed a signaling-controlled cardioidal platform representing all major compartments of the embryonic heart. In this system, three different precursor populations (aSHF, pSHF, and FHF) give rise to five major heart compartments (LV, RV, OFT, atrium, and AVC) separately or in combination, mimicking selected aspects of early human heart development. The 2D-to-3D differentiation approach ensures the specification of homogeneous precursors initially, reduces heterogeneity, and increases robustness, which remains a challenge in the organoid field. As a result, this platform is highly efficient, reproducible, functions with multiple cell lines, is screenable in high-throughput applications, is versatile, and includes single-compartment cardioids or multi-chamber cardioids.

[0377] In vivo, depending on the dose and timing of signaling, the specification of these lineages is already driven while mesoderm is induced in the primitive streak, and mesodermal cells move from the primitive streak at different times to take up defined positions within the cardiac field. Consistently, the inventors have found that specific activin / nodal and WNT signaling activation levels drive the specification to distinct SHF, AVC, and FHF precursors. After mesoderm induction, it becomes possible to determine the fate of the SHF lineage by inhibiting TGF-beta signaling, which is consistent with the signaling environment in the anterior region of the embryo. A specific combination of mesoderm induction signals and patterning signals makes it possible to mimic the properties, dynamics, and subsequent functions of the developing cardiac lineage. For example, both SHF lineages show a delay in cavitation and differentiation into CMs, and aSHF is more epithelial and highly proliferative than FHF. In contrast to aSHF, and consistent with in vivo observations, pSHF includes a more diverse range of induction and patterning conditions, which results in either an AVC phenotype or an atrial phenotype. The initial in vivo-like functions are also brought about by the contraction differences and dynamics of the atrial, AVC, LV, RV, and OFT cardioids, and a pacemaker is still absent at this developmental stage. Finally, the different induction and patterning stages allow for the analysis of precursor and compartment sorting mechanisms and cardiac chamber interactions in the fused cardioids.

[0378] The inventors have found that the role of RA signaling in the specification of different lineages is more complex with respect to dosage and timing than previously thought. The absence of RA signaling is characteristic in the initial aSHF specification and subsequent OFT differentiation. For LV specification, RA needs to be at a relatively low level, and for early atrial specification, RA needs to be at a high level. However, the specification of aSHF RV requires RA signaling at a high level at a later stage, so timing is also important. Teratogenic screening experiments have confirmed the important role of the amount of RA signaling and shown a strong effect of retinoids on the differentiation rate, specification efficiency and direction, morphogenesis, and physiological functions that result in compartment-specific defects.

[0379] Interactions between cardiac lineages during the earliest stages of heart development (such as the specification, morphogenesis, and functional differentiation of the cardiac mesoderm) are difficult to analyze in the embryo and are well known to be unavailable in early human embryos. However, this aspect is key to understanding the effects of mutations and teratogens on early heart development and how they lead to defects in specification, morphogenesis, and contractile signaling propagation, resulting in embryonic lethality. For example, how different compartments are selected and maintained in separation is unclear, but this can be addressed here using the multi-chamber cardioids platform. Another important but overlooked aspect of heart development is the ontology of contractile signaling propagation at different early stages (days 20 - 30) of human heart development. This is particularly important for understanding the cardiac causes of embryonic lethality, which can be caused by incomplete specification and morphogenesis but also by defects in initial contractile signaling propagation between heart chambers.

[0380] The fused cardioids enable comprehensive and systematic analysis of regulatory elements such as enhancers, environmental factors such as contaminants, diet, and unidentified mutations in the complex interaction between genetic and environmental factors.

Claims

1. A cardiac organoid comprising cardiac tissue having at least one lumen or central chamber, wherein the cardiac organoid comprises at least two different cardiac tissues selected from left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular duct tissue, sinoatrial node tissue, and atrioventricular node tissue, the central chamber may be shared by at least two different cardiac tissues, and the at least two different cardiac tissues have the ability to propagate electrophysiological or calcium signaling connections and / or tissue contractions. i) the cardiac organoid has the central cardiac chamber, the central cardiac chamber being shared by at least two different cardiac tissues, or ii) the cardiac organoid has at least two different cardiac tissues having the ability to propagate contractions with pulsating behavior that originates in one of the at least two different cardiac tissues and propagates or projects to adjacent cardiac tissues, or both i) and ii), The aforementioned cardiac organoid is obtained from differentiated mesodermal cells.

2. The left ventricular tissue comprises at least 60% cardiac cells selected from cardiomyocytes, endocardial cells, and epicardial cells. The right ventricular tissue contains at least 60% cardiomyocytes, The atrial tissue comprises at least 60% cardiomyocytes, The outflow tract tissue comprises at least 60% cardiomyocytes, The aforementioned atrioventricular ductal tissue contains at least 60% cardiomyocytes, The sinoatrial node tissue contains at least 60% cardiomyocytes, and / or The cardiac organoid according to claim 1, wherein the atrioventricular nodal tissue comprises at least 60% cardiomyocytes.

3. The cardiac organoid according to claim 1 or 2, wherein the lumen or central chamber is completely surrounded by tissue selected from left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular duct tissue, sinoatrial node tissue, or atrioventricular node tissue, and / or the volume of the lumen or central chamber does not lead to major blood vessels.

4. A cardiac organoid according to claim 1 or 2, having a maximum size of 0.3 mm to 50 mm.

5. Left ventricular tissue cells express one or more expression markers selected from NPPA, IRX4, and HEY2; and / or left ventricular tissue cells lack expression of one or more expression markers selected from NR2F2, TBX2, and TBX3; Right ventricular tissue cells express one or more expression markers selected from NPPA, IRX1, IRX2, and PRDX1; and / or right ventricular tissue cells lack expression of one or more expression markers selected from NR2F2, TBX2, and WNT5A. Atrial tissue cells express one or more expression markers selected from NPPA, NR2F1, NR2F2, and HEY1; and / or atrial tissue cells lack expression of one or more expression markers selected from IRX1, IRX4, and HEY2. Outflow tract tissue cells express one or more expression markers selected from WNT5A, MSX1, BMP4, WNT11, and RSPO3; and / or outflow tract tissue cells lack expression of one or more expression markers selected from TBX3, NR2F1, and NPPA. Atrioventricular ductal tissue cells express one or more expression markers selected from TBX2, MSX2, and RSPO3; and / or atrioventricular ductal tissue cells lack expression of one or more expression markers selected from IRX1, IRX4, and NPPA. Sinoatrial node tissue cells express one or more expression markers selected from SHOX2, TBX3, HCN4, ISL1, and GJC1; and / or sinoatrial node tissue cells lack expression of one or more expression markers selected from NKX2.5, IRX1, IRX4, and NPPA, and / or The cardiac organoid according to claim 1 or 2, wherein the atrioventricular nodule tissue cells express one or more expression markers selected from TBX3, TBX5, KCNE1, HCN4, and GJC1; and / or the atrioventricular nodule tissue cells lack expression of one or more expression markers selected from RSPO3, MSX2, IRX4, and NPPA.

6. The cardiac organoid according to claim 1 or 2, wherein the size at the maximum dimension of the lumen or central cardiac chamber is at least 30% of the size at the maximum dimension of the cardiac organoid.

7. A method for generating a cardiac organoid according to claim 1, comprising generating at least two different cardiac tissues in vitro, wherein the different cardiac tissues are selected from left ventricular precursor first cardiac region tissue, right ventricle / outflow tract precursor anterior second cardiac region tissue, right ventricular precursor anterior second cardiac region tissue, atrial precursor posterior second cardiac region tissue, outflow tract precursor anterior second cardiac region tissue, atrioventricular duct precursor posterior second cardiac region tissue, sinoatrial node precursor posterior second cardiac region tissue, and atrioventricular node tissue, and fusing the at least two cardiac tissues. A method comprising culturing a fusion tissue model and enabling calcium signaling connections, the ability to propagate tissue contraction, and / or the formation of a central chamber between the different cardiac tissues.

8. The aforementioned different cardiac tissues are cultured and differentiated from pluripotent cells, and the fusion occurs from the pluripotent stage to day 1 to day 7 of culture, preferably the right ventricle / outflow tract precursor anterior second cardiac region tissue, right ventricle precursor second cardiac region tissue, atrial precursor second cardiac region tissue, atrioventricular precursor second cardiac region duct tissue, sinoatrial node precursor second cardiac region tissue, and / or atrioventricular node tissue are fused on day 2 to day 5 of culture, or preferably the left ventricle precursor first cardiac region tissue is fused when the expression markers TBX5 and / or HAND1 are expressed, preferably the right ventricle / outflow tract precursor anterior second cardiac region tissue or right ventricle precursor anterior The method according to claim 7, wherein the second cardiac region tissue is fused, preferably expressing the expression markers HOXB1, TBX5 and / or OSR1, the posterior second cardiac region tissue of the atrial precursor is fused, preferably expressing the expression markers TBX3, FOXF1 and / or HOXB1, the posterior second cardiac region tissue of the atrioventricular duct precursor is fused, preferably expressing the expression markers SHOX2, TBX3, HCN4, ISL1 and / or GJC1, the posterior second cardiac region tissue of the sinoatrial node precursor is fused, and / or, preferably expressing the expression markers TBX3, TBX5, KCNE1, HCN4 and / or GJC1, the atrioventricular node tissue is fused.

9. One of the at least two different cardiac tissues is left ventricular progenitor first cardiac region tissue, and generating left ventricular progenitor first cardiac region tissue involves differentiating mesodermal cells into left ventricular progenitor cells in a culture medium containing bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, Wnt inhibitor, preferably Wnt-C59 or IWP2, and retinoic acid, wherein the retinoic acid is present at a concentration of 5 nM to 100 nM. One of the at least two distinct cardiac tissues is right ventricle / outflow tract precursor anterior second cardiac region tissue, and generating right ventricle / outflow tract precursor anterior second cardiac region tissue involves differentiating mesodermal cells into right ventricle and / or outflow tract precursor cells in a culture medium containing a TGF-beta inhibitor, preferably SB431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939. One of the at least two different cardiac tissues is an anterior second cardiac region tissue of the outflow tract precursor, and generating the second cardiac region tissue of the outflow tract precursor includes differentiating mesodermal cells into outflow tract tissue precursor cells in a culture medium containing a TGF-beta inhibitor, preferably SB431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939. One of the at least two distinct cardiac tissues is atrial progenitor posterior second cardiac region tissue, and generating the atrial progenitor posterior second cardiac region tissue involves differentiating mesodermal cells into atrial tissue progenitor cells in a culture medium comprising a TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, preferably at a concentration of 300 nM to 800 nM, and / or One of the at least two different cardiac tissues is the atrioventricular duct precursor second cardiac region tissue, and generating the atrioventricular duct precursor posterior second cardiac region tissue involves differentiating mesodermal cells into atrioventricular duct tissue precursor cells in a culture medium containing a TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably with retinoic acid at a concentration of 300 nM to 800 nM. One of the at least two different cardiac tissues is sinoatrial node precursor posterior second cardiac region tissue, and generating sinoatrial node precursor posterior second cardiac region tissue involves differentiating mesodermal cells into sinoatrial node tissue precursor cells in a culture medium containing a TGF-beta inhibitor, preferably SB431542, osteomorphonomastic protein, preferably BMP4, and retinoic acid, preferably with retinoic acid at a concentration of 300 nM to 800 nM, and / or The method according to claim 7 or 8, wherein one of the at least two different cardiac tissues is atrioventricular nodule tissue, and generating atrioventricular nodule tissue comprises differentiating mesodermal cells into atrioventricular duct tissue progenitor cells in a medium comprising a TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably the retinoic acid at a concentration of 300 nM to 800 nM, and further differentiating the atrioventricular duct precursor posterior second cardiac region tissue into atrioventricular nodule tissue by further maturation in a medium comprising a sonic hedgehog signaling activator and / or BMP.

10. The method according to claim 7 or 8, wherein fusing the at least two cardiac tissues comprises culturing them in a culture medium comprising a Wnt inhibitor, preferably Wnt-C59, bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably the retinoic acid at a concentration of 300 nM to 800 nM, particularly preferably 500 nM.

11. A method according to claim 7 or 8 for screening or testing a candidate compound for its effect on cardiac development and / or function, comprising: generating a cardiac organoid according to claim 7 or 8 while treating the cells with the candidate compound; and comparing the development of the cardiac organoid with the development and / or function of a cardiac organoid not treated with the candidate compound.

12. A method for observing the effects of suppressed, mutated, or overexpressed genes during cardiac development, comprising: generating cardiac organoids according to claim 7 or 8, wherein the cells have or overexpress a suppressed or mutated candidate gene; and comparing the development of the cardiac organoids with the development of cardiac organoids not generated using the suppressed, mutated, or overexpressed gene.

13. A method for screening or testing candidate compounds for their effects on cardiac function, comprising treating a cardiac organoid described in claim 1 or 2 with the candidate compound and comparing the function of the cardiac organoid not treated with the candidate compound.

14. A method for treating cardiac injury in a patient, comprising transplanting cells derived from cardiac organoids according to claim 1 or 2, preferably cardiomyocytes, to the injury.

15. Use of a cell culture medium comprising the following in the method of claim 7 or 8; a) Bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, the retinoic acid having a concentration of less than 100 nM. b) TGF-beta inhibitor, preferably SB431542, and Wnt inhibitor, preferably Wnt-C59 or XAV-939, c) A Wnt inhibitor, preferably Wnt-C59 or XAV-939, bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably the retinoic acid having a concentration of 50 nM to 500 nM in the culture medium. d) TGF-beta inhibitor, preferably SB431542, and Wnt inhibitor, preferably Wnt-C59 or XAV-939, e) A medium lacking a Wnt inhibitor, preferably Wnt-C59 or XAV-939, bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, and retinoic acid. f) A TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, preferably the retinoic acid at a concentration of 300 nM to 800 nM. g) Wnt inhibitor, preferably Wnt-C59 or XAV-939, bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably the retinoic acid at a concentration of 300 nM to 800 nM. h) A TGF-beta inhibitor, preferably SB431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably the retinoic acid at a concentration of 300 nM to 800 nM. i) Activin and CHIR99021, preferably the activin is at a concentration of 1 ng / mL to 8 ng / mL, preferably about 5 ng / mL, and / or the CHIR99021 is at a concentration of 1 μM to 6 μM, preferably about 3 μM, or preferably the activin is at a concentration of 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or the CHIR99021 is at a concentration of 2 μM to 8 μM, preferably about 4 μM. And / or preferably, the activin has a concentration of 6 ng / mL to 30 ng / mL, preferably about 10 ng / mL, and / or the CHIR99021 has a concentration of 0.4 μM to 4 μM, preferably about 2 μM, and / or preferably, the activin has a concentration of 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or the CHIR99021 has a concentration of 0.1 μM to 4 μM, preferably about 1 μM, or j) A Wnt inhibitor, preferably Wnt-C59, a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, and retinoic acid, preferably the retinoic acid at a concentration of 300 nM to 800 nM, particularly preferably 500 nM.

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