Multiplex cardiac tissue culture fusions

Abstract

The present invention provides: a cardiac tissue model comprising cardiac tissue having at least one internal cavity or chamber wherein the cardiac tissue model comprises at least two different cardiac tissues selected from the group consisting of left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular tube tissue, sinus node tissue and atrioventricular node tissue, wherein the central chamber may be common to at least two different cardiac tissues, and wherein the at least two different cardiac tissues comprise the ability to connect calcium signaling and / or propagate tissue contraction; methods of generating such tissue models, and uses of tissue models for screening purposes.

Description

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

[0002] background

[0003] Congenital heart disease (CHD) is the most common human developmental birth defect and the most prevalent cause of embryonic and fetal death. CHD most often occurs in specific compartments of the embryonic heart, such as the outflow tract (OFT), atria, atrioventricular canal (AVC), right ventricle (RV), and most rarely in the left ventricle (LV). For about 75% of CHD, we still do not know the underlying causes. They may originate from undiscovered genetic mutations, environmental factors, or combined effects. In order to test and study possible causes, we need models that represent all compartments of the developing human heart.

[0004] CHD occurs in the early stage of embryonic development, usually before pregnancy has been detected, which makes the characterization of defect etiology challenging. Due to the lack of genetic background and environmental interaction control during human embryonic development, these difficulties are compound. Due to complexity, speed, inaccessibility and species-specific physiological differences, it is challenging to understand the etiology of people's compartment-specific defects in animals. Therefore, the recent people's self-organized cardiac organoid model is complementary, and it provides greater accessibility, simplified dissection of mechanism and high-throughput statistical significance ability. However, these systems do not allow mechanical dissection to represent the defects of all interactive compartments (OFT, AVC, atrium, RV and LV) of human embryonic heart.

[0005] WO 2019 / 174879 A1 describes artificial cardiac tissue organoids that grow as multilayered aggregates containing a large number of non-cardiac cells (such as foregut endoderm cells).

[0006] Hofbauer et al. (Cell 2021; 184(12): 3299-3317.e22) and WO 2021 / 186044 A1 disclose the generation of left ventricular cardiac organoids (cardioids) by self-organization. Although the self-organization after initiating cardiac differentiation using specific drifting factors improved on previous artificial heart tissues, the publication still did not show the diversity of in vivo cardiac development.

[0007] Therefore, one of the goals is to provide different and more advanced artificial heart tissues that can mimic different or greater diversity of the developing heart. Summary of the invention

[0008] The present invention provides a cardiac tissue model, which comprises cardiac tissue having at least one internal cavity or central chamber, 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, wherein the central chamber can be shared by at least two different cardiac tissues, and / or wherein at least two different cardiac tissues comprise the ability of calcium signaling to connect and / or propagate tissue contraction. A cardiac tissue model is also provided, which comprises cardiac tissue having a central chamber, wherein the central chamber can be 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.

[0009] The present invention also provides a method for generating a cardiac tissue model, the method comprising the following steps: generating at least two different cardiac tissues in vitro, wherein the different cardiac tissues are selected from the first cardiac area tissue of the left ventricle progenitor, the second cardiac area tissue of the right ventricle / outflow tract progenitor, the second cardiac area tissue of the atrial progenitor, the second cardiac area tissue of the atrioventricular canal progenitor, the second cardiac area tissue of the sinoatrial node progenitor and the atrioventricular node tissue, and fusing at least two cardiac tissues, culturing the fused tissue model, and enabling calcium signal conduction connection and / or the ability to propagate tissue contraction and / or central chamber formation between different cardiac tissues.

[0010] The present invention further provides a cardiac tissue model selected from the following: a right ventricle tissue model, which comprises cells expressing expression markers IRX1, IRX2 and PRDX1; an atrial tissue model, which comprises cells expressing expression markers NR2F1, NR2F2 and HEY1; an outflow tract tissue model, which comprises cells expressing expression markers WNT5A, MSX1, BMP4 and RSPO3; an atrioventricular canal tissue model, which comprises cells expressing expression markers TBX2, MSX2 and RSPO3; a sinoatrial node tissue model, which comprises cells expressing expression markers SHOX2, TBX3, HCN4, ISL1 and GJC1; and an atrioventricular node tissue model, which comprises cells expressing expression markers TBX3, TBX5, KCNE1, HCN4 and GJC1.

[0011] The present invention further provides a method for screening or testing candidate compounds for their effects on cardiac development and / or function, the method comprising generating a cardiac tissue model according to the present invention, treating cells with the candidate compound, and comparing the development of the cardiac tissue model with the development and / or function of a cardiac tissue model not treated with the candidate compound.

[0012] Further provided are methods for observing the effects of a suppressed, mutated or overexpressed gene during cardiac development, comprising generating a cardiac tissue model according to the present invention, wherein cells have a suppressed or mutated candidate gene or overexpress the candidate gene, and comparing the development of the cardiac tissue model with the development of a cardiac tissue model generated without the suppressed, mutated or overexpressed gene.

[0013] Further provided is a method for screening or testing a candidate compound for its effect on cardiac function, comprising treating any cardiac tissue model of the invention with the candidate compound and comparing the function with that of a cardiac tissue model not treated with the candidate compound.

[0014] The present invention also provides a method of treating cardiac damage in a patient, comprising transplanting cells from the cardiac tissue model of the present invention into the damage.

[0015] The invention further provides a cell culture medium useful in any of the inventive methods or specific method steps. Such culture medium can be combined in a kit of different culture media used in the inventive methods or a kit of one or more culture media and another means, such as a carrier or mold for fusion cultured tissue.

[0016] All embodiments of the present invention, and all preferred embodiments, are described together in the following detailed description of the invention, which relates to all embodiments, aspects, methods, cardiac tissue models, organoids, uses, culture media and kits, etc. For example, culture media and kits or their components can be used for or suitable for the present inventive method. Any component used in the method can be part of a culture media or kit. The present inventive tissue model or organoid is the result of the present inventive method or can be used for the present inventive method and uses. The preferred and detailed descriptions of the inventive method read similarly in terms of the suitability of the organoids or tissue models of the present invention obtained or used. Unless otherwise indicated, all embodiments can be combined with each other. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides a cardiac tissue model, which includes cardiac tissue with at least one internal cavity or central chamber, wherein the cardiac tissue model includes 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, wherein the central chamber can be shared by at least two different cardiac tissues, and / or wherein at least two different cardiac tissues include calcium signaling to connect and / or propagate the ability of tissue contraction. A cardiac tissue model is also provided, which includes cardiac tissue with a central chamber, wherein the central chamber 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 present creative tissue model can be generated from mesoderm cells (which in turn can be generated from pluripotent cells such as induced pluripotent cells or embryonic stem cells). The tissue model includes an internal cavity in at least one of the different cardiac tissues. It is possible that the internal cavity extends to at least another of the different cardiac tissues, for example, it is directly surrounded by tissues from at least two of the different cardiac tissues. Such internal cavity is then referred to as a central chamber. The central chamber can be formed from a tissue fusion of at least two different cardiac tissues, and the at least two different cardiac tissues are selected from left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue, atrioventricular canal tissue, sinoatrial node tissue and atrioventricular node tissue or its tissue precursors, and the tissue precursor is selected from left ventricular progenitor first heart area tissue, right ventricular / outflow tract progenitor second heart area tissue, atrial progenitor second heart area tissue, outflow tract progenitor second heart area tissue, atrioventricular canal progenitor second heart area tissue and sinoatrial node progenitor second heart area tissue. Atrioventricular canal progenitor second heart area tissue is the progenitor tissue of atrioventricular canal tissue and atrioventricular node tissue. Typically, the central chamber is formed after fusion. It can start as an internal cavity in one of the different tissues or precursors, and then extend to another one or more of the different cardiac tissues. Precursor stages of the central chamber, such as small tissue bubbles or internal cavities, may already exist in different cardiac tissues before fusion.Different cardiac tissues or tissue precursors are each also aspects of the present invention.

[0019] The present creative tissue model preferably contains at least two different cardiac tissues, which include electrophysiological signaling connections, such as calcium signaling connections in particular. Electrophysiological signaling connections mean that different cardiac tissues are functionally connected and can communicate via intercellular connections through calcium / voltage signaling events. Such electrophysiological signaling connections can be carried out by gap junctions and / or ion channels between cells of different cardiac tissues. In addition, tight junctions and / or desmosomes can be present for electrophysiological signaling connections because they maintain the structural connection of cells and promote or improve signal propagation. Electrophysiological signaling can be tracked by calcium or voltage signal propagation. Calcium signals can be calcium released from ER into the cytoplasm in cells. Such signals can be propagated to neighboring cells. Calcium signaling connections can be observed by dyes or reporter lines that track calcium or voltage signal propagation. Dyes can be intracellular calcium-sensitive dyes or voltage-sensitive dyes (e.g., FluoroVolt). Dyes can enter cells by expressing calcium-sensitive or voltage-sensitive dyes as proteins or transgenics. Alternatively, dyes can be introduced into cells by cell culture medium. Another way to measure signaling events and voltage signaling connections between different cardiac tissues can be detected using multi-electrode arrays.

[0020] The present inventive tissue model may include at least two different cardiac tissues with the ability to propagate contraction. Contraction is tissue contraction in which the cells of the tissue are able to perform contraction actions. Typically, stimulation, such as by cell action potential and / or calcium signaling, can cause cell contraction. Contraction propagation means that the contraction of one of the cardiac tissues can propagate to another in a different cardiac tissue, typically a neighboring cardiac tissue. Contraction can be stimulated by contraction of the neighboring cardiac tissue, but is typically connected by calcium signaling. Therefore, these embodiments can be combined, i.e., the cardiac tissue model can include calcium signaling connections and contraction propagation between at least two different cardiac tissues. Contraction can be observed as a pulsating behavior that starts in one of the different tissues and propagates or projects into the neighboring cardiac tissue. The neighboring cardiac tissue can begin to contract or beat in response to the beating or contraction of the earlier cardiac tissue. The response may have a short time delay. Contraction propagation may be a contraction of an internal cavity or central chamber. Contraction may be a contraction through a cardiomyocyte. Cardiomyocytes are typically the main contractile cells. Other cell types such as fibroblasts and endothelial cells may be particularly involved in the signal propagation of contraction.

[0021] The inventive cardiac tissue model is an advanced tissue model that recapitulates the function of a specific cardiac compartment. Therefore, it is preferably a cardiac organoid, also called a cardiac-type organoid.

[0022] "Selected from" means that the selected category can be from any of the members grouped into the optional categories.

[0023] In this regard, the present invention provides a method for generating a cardiac tissue model, which includes generating at least two different cardiac tissues in vitro, wherein the different cardiac tissues are selected from the group consisting of left ventricular progenitor first cardiac region tissue, right ventricular / outflow tract progenitor second cardiac region tissue, right ventricular progenitor second cardiac region tissue, atrial progenitor second cardiac region tissue, outflow tract progenitor second cardiac region tissue and atrioventricular canal progenitor second cardiac region tissue, and fusing at least two cardiac tissues, culturing the fused tissue model and allowing calcium signaling connections, propagation contraction capabilities and / or central chamber formation between the different cardiac tissues. Fusion is the merging of tissues to form one (combined or fused) tissue. Fusion can be promoted by placing different cardiac tissues close to each other, preferably by contacting each other. Fusion can be achieved by growth and / or migration of cells, or can be followed by growth and / or migration of cells, thereby merging at least two different cardiac tissues. Preferably, the fusion of tissues is performed during the progenitor stage of the tissues, for example as further described herein. Preferably, the connection area between the two tissues at the fusion junction is at least 5000 μm 2 and / or a length of at least 100 μm. The fused tissue may be elongated. Preferably, the fused tissue is capable of transmitting calcium and voltage signals from one tissue to another. The right ventricular progenitor (anterior) secondary cardiac area tissue and the outflow tract (anterior) secondary cardiac area tissue are also referred to as the right ventricular / outflow tract progenitor pre-secondary cardiac area tissue. It is a progenitor tissue of the (anterior) secondary cardiac area tissue, which can differentiate into right ventricular tissue and outflow tract tissue.

[0024] In order to generate a controlled in vitro system for human heart development, the in vivo principle of the merger of all lineages that govern the construction of the heart was used. Most cardiac structures are derived from three ancestral groups that produce specific cardiomyocyte (CM) lineages. The first heart field (FHF) ancestor produces the developing left ventricle (LV) tissue, the anterior second heart field (aSHF) produces the developing right ventricle (RV) tissue and most of the outflow tract tissue (OFT), and the posterior second heart field (pSHF) produces most of the atrium, a part of the atrioventricular canal tissue (AVC), atrioventricular node tissue (AVN) and sinoatrial node tissue (SAN). The development of these structures is time-dependent; for example, FHF-derived CM forms a heart tube, which then grows into the LV, while aSHF and pSHF ancestors differentiate and form other compartments in a delayed and gradual manner. This process is choreographed by developmental signaling at specific stages of cardiogenesis through multiple pathways (WNT, activin / Nodal, BMP, RA, FGF, NOTCH, etc.). These signaling pathways control downstream key compartment-specific TFs (TBX1, TBX5, FOXF1, TBX3, ISL1, IRX4, HEY1 / 2, etc.) that direct ancestral specification, morphogenesis, and physiology at specific stages. Some of these signals are generated by developing tissues or their environment. Some signaling factors can be provided to developing tissues to guide development along developmental pathways or to produce artificial changes in development.

[0025] Preferably, the cardiac tissue model and the different cardiac tissues are mammalian tissues, preferably human or non-human primate tissues; similarly, preferably, the cardiac tissue model is a mammalian or human or non-human primate tissue culture, such as a mammalian, human or non-human primate cell aggregate. The different cardiac tissues can be derived from cultured mammalian, human or non-human primate cells, such as pluripotent cells, such as induced pluripotent cells (iPS cells) or embryonic stem cells. Human cells, tissues and cultures are particularly preferred.

[0026] "Multipotent" cells can not grow into a complete organism, but can produce cell types derived from all three germ layers (i.e., mesoderm, endoderm and ectoderm), and may be able to produce all cell types of an organism.Pluripotency can be the characteristic of the cell itself (e.g., in embryonic stem cells), or it can be artificially induced.For example, in a preferred embodiment of the present invention, multipotent (pluripotent) stem cells are derived from somatic cells, special (multipotent) cells, unipotent (unipotent) cells or progenitor cells, wherein pluripotency is induced.Such cells are referred to as induced pluripotent stem cells (iPS) cells in this article.Somatic cells, special cells, unipotent cells or progenitor cells can, for example, be from patients, which are converted into pluripotent cells, which are subjected to this inventive method.Can study the abnormality of such cells or resulting tissue culture according to this inventive method, for example, during cardiac tissue development.Patients may, for example, suffer from cardiac disease or cardiac tissue deformity.The characteristics of the disease or deformity can be repeated and investigated in this inventive tissue culture.

[0027] A "multipotent" cell is capable of producing at least one cell type from each of two or more different organs or tissues of an organism, wherein the cell types may originate from the same or different germ layers, but is not capable of producing all cell types of the organism. An example of a multipotent cell is a mesodermal cell.

[0028] In contrast, "unipotent" cells are able to differentiate into cells of only one cell lineage.

[0029] "Progenitor cells" are cells that, like stem cells, have the ability to differentiate into a specific type of cell, with limited differentiation options, usually only one target cell. Progenitor cells are usually unipotent cells, but they can also be multipotent cells.

[0030] Similar to progenitor cells, "progenitor tissues" or "precursor tissues" contain differentiated cells that, if left undisturbed, determine the developmental fate of the tissue. Examples are left ventricular progenitor first heart field tissue, which is first heart field (FHF) tissue destined to develop into left ventricular (LV) tissue; right ventricular / outflow tract progenitor second heart field tissue, which is second heart field (SHF) tissue destined to develop into right ventricular (RV) tissue or outflow tract (OFT) tissue, particularly anterior second heart field (aSHF) tissue; right ventricular progenitor second heart field tissue, which is second heart field (SHF) tissue destined to develop into right ventricular (RV) tissue or outflow tract (OFT) tissue, particularly anterior second heart field (aSHF) tissue; atrial progenitor second heart field tissue, which is second heart field (SHF) tissue destined to develop into atrial tissue, particularly In particular, it is posterior second heart field (pSHF) tissue; outflow tract progenitor second heart field tissue, which is second heart field (SHF) tissue destined to develop into outflow tract (OFT) tissue, especially anterior second heart field (aSHF) tissue; and atrioventricular canal progenitor second heart field tissue, which includes second heart field (SHF) tissue, especially posterior second heart field (pSHF) tissue, which is destined to develop into atrioventricular canal (AVC) tissue or atrioventricular node (AVN) tissue; and sinoatrial node progenitor second heart field tissue, which includes second heart field (SHF) tissue, especially posterior second heart field (pSHF) tissue, which is destined to develop into sinoatrial node (SAN) tissue.

[0031] As used herein, gene names or gene symbols refer to human genes and are described in databases such as GeneCards (www.genecards.org) or HGNC databases (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 their websites.

[0032] In the present inventive cardiac tissue model or different cardiac tissues, preferably, the left ventricular tissue or the first cardiac region of the left ventricular progenitor contains at least 60% cardiac cells selected from myocardial cells, endocardial cells and epicardial cells. Such tissues have been previously described (Hofbauer et al., Cell 2021; 184(12): 3299-3317.e22; and WO 2021 / 186044A1; both are 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 first cardiac region of the left ventricular progenitor are cardiac cells selected from myocardial cells, endocardial cells and epicardial cells. Another term for endocardial cells is cardiac endothelial cells.

[0033] In a preferred embodiment, the right ventricular tissue and / or the right ventricular progenitor secondary cardiac region tissue comprises at least 60% cardiomyocytes. Preferably, the cardiomyocyte content in these tissues is at least 70%, particularly preferably at least 80%.

[0034] In a preferred embodiment, the atrial tissue and / or the atrial progenitor secondary cardiac region tissue comprises at least 60% cardiomyocytes. Preferably, the cardiomyocyte content in these tissues is at least 70%, particularly preferably at least 80%.

[0035] In a preferred embodiment, the outflow tract tissue and / or outflow tract progenitor secondary cardiac region tissue comprises 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 right ventricular tissue and / or the right ventricular / outflow tract secondary cardiac region tissue comprises at least 60% cardiomyocytes. Preferably, the cardiomyocyte content of these tissues is at least 70%, particularly preferably at least 80%.

[0037] In a preferred embodiment, the atrioventricular canal tissue and / or atrioventricular canal progenitor secondary cardiac zone tissue comprises at least 60% cardiomyocytes. Preferably, the cardiomyocyte content in these tissues is at least 70%, particularly preferably at least 80%.

[0038] In a preferred embodiment, the atrioventricular node tissue comprises at least 60% cardiomyocytes. Preferably, the cardiomyocyte content of these tissues is at least 70%, particularly preferably at least 80%.

[0039] In a preferred embodiment, the sinoatrial node tissue and / or the sinoatrial node progenitor secondary cardiac region tissue contain at least 60% myocardial cells. Preferably, the content of myocardial cells in these tissues is at least 70%, particularly preferably at least 80%. In the subsequent development of the present inventive cardiac tissue model, the number of other cardiac cells (including endocardial cells and epicardial cells) in addition to myocardial cells in these tissues can also be increased. Therefore, the present invention also contemplates that any one of the right ventricular tissue, atrial tissue, outflow tract tissue and atrioventricular canal tissue contains at least 60% of cardiac cells selected from myocardial cells, endocardial cells and epicardial cells. Preferably, at least 70% or at least 80% of the cells of these tissues are cardiac cells selected from myocardial cells, endocardial cells and epicardial cells.

[0040] Of course, in the present creative cardiac tissue model, when present, any of these cell quantities can be combined for the corresponding tissue. The cardiac tissue model itself can comprise at least 60% cardiac cells selected from myocardial cells, endocardial cells and epicardial cells, especially if there is left ventricular tissue. Left ventricular tissue (when present) is usually the major part of the cardiac tissue model. Preferably, at least 70% or at least 80% of the cells of the cardiac tissue model are cardiac cells selected from myocardial cells, endocardial cells and epicardial cells. In the combinable embodiment with these numbers, at least 40%, preferably at least 50%, and particularly preferably at least 60% or at least 70% of the cells of the cardiac tissue model are myocardial cells.

[0041] The present creative heart tissue model comprises at least one internal cavity or one large chamber, i.e., a central chamber, which simulates the heart chamber of a natural heart. If it is functional, for example, under healthy conditions without interfering mutations or chemicals, a beating rhythm can be observed, which has a change in the volume distribution of the internal cavity or the central chamber. Since the heart tissue model is still artificial and is not connected to the blood circulation system as a pump therein, it generally lacks any large blood vessels entering (and leaving) the internal cavity or the central chamber. Preferably, the internal cavity or the central chamber is completely surrounded by different heart tissues (particularly selected from left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue or atrioventricular canal tissue). Alternatively or in combination with this, the volume of the internal cavity or the central chamber does not lead to the main blood vessels and / or is not developing blood vessels. It is possible to artificially, for example, by surgical operation, transplant blood vessels to the heart tissue model, but if there is no artificial intervention, such blood vessels will not be naturally formed from the fusion product of the present invention. "Main blood vessel" is a blood vessel similar to one of the large blood vessels (vena cava and pulmonary vein; aorta and pulmonary artery) of the mammalian heart. Such a main vessel may have a diameter that is 10% or greater than the diameter of one of the different cardiac tissues forming the cardiac tissue model. One of the different cardiac tissues is mentioned because such a main vessel will typically enter the interior cavity or central chamber at one tissue. Preferably, the volume of the interior cavity or central chamber is not directed to the blood vessel.

[0042] The present inventive tissue model is artificial and grown in culture using natural developmental principles, but is not a heart grown in vivo at the development site of any in vivo heart. Due to the culture process, the size is usually limited. In a preferred embodiment of the present invention, the cardiac tissue model has the following size: 0.3mm to 50mm, even more preferably 1mm to 40mm or especially preferably 2mm to 30mm in its maximum dimension. Due to the fusion of different cardiac tissues, the cardiac tissue model can have an elongated shape. As a reference, the maximum dimension of the shape is used to determine the size of the cardiac tissue model.

[0043] The inner cavity or central chamber is a cavity in the heart tissue model and is quite large (compared with the previous heart tissue model) and occupies the main part of the volume of the tissue model. Preferably, the size of the inner cavity or central chamber at its maximum size is at least 30% of the size of the heart tissue model at its maximum size. If there are more cavities in addition to the inner cavity or central chamber, this is only applied to the largest inner cavity or central chamber (and surrounding tissue, not extending to the tissue layer around another inner cavity). In a preferred embodiment, the size of the inner cavity or central chamber at its maximum size is at least 40% of the size of the heart tissue model at its maximum size, more preferably at least 50%, and even more preferably at least 60%. Due to the fusion of different heart tissues and the extension of the central chamber into different heart tissues, both the central chamber and the heart tissue model can have an elongated shape. Similar to the above, the maximum size in the shape is used.

[0044] In some embodiments (e.g., multi-cavity heart-type organoids), at least two, e.g., three or more, of the different cardiac tissues have an internal cavity. The internal cavities of the different cardiac tissues can be as described above, e.g., having a size of at least 30% of the size of the cardiac tissue model. Any of the internal cavities can also be smaller, especially when confined to a single different cardiac tissue. The internal cavity at its maximum dimension can have a size of at least 20% of the size of the cardiac tissue model at its maximum dimension. In addition, the size of the internal cavity at its maximum dimension can be at least 30%, preferably at least 40%, of the size of the cardiac tissue at its maximum dimension. The cardiac tissue here only considers the following portion of the tissue model, which belongs to a specific cardiac tissue that limits the internal cavity.

[0045] Cardiomyocyte is cardiac muscle cell, and is mainly responsible for the pulsating activity or tissue contraction of cardiac tissue model or different cardiac tissues.They are the main component of this creative tissue model, and form the layer around inner cavity or central chamber in tissue model.In the tissue of early development, they can directly face inner cavity or central chamber, but in the tissue later and more developed, inner layer is formed by endocardial cell.Therefore, in preferred embodiments, cardiomyocyte or endocardial cell directly face inner cavity or central chamber of cardiac tissue model and / or different cardiac tissues (especially preferably selected from left ventricle tissue, right ventricle tissue, atrial tissue, outflow tract tissue, atrioventricular canal tissue, sinoatrial node tissue and atrioventricular node tissue).

[0046] The cardiac tissue model and its different cardiac tissues and different cardiac tissues themselves (before fusion) forming another aspect of the present invention, as well as precursor tissues, including FHF, aSHF, pSHF or special or multipotent stem cells, can be characterized by specific expression patterns or expression markers. These expression patterns belong to the cells constituting cardiac tissue / tissue model / precursor. Gene names or gene symbols (see above) are used to characterize expression markers. Such patterns are shown in the accompanying drawings. The expression of cell markers can be determined by any suitable technology, including immunocytochemistry, immunofluorescence, RT-PCR, immunoblotting, fluorescence activated cell sorting (FACS), RNA sequencing, single cell RNA sequencing and enzymatic analysis. Some characteristic expression markers are provided below. Some markers can be overexpressed or underexpressed (underexpressed), which is lower than the detection limit in some cases. Preferably, overexpression is an expression that is increased compared with the expression in human embryonic stem cells. Preferably, underexpression is an expression that is reduced compared with the expression in human embryonic stem cells. Underexpression can also constitute undetectable expression.

[0047] The term "expression marker" means that the expression of the mRNA encoding the marker can be detected above the background level using RT-PCR or RNA sequencing or single cell RNA sequencing, preferably RT-PCR. The expression level of the expression marker can be compared with the expression level obtained from the negative control (i.e., cells known to lack the marker) or by isotype control (i.e., a control antibody that does not have relevant specificity and only non-specifically binds to cell proteins, lipids or carbohydrates). Therefore, the cell "expressing" the marker has an expression level that can be detected to be higher than the expression level determined by the negative control for the marker. Alternatively, using immunofluorescence microscopy or flow cytometry methods, such as fluorescence activated cell sorting (FACS), cell surface markers above background levels can be detected on cells.

[0048] The terms "lack of expression", "no expression" and "absence of a marker" mean that the expression of the mRNA of the expression marker above the background level cannot be detected using RT-PCR or RNA sequencing or single cell RNA sequencing, preferably RT-PCR. The expression level of the expression marker can be compared with the expression level obtained from a negative control (i.e., cells known to lack the marker) or by an isotype control (i.e., a control antibody that does not have the relevant specificity and only non-specifically binds to cellular proteins, lipids or carbohydrates). Therefore, a cell "lacking expression" of the marker looks similar to a negative control with respect to the marker. Alternatively, using immunofluorescence microscopy or flow cytometry methods, such as fluorescence activated cell sorting (FACS), cell surface markers above the background level cannot be detected on the cell.

[0049] 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. After the left ventricular tissue matures, NR2F2, TBX2 and TBX3 may not exist or be underexpressed.

[0050] 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. After the right ventricular tissue matures, NR2F2, TBX2 and WNT5A may not exist or be insufficiently expressed.

[0051] 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. After the atrial tissue matures, IRX1, IRX4 and HEY2 may not exist or be underexpressed.

[0052] 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. After the outflow tract tissue matures, TBX3, NR2F1 and NPPA may not exist or be underexpressed.

[0053] 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. After the atrioventricular canal tissue matures, IRX1, IRX4 and NPPA may not exist or be underexpressed.

[0054] 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. After the sinoatrial node tissue matures, NKX2.5, IRX1, IRX4 and NPPA may not be present or under-expressed.

[0055] Preferably, the atrioventricular node tissue cells express one or more expression markers selected from TBX3, TBX5, KCNE1, HCN4 and GJC1; and / or the sinoatrial node tissue cells lack the expression of one or more expression markers selected from RSPO3, MSX2, IRX4 and NPPA. After the atrioventricular node tissue matures, RSPO3, MSX2, IRX4 and NPPA may not be present or under-expressed.

[0056] In a further aspect, the present invention provides a method for generating a cardiac tissue model of the present invention. The method includes generating at least two different cardiac tissues in vitro, wherein the different cardiac tissues are selected from left ventricular progenitor first cardiac area tissue, right ventricular / outflow tract progenitor second cardiac area tissue, atrial progenitor second cardiac area tissue, outflow tract progenitor second cardiac area tissue, atrioventricular canal progenitor second cardiac area tissue, sinoatrial node progenitor second cardiac area tissue and atrioventricular node tissue. These at least two different cardiac tissues can be provided as pre-grown tissues, for example in a frozen state. Then, the method uses thawed and viable cardiac tissue. The method further contains the following steps: fusing at least two cardiac tissues; culturing the fused tissue model, and allowing calcium signaling connections, tissue contraction propagation behavior and / or central chamber formation between different cardiac tissues.

[0057] The present invention also provides a cardiac tissue model obtainable by any method of the present invention. The obtained cardiac tissue model may have any structural elements as described above.

[0058] Fusion of at least two cardiac tissues may include placing different cardiac tissues in contact with each other and fusing the tissues. Fusion may include cell growth and / or cell migration. Fusion may be achieved by placing two or more cardiac tissues (preferably in the ancestral stage of left ventricular tissue, right ventricular tissue, atrial tissue, outflow tract tissue or the later stage of pacemaker cells (sinoatrial node tissue and atrioventricular node tissue)) in a position close to each other, preferably placed in a plate. Usually within one day after fusion, the tissues form structural connections to each other, such as the areas of the above-mentioned connections. After a few days, the structural connections lead to functional interactions. Preferably, when more than two cardiac tissues are being fused, a fusion mold of an elongated shape is used to have control over the arrangement of cardiac tissues. The viable cells of different cardiac tissues promote tissue fusion to form a cardiac tissue model. Once fused, further growth leads to the formation of a central chamber. For fusion, the at least two different cardiac tissues preferably also do not contain a major central chamber or internal cavity, as this would compromise fusion efficiency and subsequent central chamber formation across more than one of the at least two different cardiac tissues (except for the left ventricular progenitor region tissue, which, when fused, may contain an internal cavity or central chamber and still allow for effective fusion and formation of an internal cavity or central chamber extending within another of the two cardiac tissues). One or more of the different cardiac tissues may include an internal cavity when placed for fusion or during fusion.

[0059] After fusion, calcium signaling connections and / or contractile propagation can develop between different cardiac tissues.

[0060] It is preferred to grow at least two different cardiac tissues in situ before fusion, because the developmental stages can be better controlled. Some developmental stages are described by days in culture. These days in culture refer to continued growth in culture at the optimal temperature (for a given cell, for example 37° C. for human cells) and in growth medium. Freezing of the tissue or cells from which the tissue is derived interrupts the count of days in culture. Thus, "days in culture" refers to the time corresponding to culture under optimal growth conditions for a given cell type.

[0061] In a preferred embodiment, the different cardiac tissues have been cultured and differentiated from pluripotent cells. That is, pluripotent cells are grown and differentiated to produce different cardiac tissues. Starting from the culture of pluripotent cells, the fusion is preferably on the 1st to 7th day of culture starting from the pluripotent stage.

[0062] In a preferred embodiment, the right ventricle / outflow tract progenitor second heart zone tissue, the atrial progenitor second heart zone tissue, the atrioventricular progenitor second heart zone tissue are fused on culture day 2 to 5 (again counting from the pluripotent stage). Preferably, the sinoatrial node progenitor second heart zone tissue and / or the atrioventricular node tissue are fused on culture day 6-10, particularly preferably with tissues of similar age. Alternatively, or in combination, the optimal fusion time is determined by certain expression markers of the tissues. Preferably, the left ventricle progenitor first heart zone tissue is fused when the expression markers TBX5 and / or HAND1 are expressed. Preferably, the right ventricle / outflow tract progenitor second heart zone tissue is fused when the expression markers TBX1, FOXC1 and / or FOXC2 are expressed. Preferably, the atrial tissue is fused when the expression markers HOXB1, TBX5 and / or OSR1 are expressed. Preferably, the atrioventricular canal progenitor second heart zone tissue is fused when the expression markers TBX3, FOXF1 and / or HOXB1 are expressed. Preferably, when the expression markers SHOX2, TBX3, HCN4, ISL1 and / or GJC1 are expressed, the sinoatrial node progenitor secondary cardiac region tissue is fused. Preferably, when the expression markers TBX3, TBX5, KCNE1, HCN4 and / or GJC1 are expressed, the atrioventricular node tissue is fused. These markers indicate development to conditions suitable for effective fusion.

[0063] Can use culture medium and / or growth factor and / or differentiation factor to carry out generation different tissues.In the embodiment part of this paper, especially embodiment 1 and 2, provide example.Wherein any described differentiation factor can be used for this creative method with any combination and individually or together with other differentiation markers realizing identical differentiation development.Below described some preferred components for cultivating for specific tissue.

[0064] In a particularly preferred embodiment, one of the at least two different cardiac tissues is left ventricular progenitor first heart region tissue. Generating left ventricular progenitor first heart region tissue can include differentiating mesodermal cells into left ventricular precursor cells in a culture medium comprising bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, Wnt inhibitor, preferably Wnt-C59 or IWP2, and retinoic acid, wherein retinoic acid has a concentration of 5nM to 100nM in the culture medium. Cultivation with this culture medium is preferably carried out on about the second day of culture, for example, during the 1.5 to 3 days of culture.

[0065] Particularly preferably, one of the at least two different cardiac tissues is right ventricular preprogenitor second heart zone tissue or right ventricular / outflow tract preprogenitor second heart zone tissue. Generating right ventricular preprogenitor second heart zone tissue or right ventricular / outflow tract preprogenitor second heart zone tissue may include differentiating mesodermal cells into right ventricular progenitor cells in a culture medium containing a TGF-β inhibitor, preferably SB 431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939. Cultivation with the culture medium is preferably performed on about day 2 of culture, for example, during day 1.5 to day 3 of culture. Generating right ventricular preprogenitor second heart zone tissue and in particular further developing it into right ventricular tissue may further include culturing in a culture 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 retinoic acid having a concentration of 50 nM to 500 nM in the culture medium.

[0066] Preferably, one of the at least two different cardiac tissues is the second cardiac zone tissue before the outflow tract progenitor. Generating the second cardiac zone tissue before the outflow tract progenitor may include differentiating mesodermal cells into outflow tract tissue precursor cells in a culture medium containing a TGF-β inhibitor, preferably SB 431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939. Cultivation with the culture medium is preferably performed on about the second day of culture, for example, during the 1.5 to 3 days of culture. Generating the second cardiac zone tissue before the outflow tract progenitor or further to the outflow tract tissue may further include culturing in a culture 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.

[0067] Preferably, one of the at least two different cardiac tissues is the second cardiac region tissue after the atrial progenitor. Generating the second cardiac region tissue after the atrial progenitor may include differentiating mesoderm cells into atrial tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, preferably at a retinoic acid concentration of 300nM to 800nM. Cultivation with the culture medium is preferably performed on about the second day of culture, for example, during the 1.5 to 3 days of culture. Generating the second cardiac region tissue after the atrial progenitor or further differentiating it to the atrial progenitor tissue may include further culturing in a culture medium comprising 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 retinoic acid concentration of 300nM to 800nM.

[0068] Preferably, one of the at least two different cardiac tissues is the second heart zone tissue after the atrioventricular canal progenitor. Generating the second heart zone tissue after the atrioventricular canal progenitor may include differentiating mesoderm cells into atrioventricular canal tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably a retinoic acid concentration of 300nM to 800nM. Cultivation with the culture medium is preferably performed on about the second day of culture, for example, during the 1.5 to 3 days of culture. Generating the second heart zone tissue after the atrioventricular canal progenitor or further differentiating it into atrioventricular canal tissue may include further culturing in a culture medium comprising 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 a retinoic acid concentration of 300nM to 800nM.

[0069] Preferably, one of the at least two different cardiac tissues is the second cardiac region after the sinoatrial node progenitor, and generating the second cardiac region after the sinoatrial node progenitor comprises differentiating mesodermal cells into sinoatrial node tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a retinoic acid concentration of 300nM to 800nM.

[0070] Preferably, one of the at least two different cardiac tissues is atrioventricular node tissue, and generating atrioventricular node tissue comprises differentiating mesodermal cells into atrioventricular canal tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a retinoic acid concentration of 300 nM to 800 nM. Generating atrioventricular node tissue may further comprise differentiating atrioventricular canal progenitor secondary cardiac region tissue into atrioventricular node tissue by further culturing in a culture medium containing an activator or inhibitor of sonic hedgehog signaling and / or bone morphogenetic protein.

[0071] The fusion of cardiac tissues is carried out under culture conditions that allow fusion and do not hinder the differentiation of progenitor tissues into their target tissues during or after fusion. Preferably, the fusion of at least two cardiac tissues comprises culturing in a culture medium comprising 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 retinoic acid concentration of 300 nM to 800 nM, particularly preferably 500 nM. These are excellent culture conditions for the cardiac tissues described herein.

[0072] The above are mesodermal cells as ancestors for the present inventive tissue differentiation. Mesodermal cells can be derived from pluripotent cells. Preferably, the in vitro generation of at least two different cardiac tissues includes the generation of mesodermal cells. Mesodermal cells can be generated by differentiating pluripotent cells in a culture medium comprising activin and Wnt activator, preferably CHIR99021; preferably at least until the differentiated cells express expression markers selected from BRA, EOM, MIXL1, FOXA2, preferably in the absence of SOX2. Preferably, the culture medium for generating mesodermal cells further comprises a PI3 kinase inhibitor, such as LY294002. The use of PI3 kinase inhibitors is particularly preferred because it results in the cleanest and most homogeneous tissue model. PI3 kinase inhibitors have the following effects: many pluripotent cells exit pluripotency and differentiate into mesodermal lineages, which determines a highly homogenous cardiac tissue model in the future. LY294002 is preferably used at a concentration of 3 μM to 15 μM.

[0073] Already at the stage of mesodermal cells, there is a general prevalence of differentiation to specific tissues. For example, mesodermal cells can be the ancestors of the first heart field (FHF) or the second heart field (SHF) (including aSHF and pSHF).

[0074] For mesodermal cells further differentiated into left ventricular tissue (FHF), the preferred concentration of activin is 1 ng / mL to 8 ng / mL, more preferably about 5 ng / mL, and / or the preferred concentration of CHIR99021 is 1 μM to 6 μM, more preferably about 3 μM. For mesodermal cells further differentiated into right ventricular tissue, atrial tissue or outflow tract tissue (a / pSHF), the preferred concentration of activin is 30 ng / mL to 100 ng / mL, more preferably about 50 ng / mL, and / or the preferred concentration of CHIR99021 is 2 μM to 8 μM, more preferably about 4 μM. For mesodermal cells further differentiated into atrioventricular canal tissue (partial pSHF) or atrioventricular node tissue, the preferred concentration of activin is 6 ng / mL to 30 ng / mL, more preferably about 10 ng / mL, and / or the preferred concentration of CHIR99021 is 0.4 μM to 4 μM, more preferably about 2 μM. For further differentiation of mesodermal cells into sinoatrial node tissue, preferably the activin concentration is 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or the CHIR99021 concentration is 0.1 μM to 4 μM, preferably about 1 μM.

[0075] Preferably, the pluripotent stem cells have been grown in a culture medium comprising 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.

[0076] Preferably, the culture of pluripotent stem cells and their differentiation into mesodermal cells is in 2D culture. Subsequent steps, such as the culture of mesodermal cells and their differentiation into progenitor cells and the generation of different cardiac tissues and tissue models are in 3D culture.

[0077] 2D culture can include growing or maintaining cells on a surface such as a tissue culture plate. Such a surface can be coated with a suitable culture matrix such as vitronectin.

[0078] In 3D culture, cells or aggregates are not attached to the surface, but cells are allowed to aggregate with each other or remain free-floating under suspension conditions, so that expansion in all 3D directions is uniformly possible. Such culture is in low-attachment culture, so that cells or aggregates are not attached to the culture vessel wall. Preferably, low-attachment culture includes culturing cells in a container with a low-attachment surface that inhibits cell adhesion to the surface. The low-attachment surface is preferably hydrophilic, neutrally charged or non-ionic. They can have a hydrogel layer or coating that repels cell attachment, which forces cells into a suspended state. Such low-attachment culture containers are known in the art, for example, described in WO 2019 / 014635 or WO 2019 / 014636. Preferably, the bottom of the culture container is round, especially concave. Alternatively, it can be flat or have a V-shaped bottom shape.

[0079] Preferably, the pluripotent cells can then be dissociated cells for initiating culture. They are then allowed to form aggregates. Mesodermal cells are preferably cultures that are already in an aggregate state, for example as obtained from culturing and differentiating pluripotent cells. Aggregates then lead to the formation of different tissues and tissue models as the aggregates expand.

[0080] Preferably, a cell culture medium such as E8 culture medium is used. The cell culture medium used in the process of the present invention preferably comprises amino acids required for cell growth, and energy sources such as carbohydrates, particularly preferably glucose. It should further comprise salts and ions required for cell growth, such as Ca, Fe, Mg, K, Na, Zn, Cl, SO4, NO3, PO4 ions. Further preferred components of the culture medium are vitamins, such as vitamin B-12, biotin, choline, folic acid, inositol, nicotinamide, pantothenic acid, pyridoxine, riboflavin, thiamine.

[0081] Preferred amino acids include essential amino acids, and any one of the following amino acids is also preferred: alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.

[0082] In preferred embodiments, the cells have been cultured or treated in / with such a culture medium (especially a culture medium comprising albumin and / or fibroblast growth factors) for at least 1 day, preferably at least 2 days, for example 1 to 30 days, such as 2 to 20 days, while of course maintaining pluripotency.

[0083] A preferred bone morphogenic protein (BMP) is BMP4, in particular human BMP4. Preferably, the culture medium with BMP comprises at least 8 ng / ml bone morphogenic protein, preferably BMP4.

[0084] Some culture media or cultures contain FGF (fibroblast growth factor), preferably FGF2, FGF4, FGF8, FGF17, FGF18. FGF2 is particularly preferred. Unless otherwise specified, FGF2 is preferably used at a concentration of at least 6 ng / ml.

[0085] WNT inhibitors are known in the art. WNT inhibitors are disclosed, for example, in Nusse and Clevers, Cell 169, 2017: 985-999. Other WNT activators are disclosed at 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 a combination thereof. Particularly preferred WNT inhibitors are IWP-2 and XAV.

[0086] In any culture medium with Wnt inhibitors or activators, albumin is preferably present. The albumin may be bovine serum albumin (BSA). Albumin protects cells from the toxicity of small molecules such as WNT activators / inhibitors and is therefore recommended.

[0087] The TGF-β inhibitor can be a TGF-β signaling pathway inhibitor because it inhibits TGF-β function. It can be an inhibitor of the TGF-β superfamily pathway. Preferably one or more preferably two or even more TGF-β inhibitors are used. In some preferred embodiments, it includes or comprises one or more inhibitors of TGF-β receptors or TGF-β pathways. In a preferred embodiment, at least one TGF-β (pathway) inhibitor is at least one: SMAD inhibitor preferably DMH1 (dorsomorphin homolog 1) and / or SB431542 (4-[4-(2H-1,3-benzodioxol (benzodioxol)-5-yl)-5-(pyridin-2-yl)-1H-imidazole-2-yl] benzamide). Further preferred TGF-β inhibitors (which may replace or be combined with those mentioned above) are Noggin (a protein that binds and inactivates BMP proteins, which belong to the TGF-β superfamily), A83-01 (a small molecule), LDN 193189 (a small molecule) and / or Dorsomorphine. Further TGF-β inhibitors are known in the art, for example as disclosed at www.medchemexpress.com / Targets / TGF-(beta)%20Receptor.html.

[0088] Activins, such as activin A, can be used to determine cardiac tissue precursors toward LV, a / pSHF and SAN or AVC fates. Typically, high activin (e.g., above 10 ng / mL) directs tissue models toward a / pSHF and SAN fates, medium activin (e.g., about 10 ng / mL) directs tissue models toward atrioventricular fates, and low activin (e.g., below 10 ng / mL) directs tissue models toward left ventricular fates. These example concentrations can vary, depending on the cell type used.

[0089] Wnt activators are known in the art and are described in, for example, Nusse and Clevers, Cell 169, 2017: 985-999. Nusse and Clevers discussed the Wnt / b-catenin signaling pathway and its operation in stem cells. Wnt activators can be GSK3 inhibitors. Other 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). Further preferred Wnt activators are WAY-316606, ABC99, IQ1, QS11, SB-216763.

[0090] The activator of sonic hedgehog (SHH) signaling is preferably a Smoothened agonist, particularly preferably SAG (N-methyl-N′-(3-pyridylbenzyl)-N′-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane), cyclopamine or purmorphamine (PMA) or a combination thereof. The inhibitor of sonic hedgehog (SHH) signaling is preferably cyclopamine.

[0091] The present invention further provides the above-mentioned different cardiac tissues and their precursors as tissue models.

[0092] Therefore, in a further aspect, a right ventricular tissue model is provided. The right ventricular tissue model may comprise cells expressing the markers IRX1, IRX2 and PRDX1. The entire disclosure herein regarding right ventricular tissue also applies to the right ventricular tissue model.

[0093] Atrial tissue models are further provided. The atrial tissue model may comprise cells expressing markers NR2F1, NR2F2 and HEY1. The entire disclosure herein regarding atrial tissue also applies to the atrial tissue model.

[0094] Further provided is an outflow tract tissue model. The outflow tract tissue model may include cells expressing markers WNT5A, WNT11, MSX1, BMP4 and RSPO3. The entire disclosure herein about outflow tract tissue also applies to the outflow tract tissue model.

[0095] Further provided is an atrioventricular canal tissue model. The atrioventricular canal tissue model may include cells expressing markers TBX2, MSX2, and RSPO3. The entire disclosure herein about atrioventricular canal tissue also applies to the atrioventricular canal tissue model.

[0096] A sinoatrial node tissue model is further provided. The sinoatrial node tissue model may comprise cells expressing markers SHOX2, TBX3, HCN4, ISL1 and GJC1. The entire disclosure herein regarding sinoatrial node tissue also applies to the sinoatrial node tissue model.

[0097] An atrioventricular node tissue model is further provided. The atrioventricular node tissue model may comprise cells expressing markers TBX3, TBX5, KCNE1, HCN4 and GJC1. The entire disclosure herein regarding atrioventricular node tissue also applies to the atrioventricular node tissue model.

[0098] The right ventricle tissue model, the atrial tissue model, the outflow tract tissue model, the atrioventricular canal tissue model, the sinoatrial node tissue model, and the atrioventricular node tissue model (also collectively referred to as tissue models) may not be fused or they may be fused to another tissue. The tissue model recapitulates a specific part of the heart and, together with the left ventricular tissue in WO 2021 / 186044A1, is also considered a cardiac organoid or a cardiac-type organoid.

[0099] Any tissue model can undergo cavitation and then itself contain an internal cavity or central chamber - similar to the fused tissue models described above. Preferably, the size of the internal cavity or central chamber at its largest dimension is at least 30% of the size of the tissue model at its largest dimension. If there are more cavities in addition to the central chamber or largest internal cavity, this only applies to the largest internal cavity or central chamber (and surrounding tissue, not extending to tissue layers surrounding another internal cavity). In a preferred embodiment, the size of the internal cavity or central chamber at its largest dimension is at least 40%, more preferably at least 50%, and even more preferably at least 60% of the size of the tissue model at its largest dimension.

[0100] Tissue models can include calcium signaling or tissue contractile behavior or the ability of a tissue to contract. As described above, for the fused heart tissue model, calcium signaling and contraction can be observed - except, of course, that 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 cells in this tissue model.

[0101] Preferably, the tissue model comprises at least 60% cardiac cells selected from the group consisting of cardiomyocytes, endocardial cells and epicardial cells. Preferably, at least 70% or at least 80% of the cells of the tissue model are cardiac cells selected from the group consisting of cardiomyocytes, endocardial cells and epicardial cells. Particularly preferred is that the tissue model comprises at least 60% cardiomyocytes.

[0102] Due to the culture process, usually the size of these tissue models is limited. In a preferred embodiment of the invention, the tissue model has a size of 0.2 to 30 mm, even more preferably 0.5 to 25 mm or especially preferably 1 to 20 mm in its largest dimension.

[0103] The cardiac tissue model can be obtained or generated by the above methods for different cardiac tissues without fusing to another one of the different cardiac tissues. In particular, the cardiac tissue model can be obtained by the above culture methods for different cardiac tissues.

[0104] The present invention, i.e., cardiac tissue models, methods, and different tissue models can be used to screen the effects of candidate compounds and / or the effects of genetic modifications or environmental factors. Such methods can include generating a cardiac tissue model according to the present invention, during which cells (at any stage during development) are treated with a candidate compound. In addition, the final tissue model can be used to test the candidate compound.

[0105] For example, the present invention provides a method for screening or testing a candidate compound for its effect on cardiac development and / or function, the method comprising generating a cardiac tissue model according to the method of the present invention, treating cells with a candidate compound, and comparing the development of the cardiac tissue model with the development and / or function of a cardiac tissue model not treated with the candidate compound.

[0106] Further provided are methods for observing the effects of a suppressed, mutated or overexpressed gene during cardiac development, comprising generating a cardiac tissue model according to the present invention, wherein cells have a suppressed or mutated candidate gene or overexpress the candidate gene, and comparing the development of the cardiac tissue model with the development of a cardiac tissue model generated without the suppressed, mutated or overexpressed gene.

[0107] Of course, these methods of screening / testing candidate compounds and genetic alterations can be combined. For example, methods of screening or testing candidate compounds for their effects on cardiac development and / or function according to the present invention are provided, combined with observing the effects of genes suppressed or overexpressed during cardiac development according to the present invention.

[0108] Also provided is a method for screening or testing a candidate compound for its effect on cardiac function, which comprises treating a cardiac tissue model according to the present invention with the candidate compound and comparing the function with that of a cardiac tissue model not treated with the candidate compound.

[0109] Method can include comparing the development or function of the cardiac tissue model with the development or function of the cardiac tissue model not treated with the candidate compound. For comparison, of course, except being treated with the candidate compound, all processing steps should be identical. Example function is, for example, any cardiac function, such as morphological changes or metabolic renewal or genetic expression of a gene of interest that can be affected by the candidate compound, such as beating behavior (e.g., intensity and / or rhythm, such as arrhythmia) or can be affected by the candidate compound. In the internal cavity or central chamber, fluid flow and / or electric current in the tissue around the internal cavity or central chamber can also be observed. The development of function can be toxic development or function that can be caused by the candidate compound. Therefore, this inventive method or tissue model can be used for toxicity testing or screening.

[0110] Genetic changes can be tested. The present invention provides methods for observing the effects of mutated (e.g., disease-related), suppressed, mutated, or overexpressed genes during cardiac development, including generating a cardiac tissue model according to the present invention, wherein the cell has a suppressed or mutated candidate gene or overexpresses the candidate gene, and comparing the development of the cardiac tissue model with the development of a cardiac tissue model that is not generated with a suppressed, mutated, or overexpressed gene. Here, suppression or overexpression is related to a normal state (modification without the expression or mutation of the candidate gene). Mutation, overexpression, or expression inhibition can be performed with any method known in the art, such as by gene knockout, siRNA inhibition, or inhibition based on CRISPR / Cas inactivation. Overexpression can be performed by introducing a transgenic or applying a gene activator that causes upregulation of gene expression. Such mutant tissues or methods using mutations in cells and using these cells to generate this creative cardiac tissue can also be used to screen or test candidate compounds as described above. Therefore, the present invention provides the use of this creative cardiac tissue or method as a disease model, such as a heart disease model, particularly for studying cardiac function. Therefore, the method of screening or testing candidate compounds can also be combined with observing the effects of suppressed or overexpressed genes. In a combined approach, comparisons can be made between variants of a tissue or approach (both with mutations) with ("drugged") or without ("undrugged") the candidate treatment, or between mutant and non-mutated variants (both formulated with drugs), or between all four states (mutated + drugged, mutant + undrugged, non-mutated + drugged, non-mutated + undrugged).

[0111] The present inventive cardiac tissue can be used to reproduce a specific normal or abnormal condition of the heart, such as a disease, illness or injury, and to recover from such a condition. Disease can be, for example, a congenital defect or myopathy or hypertrophy of the heart. For example, by making cells or aggregates susceptible or exposed to such factors during the method or tissue model, the factors to be adjusted are, for example, genetic defects, environmental conditions, maternal diabetes, toxins, such as plastic particles, such as microplastics. The present inventive cardiac tissue model can include damage or healing damage. The present inventive method can include causing damage to the tissue model, and optionally further recovering from such damage. Agents (such as test compounds) can be tested to cause such conditions or to test the effects of compounds during damage, as well as for the effects of recovery and / or healing, or during recovery and / or healing. Example damage-related effects to be studied are fibrosis after damage and the healing of such fibrosis, for example, by treating with candidate compounds.

[0112] The present inventive tissue model, in particular when comprising LV tissue and / or RV tissue, can be used to study chamber dilatation, fibrillation or to study communication between RV and LV tissue. When comprising atrial tissue or sinoatrial node tissue, it can be used to study atrial fibrillation. Typically, drug effects or environmental effects have compartment-specific effects on cardiac function. Such effects can be studied in the present inventive tissue, which comprises LV, RV, OFT, atrium, AVC, SAN and / or AVN tissue. In particular, in fused tissue models with different tissues, the effects on these tissues can be studied by comparing them with the effects on other tissues within the tissue model.

[0113] Further provided is a method for treating a patient's heart injury, comprising transplanting cells from a cardiac tissue model of the present invention to the injury. The cell may preferably be a cardiomyocyte, a fibroblast, an endothelial cell, a smooth muscle cell or a pacemaker cell, or a combination or mixture thereof. Particularly preferred is a cardiomyocyte. Also preferred is a pacemaker cell. The pacemaker cell is preferably obtained from a sinoatrial node tissue or a sinoatrial node tissue model. Possibly, a tissue model of the present invention is provided, cells required for separation and regeneration of the injury, such as cardiomyocytes or precursor cells, are provided to the site of the injury. Integrating the cells of the tissue model into the wound generally comprises a regeneration process, which is improved by the presence of the cells of the present creative tissue model. The cell can be connected to the injured heart of the patient and improve its regeneration. An example of cardiac injury is an infarcted heart.

[0114] The present invention further includes a cell culture medium for carrying out the method of the present invention. The culture medium can be used in the steps as described above. Any specific combination of compounds as described above can be provided in the culture medium. In particular, a cell culture medium is provided, which comprises

[0115] a) a bone morphogenetic protein, preferably BMP4, a 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;

[0116] b) TGF-β inhibitor, preferably SB 431542, and Wnt inhibitor, preferably Wnt-C59 or XAV-939;

[0117] 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 culture medium;

[0118] d) the TGF-β inhibitor is preferably SB 431542 and the Wnt inhibitor is preferably Wnt-C59 or XAV-939;

[0119] e) the Wnt inhibitor is preferably Wnt-C59 or XAV-939, the bone morphogenetic protein is preferably BMP4, the fibroblast growth factor is preferably FGF2, insulin, and the culture medium lacks retinoic acid;

[0120] f) a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, preferably retinoic acid alone at a concentration of 300 nM to 800 nM;

[0121] 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 concentration of retinoic acid is 300nM to 800nM;

[0122] h) a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a retinoic acid concentration of 300 nM to 800 nM;

[0123] i) activin and CHIR99021; preferably, activin concentration is 1 ng / mL to 8 ng / mL, preferably about 5 ng / mL, and / or CHIR99021 concentration is 1 μM to 6 μM, preferably about 3 μM; or preferably, activin concentration is 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or CHIR99021 concentration is 2 μM to 8 μM, preferably about 4 μM; or preferably, activin concentration is 6 ng / mL to 30 ng / mL, preferably about 10 ng / mL, and / or concentration is 0.4 μM to 4 μM, preferably about 2 μM; or preferably, activin concentration is 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or CHIR99021 concentration is 0.1 μM to 4 μM, preferably about 1 μM; or

[0124] j) Wnt inhibitor, preferably Wnt-C59, bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin and retinoic acid, preferably the concentration of retinoic acid is 300 nM to 800 nM, particularly preferably 500 nM.

[0125] Any culture medium may contain amino acids required for cell growth, and energy sources such as carbohydrates, particularly preferably glucose. Further, they may contain salts and ions required for cell growth, such as Ca, Fe, Mg, K, Na, Zn, Cl, SO4, NO3, PO4 ions. Further preferred components of the culture medium are vitamins, such as vitamin B12, biotin, choline, folic acid, inositol, nicotinamide, pantothenic acid, pyridoxine, riboflavin, thiamine.

[0126] Preferred amino acids include essential amino acids, and any one of the following amino acids is also preferred: alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.

[0127] Any of these culture media can be combined in a kit of different culture media used in the present inventive method or a kit of one or more culture media and another means, such as a carrier or mold for fusing cultured tissues. The culture media can be provided in a kit that further comprises instructions for carrying out the present inventive method. Such instructions can be in printed form or in a computer readable format on a suitable data carrier. The carrier or mold can be a plate with a recess, the shape of the recess being used to fuse different tissues as described above. An example is an elongated recess so that the linear arrangement of different cardiac tissues contacts each other.

[0128] According to the invention, the following numbered embodiments are preferred. Any numbered embodiment can of course be combined with the embodiments and preferences described above, or with the corresponding elements of the Examples section.

[0129] 1. A cardiac tissue model comprising cardiac tissue having at least one internal cavity or central chamber, 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; wherein the central chamber can be shared by at least two different cardiac tissues, and wherein at least two different cardiac tissues comprise electrophysiological signaling connections or calcium signaling connections or the ability to propagate tissue contraction.

[0130] 2.1 heart tissue model, where

[0131] The left ventricular tissue comprises at least 60% cardiac cells selected from the group consisting of cardiomyocytes, endocardial cells, and epicardial cells;

[0132] Right ventricular tissue contains at least 60% myocytes;

[0133] Atrial tissue contains at least 60% cardiomyocytes;

[0134] Outflow tract tissue comprises at least 60% cardiomyocytes; atrioventricular canal tissue comprises at least 60% cardiomyocytes; sinoatrial node tissue comprises at least 60% cardiomyocytes; and / or

[0135] The atrioventricular node tissue contains at least 60% of myocardial cells.

[0136] 3. The cardiac tissue model of 1 or 2, wherein the internal cavity or central chamber 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 wherein the volume of the internal cavity or central chamber is not directed into a main blood vessel.

[0137] 4. The cardiac tissue model of any one of 1 to 3, which has a size of 0.3 mm to 50 mm in its largest dimension.

[0138] 5. The cardiac tissue model of any one of 1 to 4, wherein:

[0139] 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;

[0140] 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;

[0141] 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;

[0142] 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;

[0143] 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;

[0144] 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 expression of one or more expression markers selected from NKX2.5, IRX1, IRX4 and NPPA; and / or

[0145] 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.

[0146] 6. The cardiac tissue model of any one of 1 to 5, wherein the size of the internal cavity and / or central chamber at its largest dimension is at least 30% of the size of the cardiac tissue model at its largest dimension.

[0147] 7. The cardiac tissue model of any one of 1 to 6, wherein the cardiomyocytes or endocardial cells directly face the inner cavity and / or the central chamber.

[0148] 8. The method for generating a cardiac tissue model according to any one of items 1 to 7, comprising generating at least two different cardiac tissues in vitro, wherein the different cardiac tissues are selected from the group consisting of left ventricular progenitor first cardiac area tissue, right ventricular / outflow tract progenitor second cardiac area tissue, atrial progenitor second cardiac area tissue, outflow tract progenitor second cardiac area tissue, atrioventricular canal progenitor second cardiac area tissue, sinoatrial node tissue and atrioventricular node tissue, and fusing the at least two cardiac tissues,

[0149] Culture confluent tissue models and enable calcium signaling connections between different cardiac tissues, the ability to propagate contractions, and / or central chamber formation.

[0150] 9. The method of claim 8, wherein fusing the at least two cardiac tissues comprises placing different cardiac tissues in contact with each other and allowing the tissues to fuse, preferably by cell growth.

[0151] 10. The method of 8 or 9, wherein the different cardiac tissues have been cultured and differentiated from pluripotent cells, and wherein the fusion is at culture day 1 to 7 from the pluripotent stage; preferably, the right ventricle / outflow tract progenitor second cardiac zone tissue, the atrial progenitor second cardiac zone tissue, the atrioventricular progenitor second cardiac zone tube tissue, the sinoatrial node progenitor second cardiac zone tissue and / or the atrioventricular node tissue are fused at culture day 2 to 5; or preferably, the left ventricular progenitor first cardiac zone tissue is fused when it expresses the expression markers IRX4, TBX5 and / or HEY2; preferably, the right ventricular progenitor second cardiac zone tissue is fused when it expresses the expression markers TBX1, FOXC1 and / or FOXC2; preferably, the second cardiac zone tissue of the atrial progenitors is fused when it expresses the expression markers HOXB1, TBX5 and / or OSR1; preferably, the second cardiac zone tissue of the atrioventricular canal progenitors is fused when it expresses the expression markers TBX3, FOXF1 and / or HOXB1; preferably, the second cardiac zone tissue of the sinoatrial node progenitors is fused when it expresses the expression markers SHOX2, TBX3, HCN4, ISL1 and / or GJC1; and / or preferably, the atrioventricular node tissue is fused when it expresses the expression markers TBX3, TBX5, KCNE1, HCN4 and / or GJC1.

[0152] 11. The method of any one of items 8 to 10, wherein one of the at least two different cardiac tissues is left ventricular progenitor first cardiac zone tissue, and generating left ventricular progenitor first cardiac zone tissue comprises differentiating mesodermal cells into left ventricular precursor cells in a culture medium comprising bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin, a Wnt inhibitor, preferably Wnt-C59 or IWP2, and retinoic acid, wherein the retinoic acid has a concentration of 5 nM to 100 nM in the culture medium.

[0153] 12. The method of any one of claims 8 to 11, wherein one of the at least two different cardiac tissues is right ventricle / outflow tract progenitor secondary cardiac zone tissue, and generating right ventricle / outflow tract progenitor secondary cardiac zone tissue comprises differentiating mesodermal cells into right ventricular progenitor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939; optionally, subsequently culturing in a culture medium comprising 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 wherein the retinoic acid has a concentration of 50 nM to 500 nM in the culture medium.

[0154] 13. The method of any one of claims 8 to 12, wherein one of the at least two different cardiac tissues is an outflow tract progenitor secondary cardiac zone tissue, and generating an outflow tract progenitor secondary cardiac zone tissue comprises differentiating mesodermal cells into outflow tract tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939; optionally, subsequently culturing in a culture medium comprising 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.

[0155] 14. The method of any one of items 8 to 13, wherein one of the at least two different cardiac tissues is atrial progenitor second cardiac zone tissue, and generating atrial progenitor second cardiac zone tissue comprises differentiating mesodermal cells into atrial tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, preferably at a retinoic acid concentration of 300 nM to 800 nM; optionally, subsequently culturing in a culture medium comprising 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 retinoic acid concentration of 300 nM to 800 nM.

[0156] 15. The method of any one of claims 8 to 14, wherein one of the at least two different cardiac tissues is atrioventricular canal progenitor second cardiac zone tissue, and generating atrioventricular canal progenitor second cardiac zone tissue comprises differentiating mesoderm cells into atrioventricular canal tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a retinoic acid concentration of 300 nM to 800 nM; optionally, subsequently culturing in a culture medium comprising 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 retinoic acid concentration of 300 nM to 800 nM.

[0157] 16. The method of any one of items 8 to 15, wherein one of the at least two different cardiac tissues is sinoatrial node progenitor second cardiac zone tissue, and generating sinoatrial node progenitor second cardiac zone tissue comprises differentiating mesodermal cells into sinoatrial node tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a retinoic acid concentration of 300 nM to 800 nM.

[0158] 17. The method of any one of claims 8 to 16, wherein one of the at least two different cardiac tissues is atrioventricular node tissue, and generating the atrioventricular node tissue comprises differentiating mesoderm cells into atrioventricular canal tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a retinoic acid concentration of 300 nM to 800 nM; and wherein the atrioventricular canal progenitor secondary cardiac zone tissue is further differentiated into atrioventricular node tissue by further maturation in a culture medium containing an activator and / or inhibitor of sonic hedgehog signaling and / or BMP.

[0159] 18. The method of any one of 17 to 18, wherein fusing at least two cardiac tissues comprises culturing in a culture medium comprising 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 retinoic acid concentration of 300 nM to 800 nM, particularly preferably 500 nM.

[0160] 19. The method of any one of 8 to 18, wherein the in vitro generation of at least two different cardiac tissues comprises generating mesodermal cells by differentiating pluripotent cells in a culture medium comprising activin and a Wnt activator, preferably CHIR99021; preferably at least until the differentiated cells express an expression marker selected from the group consisting of BRA, EOM, MIXL1, FOXA2, preferably in the absence of SOX2; and / or preferably wherein

[0161] For further differentiation of mesodermal cells into left ventricular tissue, the concentration of activin is 1 ng / mL to 8 ng / mL, preferably about 5 ng / mL, and / or the concentration of CHIR99021 is 1 μM to 6 μM, preferably about 3 μM;

[0162] For further differentiation of mesodermal cells into right ventricular tissue, atrial tissue or outflow tract tissue, the concentration of activin is 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or the concentration of CHIR99021 is 2 μM to 8 μM, preferably about 4 μM; and / or

[0163] For further differentiation of mesodermal cells into atrioventricular canal tissue, the concentration of activin is 6 ng / mL to 30 ng / mL, preferably about 10 ng / mL, and / or the concentration of CHIR99021 is 0.4 μM to 4 μM, preferably about 2 μM; and / or

[0164] For further differentiation of mesodermal cells into sinoatrial node tissue, preferably the activin concentration is 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or the CHIR99021 concentration is 0.1 μM to 4 μM, preferably about 1 μM.

[0165] 20. A right ventricle tissue model comprising cells expressing the markers IRX1, IRX2 and PRDX1.

[0166] 21. An atrial tissue model comprising cells expressing markers NR2F1, NR2F2 and HEY1.

[0167] 22. An outflow tract tissue model comprising cells expressing markers WNT5A, MSX1, BMP4 and RSPO3.

[0168] 23. An atrioventricular canal tissue model comprising cells expressing markers TBX2, MSX2 and RSPO3.

[0169] 24. A sinoatrial node tissue model comprising cells expressing markers SHOX2, TBX3, HCN4, ISL1 and GJC1.

[0170] 25. An atrioventricular node tissue model comprising cells expressing markers TBX3, TBX5, KCNE1, HCN4 and GJC1.

[0171] 26. The tissue model of any one of 20 to 25, comprising at least 60% cardiac cells selected from the group consisting of cardiomyocytes, endocardial cells and epicardial cells.

[0172] 27. The tissue model of any one of 20 to 26, comprising a size of 0.2 mm to 30 mm in its largest dimension.

[0173] 28. The tissue model of any one of 20 to 27, comprising an internal cavity.

[0174] 29. The method of any one of 8 to 19, which is used to screen or test a candidate compound for its effect on cardiac development and / or function, the method comprising generating a cardiac tissue model according to any one of 8 to 19, treating cells with a candidate compound, and comparing the development of the cardiac tissue model with the development and / or function of a cardiac tissue model not treated with the candidate compound.

[0175] 30. A method for observing the effects of a mutated, suppressed or overexpressed gene during cardiac development, comprising generating a cardiac tissue model according to any one of 8 to 19, wherein cells have a suppressed or mutated candidate gene or overexpress the candidate gene, and comparing the development of the cardiac tissue model with the development of a cardiac tissue model generated without the suppressed, mutated or overexpressed gene.

[0176] 31. The method of screening or testing candidate compounds for their effects on cardiac development and / or function according to 29, which is combined with observing the effects of suppressed, mutated or overexpressed genes during cardiac development according to 30.

[0177] 32. A method for screening or testing a candidate compound for its effect on cardiac function, comprising treating a cardiac tissue model according to any one of 1 to 7 or 20 to 28 with the candidate compound, and comparing the function with that of the cardiac tissue model not treated with the candidate compound.

[0178] 33. A method of treating cardiac damage in a patient, comprising transplanting one or more cells, preferably cardiomyocytes, from the cardiac tissue model of any one of 1 to 7 or 20 to 28 into the damage.

[0179] 34. A cell culture medium comprising

[0180] a) a bone morphogenetic protein, preferably BMP4, a 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;

[0181] b) TGF-β inhibitor, preferably SB 431542, and Wnt inhibitor, preferably Wnt-C59 or XAV-939;

[0182] 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 culture medium;

[0183] d) the TGF-β inhibitor is preferably SB 431542 and the Wnt inhibitor is preferably Wnt-C59 or XAV-939;

[0184] e) the Wnt inhibitor is preferably Wnt-C59 or XAV-939, the bone morphogenetic protein is preferably BMP4, the fibroblast growth factor is preferably FGF2, insulin, and the culture medium lacks retinoic acid;

[0185] f) a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, preferably retinoic acid alone at a concentration of 300 nM to 800 nM;

[0186] 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 concentration of retinoic acid is 300nM to 800nM;

[0187] h) a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a retinoic acid concentration of 300 nM to 800 nM;

[0188] i) activin and CHIR99021; preferably, activin at a concentration of 1 ng / mL to 8 ng / mL, preferably about 5 ng / mL, and / or CHIR99021 at a concentration of 1 μM to 6 μM, preferably about 3 μM; or preferably, activin at a concentration of 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or CHIR99021 at a concentration of 2 μM to 8 μM, preferably about 4 μM; and / or preferably, activin at a concentration of 6 ng / mL to 30 ng / mL, preferably about 10 ng / mL, and / or CHIR99021 at a concentration of 0.4 μM to 4 μM, preferably about 2 μM; and / or preferably, activin at a concentration of 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or CHIR99021 at a concentration of 0.1 μM to 4 μM, preferably about 1 μM;

[0189] j) Wnt inhibitor, preferably Wnt-C59, bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin and retinoic acid, preferably the concentration of retinoic acid is 300 nM to 800 nM, particularly preferably 500 nM.

[0190] Throughout this disclosure, the articles “a,” “an,” and “the” are used herein to refer to one or to more than one (ie, to at least one) of the grammatical objects of the article.

[0191] As used herein, approximating words such as, but not limited to, "about," "substantially," or "substantially" refer to conditions that, when so modified, are understood to not necessarily be absolute or perfect, but would be considered close enough to warrant designation of the condition as existing by one of ordinary skill in the art. The degree to which the description can vary will depend on how large a variation can be made and still allow one of ordinary skill in the art to recognize that the modified feature still has the desired properties and capabilities of the unmodified feature. In general, but subject to the foregoing discussion, numerical values ​​modified herein by approximating words such as "about" can vary from the value by, for example, ±10%.

[0192] As used herein, the words "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 used in combination with a numerical range of values ​​for an element, the expression "comprising" means that the element is limited to the range, while "comprising" still involves the optional presence of other elements. For example, an element with a range may be subject to an implicit proviso that excludes the presence of the element in amounts outside of the range. As used herein, the phrase "consisting essentially of" requires the specified integer or steps and those that do not materially affect the feature or function of the claimed invention. As used herein, the enclosed term "consisting of" is used only to indicate the presence of the stated elements.

[0193] The invention is further illustrated by the following figures and examples, without limiting the invention to these embodiments.

[0194] Attached photos

[0195] Figure 1 : aSHF and pSHF progenitors express typical markers and form functional cardiac organoids.

[0196] A. Differentiation scheme towards three major cardiac lineages: first HF, anterior second HF (aSHF), and posterior second HF (pSHF).

[0197] B. Real-time qPCR of TBX1 and TBX5 levels in FHF and aSHF progenitors at d3.5 in 2D, 3D and 2D->3D protocols. Fold changes were normalized to housekeeping genes and pluripotency.

[0198] C. RNAscope staining of TBX1 and TBX5 in cryosections of aSHF cardiac organoids at d3.5 showed increased TBX1 expression and loss of TBX5 expression in the 2D->3D approach compared to 3D differentiation.

[0199] D. Volcano plots of differentially expressed genes between FHF and aSHF ancestors (upper) and between aSHF and pSHF ancestors (lower) at d3.5.

[0200] E. Heatmap reveals upregulation of aSHF and pSHF genes in the corresponding differentiation protocols at d3.5. General cardiac genes are more highly expressed in FHF compared to aSHF and pSHF.

[0201] F. RNAscope staining (TBX1, TBX5 and HOXB1) of FHF, aSHF and FHF ancestor at d3.5.

[0202] G. Immunostaining of organoids for aSHF markers (FOXC2) and pSHF markers (FOXF1) (G') and quantification of staining (G") at d3.5 (N=3, n=3-4). Mean ± SD.

[0203] H. Venn diagram showing the degree of intersection between aSHF, pSHF, and FHF for genes upregulated compared to pluripotency.

[0204] Three biological replicates at I.d9.5 showed highly robust and efficient differentiation into FHF, aSHF, and pSHF cardiac organoids.

[0205] Cryosections of FHF, aSHF, and pSHF cardiac organoids at J.d9.5 showed expression of the CM-specific marker MYL7.

[0206] K. Quantification of TNNI1-GFP+ cells in cardiac organoids by flow cytometry on day 9.5. Mean + / - standard deviation. (N=3, n=8)

[0207] L. Representative flow cytometry plots of FHF-, aSHF-, and pSHF-derived CMs using reporter and WT lines.

[0208] 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.

[0209] Figure 2 a / pSHF-derived cardiac organoids differ in morphology and gene expression

[0210] A. Time course of cardiac organoids of the three major lineages from d2.5 to d9.5 revealed delayed cavity formation and delayed TNNI-GFP expression in SHF compared to FHF lineage.

[0211] B. Quantification of changes in cardiac organoid area during differentiation in all three protocols. (N=3, n=32)

[0212] C. and D. Cryosections of cardiac organoids from d2 to d5.5 show delayed cavity initiation (white arrows) and cavity formation (yellow arrows) in the SHF lineage. Immunostaining (C) and quantification of mRNA expression (D) of the proliferation marker Ki67 over time show that aSHFs have higher proliferation at d4.5.

[0213] E. Principal component analysis (PCA) plot of vst using the top 1000 variable genes. VST: variance stabilizing count transformation.

[0214] F. Expression of lineage-specific genes over time. aSHF cardiac organoids express RV-specific genes, while pSHF cardiac organoids express atrial-specific genes.

[0215] G. Volcano plots showing differentially expressed genes in FHF versus aSHF (upper) and FHF versus pSHF (lower) cardiac organoids at d9.5.

[0216] H. Lineage-specific staining of IRX1 (RV marker) and NR2F2 (atrial marker) at d14 and quantification of staining (H'). Each data point represents one organoid. (N=3, n=3-4)

[0217] All scale bars in this figure are 200 mm in length.

[0218] Figure 3 :Optimization of SHF protocol for OFT and AVC cardiac organoids.

[0219] A. Differentiation scheme of aSHF progenitors into RV and OFT cardiac organoids by adding or not adding RA during patterning stage 2.

[0220] B. RNAseq time courses of developing RV and OFT cardiac organoids demonstrate expression of lineage-specific genes.

[0221] C. Global gene expression differences between RV and OFT cardiac organoids at d9.5.

[0222] D. and E. On day 14, OFT cardiac organoids highly expressed OFT markers ISL1 (D) and WNT5A (E) compared to RV cardiac organoids.

[0223] F. Whole mount images of RV, OFT, atrium, and AVC cardiac organoids from three biological replicates at d9.5.

[0224] G. Differentiation scheme of atrial and AVC cardiac organoids using different induction conditions and BMP addition during patterning stage 2 of AVC.

[0225] H. Differentially expressed genes among AVC, FHF and SHF ancestors at d1.5.

[0226] I. Volcano plot showing the most differentially expressed genes in AVC compared with atrial progenitors at d3.5

[0227] J. AVC expression in organoids over time.

[0228] K. AVC cardiac organoids upregulate TBX2 but still express TBX3 compared to atrial cardiac organoids.

[0229] L. Addition of RA and FGF and inhibition of NOTCH and BMP in atrial cardiac organoids from d7.5 onwards resulted in upregulation of chamber markers.

[0230] RV and LV cardiac organoids maintained in published maturation media after M.d7.5 showed an increase in chamber-specific markers. Atrial cardiac organoids maintained in RA, FGF, NOTCHi, and BMPi followed by published maturation media downregulated AVC-specific markers compared to cardiac organoids in CDMI.

[0231] All scale bars in the figures have a length of 200 mm.

[0232] Figure 4: Functional characterization of cardiac organoid subtypes

[0233] A. Bar graph showing the percentage of organoid contraction within 1 minute recorded on both day 6 and day 9.

[0234] B. Quantification of beats per minute (BPM) of different organoid types at both day 6 and day 9.

[0235] C. Quantification of the number of pixels moved during contraction divided by the organoid area (extent of contraction). This is an indicator of how far the edge of the cardiac organoid moves during one contraction. Non-beating organoids were not included.

[0236] AC: All were performed with N=2-7 and for each biological replicate there were 16 technical replicates, generating 80, 65, 48, 48 and 33 organoids for LV, RV, OFT, atrium and AVC, respectively.

[0237] D. RNA expression of HCN4 throughout differentiation quantified by bulk RNA-seq. Each point represents the mean (N=3) and the error bars represent the standard deviation. Lines connect the points to facilitate tracking of trends.

[0238] E. Bulk RNA-seq showing both L-type and T-type calcium channels at D9.5 of differentiation.

[0239] F. Representative calcium signal propagation throughout LV, RV, and atrial cardiac organoids for one beat. Color map at 50% of peak intensity at each pixel.

[0240] Zoomed heatmap of RNA-seq at G.D9.5 showing expression of key ion channels involved in contraction

[0241] H. Patch clamp analysis of cells dissociated from different RV and atrial cardiac organoids. Shown are representative AP curves for each cell type. Top: RV Bottom: atrium.

[0242] I. Action potential duration measured from peak to 90% repolarization (APD90).

[0243] J. Amplitude of AP measured from peak.

[0244] K. Resting membrane potential of each cell type.

[0245] KM: Each point represents the mean of one cell. Bars represent the mean of the cells and error bars represent the standard deviation.

[0246] *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0247] Figure 5 Fusion of developing heart organoids to study electrochemical signal propagation during development

[0248] A. 2D cardiac progenitors were dissociated at d3.5 and mixed to form cardiac organoids of the same or different progenitors.

[0249] B. Different types of cardiac progenitors marked by H2B-GPF or LMNB1-RFP were selected at d7.5, but progenitors of the same type were mixed.

[0250] C. Cardiac organoids maintain their identity upon mixing. RV progenitors continue to differentiate into RV CMs as shown by IRX1 staining.

[0251] D. CDH1 staining of cardiac organoids at d4.5, 24 hours after mixing of different or the same progenitors.

[0252] E. Cardiac organoids can be fused together in different combinations at D3.5 by fusing two or three different compartments together. By doing this using fluorescent green and red lines, we can track exactly how the different compartments interact with each other.

[0253] F. Representative brightfield images of atrial (A), LV, and RV cardiac organoids from confluent cardiac organoids at d6.5.

[0254] G. Representative calcium signal propagation across a 3-chamber cardiac organoid for one beat. The plot is colored by the time it takes each pixel to reach 50% of its peak intensity.

[0255] H. Cardiac organoids maintain their identity after confluence, and all compartments uniformly express the CM marker TNNT2.

[0256] I. Percentage of organoids beating for each confluency type for d6 and d9.

[0257] J. Beats per minute (BPM) for different fusion types are shown on both d6 and d9.

[0258] K. Shows in which cardiac organoid the beat was initiated (indicated by color) as a percentage of all recorded cardiac organoids. For each beat, Mix indicates that the beat was initiated by a different organoid, and None indicates that the fused cardiac organoids did not beat at all, or that there was no interaction between the fused cardiac organoids. Both day 6 and day 9 are shown.

[0259] The timing of cardiac progenitor fusion was then optimized to promote the formation of a shared cavity between cardiac organoids by aggregating FHF progenitors at d1.5 and a / pSHF progenitors at d3.5 and fusing the aggregates 4 h after aggregation.

[0260] M. Representative images of cardiac organoids with three compartments using the protocol depicted in M. The atria are labeled in red, the LV in grey, and the RV in green.

[0261] N. Representative cryosections of triple-fused cardiac organoids using the protocol depicted in M, stained with the pan-cardiac marker TNNT2. Arrows indicate shared cavities between chambers.

[0262] O. Cryosection of two different compartments of a cardiac organoid fused using the protocol depicted in M. The fused cardiac organoids share some cavities indicated by blue arrows and express TNNT2.

[0263] All scale bars in the figures have a length of 200 mm. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0264] Figure 6 : Validation of cardiac organoid subtypes by different KO phenotypes

[0265] A. SL1 KO cardiac organoids show a dramatic reduction in size using the atrial and OFT protocols, and a mild reduction in size using the LV and RV protocols at d9.5.

[0266] B. Cross-sections of cardiac organoids show reduced TNNT2 expression in the RV, atrium, and OFT in ISL1 KO compared to WT.

[0267] C. RNAseq analysis shows misregulated genes in ISL1 KO cardiac organoids compared to WT at d9.5.

[0268] D. OFT ISL1 KO cardiac organoids show identity switching and express the atrial marker NR2F2. Compared with WT, atrial and OFT cardiac organoids in ISL1 KO lines express less TNNT2.

[0269] E. Contraction analysis at d9 showed that atrial WT cardiac organoids had more beats per minute (BPM) compared to atrial ISL1 KO. At d14, atrial ISL1 KO cardiac organoids had a higher contraction rate compared to WT. At d14, OFT ISL1 KO cardiac organoids began to contract, while OFT WT cardiac organoids did not contract. (N=1, n=24)

[0270] F. Global gene expression differences in OFT cardiac organoids using the ISL1 KO line compared to the WT line at d9.5.

[0271] G. Atrial and AVC progenitors in TBX5 KO do not express HOXB1, a pSHF marker, compared to WT.

[0272] H. Representative whole mount images of TBX5 KO and WT cardiac organoids at d9.5 and quantification of cardiac organoid area (H′) (N=3, n=8).

[0273] I. TBX5 KO cardiac organoids downregulate NPPA and TNNT2 in both LV and RV regimes compared to WT. Atrial and AVC TBX5 KO cardiac organoids fail to differentiate into CMs.

[0274] J. RNAseq analysis showed differential expression of chamber-specific genes (NPPA, NPPB) and identity genes in TBX5 KO compared with WT cardiac organoids at d9.5.

[0275] K. Global gene analysis in cardiac progenitors at d3.5 revealed misregulated genes in FOXF1 KO compared with WT.

[0276] L. Time course of atrial and AVC cardiac organoids in FOXF1 KO and WT.

[0277] M. Representative real-time qPCR comparing atrial and AVC progenitors at d3.5 using FOXF1 KO and WT lines. Fold changes were normalized relative to housekeeping genes and pluripotency.

[0278] N. Cardiac organoid size is reduced in FOXF1 KO cardiomyocytes using the LV and AVC protocols compared to WT cardiomyocytes at d9.5. RV and atrial cardiomyocytes in the FOXF1 KO and WT lines were of the same size at d9.5.

[0279] O. Contraction analysis of LV, atrial and AVC cardiac organoids at d6.5 and d9.5 showed reduced BMP in FOXF1 KO compared to WT at d6.5. (N=1, n=16)

[0280] All scale bars in the figures have a length of 200 mm. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0281] Figure 7 A multi-chambered cardiac organoid platform for screening teratogen-induced cardiac defects

[0282] A. Whole mount images of cardiac organoids treated with different concentrations of thalidomide from the beginning of mesoderm induction compared to control cardiac organoids (d9.5). A′. Quantification of cardiac organoid area at d9.5 treated with thalidomide. (N=1, n=8)

[0283] B. Representative real-time qPCR of d9.5 cardiac organoids treated with thalidomide showing misregulation of lineage-specific genes. Fold changes are normalized to housekeeping genes and pluripotency.

[0284] C. Comparison of size and TNNI-GFP expression in d4.5 cardiac organoids treated with different concentrations of Acitretin.

[0285] D. Inefficient CM differentiation and morphological changes in cardiac organoids treated with acitretin.

[0286] E. Representative real-time qPCR of d3.5 and d9.5 cardiomyocyte organoids treated with acitretin. Fold changes were normalized to housekeeping genes and pluripotency.

[0287] F. Whole mount images of cardiac organoids treated with BPA (B), PFO (P) and nanoplastics (NP) and control cardiac organoids (d9.5).

[0288] All cardiac organoids were treated with teratogens from mesoderm induction (day 0) to day 9.5. All scale bars in this figure are 200 mm in length. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0289] Figure 8

[0290] A. Immunostaining of TBX5 and FOXC2 in day 3.5 aSHF cells using 3D and 2D->3D protocols.

[0291] B. SOX2 and EOMES staining after aSHF (d1.5) mesoderm induction in 3D and 2D->3D schemes.

[0292] C. Heatmap of head mesoderm markers for all ancestral groups at d3.5.

[0293] D. Real-time qPCR of TBX1 and TBX5 levels of tested factors important for aSHF development alone and in combination.

[0294] E. Optimization of the aSHF protocol by testing different BMP and RA concentrations during the patterning 2 phase.

[0295] F. Optimization of pSHF testing different factors during patterning 2 phases in 2D and 3D (Cyclo: Cyclopamin, CH: CHIR99021, B: BMP4, I: Insulin).

[0296] G. RNAscope staining of TBX1 and TBX5 in aSHF progenitors during mesoderm induction using different activin concentrations.

[0297] H. TBX5 staining of all three progenitors at d3.5.

[0298] I. Uniform NKX2-5 expression and absence of SOX2+ cells in all progenitors at d3.5.

[0299] J. Low cell density during mesoderm induction leads to uniform TNNI expression at d9.5 in aSHF-derived cardiac organoids. J'. SOX1 / 2+ nuclei in aSHF cardiac organoids using high cell density during mesoderm induction.

[0300] K. Cardiac organoids of all lineages showed very few cells expressing PECAM1 and FOXA2, and lack of COL1A1 and SOX2

[0301] L. 2D 24-well plate endothelial differentiation of all three progenitor populations.

[0302] All scale bars in the figures have a length of 200 mm.

[0303] Fig. 9

[0304] A. Lineage-specific staining of HEY2 (LV marker) in LV, RC, and atrial cardiac organoids.

[0305] B. RV (IRX1) and atrium (NR2F2) specific staining of cardiac organoids derived from the hESC line H9.

[0306] C. Venn diagram showing the degree of intersection between LV, RV, and atrium at d9.5 for genes upregulated compared to pluripotency.

[0307] D. Volcano plot of differentially expressed genes between RV and atrium at d9.5.

[0308] All scale bars in the figures have a length of 200 mm.

[0309] Fig.10

[0310] A. GO term analysis of RV and OFT cardiac organoids at d4.5.

[0311] B. Optimization of RA concentration during patterning phase 2 revealed that RA500 resulted in higher upregulation of RV and chamber-specific genes.

[0312] C. High protein expression of HAND1 and HAND2 in OFT cardiac organoids compared to RV cardiac organoids.

[0313] D. Quantification of changes in cardiac organoid area during differentiation in all five protocols. (N=3, n=32)

[0314] All scale bars in the figures have a length of 200 mm.

[0315] Fig.11

[0316] A. Representative curves of contraction rate tracked using brightfield imaging and MuscleMotion software.

[0317] B. Representative images showing the extent of contraction in different organoid types. Red pixels represent pixels that change during contraction.

[0318] C. Calcium traces show f / f0 over a 30 sec time span, with each cardiac organoid having a distinct beating pattern.

[0319] D. FluoroVolt-AP curves recorded from 3D cardiac organoids of FHF, RV, atrium, and AVC. Each column represents a different cardiac organoid, and a different location of the cardiac organoid is superimposed in each quadrant.

[0320] EG. APD90, APD50, and APD30 from Fluoro Volt in 3-D cardiac organoids. Each point represents one cardiac organoid, and the APD has been averaged across beats and locations in cardiac organoids. Error bars represent standard deviations across cardiac organoid types, N=2n=5 for LV and atrium, and N=1n=3 for RV and AVC.

[0321] H.cAPD90 patch clamp data Figure 4 K. Correction using Fridericia correction method

[0322] I. Figure 4 The beating intervals of cells recorded in K.

[0323] Fig.12

[0324] A. Representative widefield image of ancestral populations 1 day after admixture, showing some separation between different ancestral populations.

[0325] B. Cross-sections of cardiac organoids from each mixing condition, stained for TNNT2.

[0326] C. Differential expression of E-cadherin in control LV, RV, and atrial cardiac organoids over time.

[0327] D. Control conditions for CDH2 and CDH1 staining of LV, RV, and atrial progenitor cardiac organoids at D3.5 of differentiation.

[0328] E. Cardiac organoids were fused together at different times during differentiation. Cardiac organoids at d3.5 or D5.5 were combined. Images were then taken at D9.5. (R = biological replicate). Cardiac organoids fused at D3.5 were more closely connected to each other than at D5.5.

[0329] F. One day after cardiac organoid fusion (day 4.5). The right side has LMN1-RFP-labeled RV and H2B-GFP-labeled FHF, and the left side is the opposite.

[0330] G. Time-lapse of calcium signaling across a three-chambered cardiac organoid.

[0331] H. Time-lapse of contraction of a two-chambered heart organoid.

[0332] I.2 ICC staining of ventricular organoids. LV organoids were stained in grey, RV organoids in green, and atrial organoids in red. Staining for NR2F2, IRX1, and TNNT2.

[0333] All scale bars in the figures have a length of 200 mm.

[0334] Fig.13

[0335] A. Validation of ISL1 KO lines for all protocols at d3.5.

[0336] B. RNAseq analysis showing misregulated genes in ISL1 KO vs WT at d3.5 in all protocols.

[0337] C. WNT5A (OFT marker) was strongly downregulated in OFT ISL1 KO cardiac organoids compared to WT at d14.5.

[0338] D. Analysis of the extent of contraction revealed that atrial ISL1 KO cardiac organoids contracted less compared to WT at d9.5 but had similar contractions at d14.5.

[0339] E. Time course of RV, atrial, and OFT cardiac organoid formation using ISL1 KO and WT lines

[0340] F. Quantification of cardiac organoid area of ​​ISL1 KO and WT cardiac organoids at d3.5 (N=4, n=8-24) and 9.5 (N=2, n=8-24).

[0341] G. RNAseq analysis of TBX5 KO and WT cardiac organoids revealed misregulation of aSHF- and pSHF-specific genes.

[0342] H. Validation of TBX5 KO lines of LV, RV, atrial and AVC cardiac organoids at d3.5.

[0343] I. Representative RT-qPCR of atrial and AVC TBX5 KO cardiac organoids at d3.5 and d9.5 compared to WT. Fold changes were normalized to housekeeping genes and pluripotency.

[0344] J. Validation of FOXF1 KO lines in atrial and AVC cardiac organoids at d3.5.

[0345] K. Area analysis of WT and FOXF1 KO organoids for all protocols at d9.5. (N=3-4 (LV, RV chamber, atrium, AVC) and N=1 (OFT), n=8-16)

[0346] L. TNNT2 expression in FOXF1 WT and KO cardiac organoids at d9.5.

[0347] M. RNAseq analysis of LV RV and atrial cardiac organoids at d9.5.

[0348] N. Representative RT-qPCR of atrial and AVC cardiac organoids from FOXF1 KO and WT. Fold changes were normalized to housekeeping genes and pluripotency.

[0349] All scale bars in the figures have a length of 200 mm. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0350] Fig.14

[0351] A. Whole mount images of cardiac organoids treated with aspirin show no differences in size and TNNI-GFP expression compared to untreated cardiac organoids.

[0352] B. Real-time qPCR of cardiac organoids treated with aspirin showed no obvious gene expression differences compared to control cardiac organoids.

[0353] C. Representative whole mount images of cardiac organoids treated with different concentrations of Acitretin and quantification of cardiac organoid area at d9.5 (H') (N=1, n=8).

[0354] D. OFT TNNI-GFP signal identification showed that at d4.5 and d9.5, the GFP signal in OFT cardiac organoids treated with 5nM and 10nM acitretin was higher than that in untreated cardiac organoids. (N=1, n=8)

[0355] E. Whole mount images and quantification (C') of cardiac organoids treated with all-trans retinol at d9.5 revealed no size differences between cardiac organoids treated and untreated within one protocol, with the exception of LV cardiac organoids treated with 50 nM compared to untreated.

[0356] F. Real-time qPCR shows downregulation of OFT and upregulation of ventricular genes in OFT cardiac organoids treated with all-trans retinol.

[0357] G. Whole-mount images show low CM differentiation efficiency in RV and atrial cardiac organoids when treated with different combinations of plastic residues.

[0358] All cardiac organoids were treated with teratogens from mesoderm induction (day 0) to day 9.5. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0359] Fig.15

[0360] A. Differentiation scheme of three cardiac lineages derived from pSHF: atrium, sinoatrial node (SAN), and atrioventricular canal. Demonstrated that previously described atrial cardiac organoids can be pushed into a more mature state, novel differentiation of the SAN by altering both induction and patterning stages, and showed how atrioventricular canal cardiac organoids can be pushed toward atrioventricular node identity.

[0361] B. Validation of the sinus node protocol showing protein expression of SHOX2 (key SAN marker), NKX2.5 (absent in some parts of the SAN), and HCN4 (key SAN marker)

[0362] C. Heat map showing the bulk RNA expression of SAN at day 3.5 of differentiation, at which time it has a similar gene profile to the atrium.

[0363] D. Heat map showing bulk RNA expression at day 9.5 of SAN differentiation and upregulation of SAN markers compared to atrial protocols.

[0364] E. Contraction rate analysis shows that SANs beat faster than other cardiac organoids and more closely match the rate of the paced heart.

[0365] Fig.16

[0366] A. Immunostaining of NKX2-5 and SOX2 in cross-sections of FHF, aSHF, and pSHF cardiac organoids at day 3.5 (see Figure 8 I).

[0367] B and B'. Optimization of the aggregation density (day 3.5) of FHF, aSHF, and pSHF progenitors resulted in different cardiac organoid formation efficiencies later on.

[0368] C. Beating frequency of LV, RV and atrial cardiac organoids in the 178 / 5hPSC line.

[0369] D. OFT progenitors give rise to α-SMA-, SM22-, and calmodulin-positive smooth muscle cell progenitors.

[0370] Fig.17

[0371] A. Time course of cell counts and cell size in different cardiac organoid subtypes. (E) Expression of lineage-specific genes over time as shown by bulk RNA-seq.

[0372] B. Specific NR2F2 immunostaining in atrial cardiac organoids.

[0373] C. LV, RV and atrial cardiac organoids were formed from the 178 / 5hiPSC cell line.

[0374] D. Overview of cardiac organoid maturation conditions.

[0375] E. Expression of MYL2, NPPA, and NPPB compartment markers during maturation of RV and LV cardiac organoids.

[0376] F. Marker expression during atrial cardiac organoid specification and maturation.

[0377] G. Immunostaining of the cardiac maturation marker MYL2 in mature LV and RV cardiac organoids.

[0378] Fig.18

[0379] A. Global cardioplastic organoid TNNI expression in mature RV and LV cardioplastic organoids.

[0380] B. Increased MYH7 / MYH6 ratio in mature cardiac organoids.

[0381] C. Myometrial structure in mature cardiac organoids, visualized by α-actin immunostaining.

[0382] D. Contraction analysis of mature cardiac organoids.

[0383] Fig.19

[0384] A. scRNAseq of multicompartmental cardiac organoids confirms compartment-specific identities. 10x Genomics scRNAseq of five cardiac organoid subtypes, clustered and showing key markers for each compartment.

[0385] B. scRNAseq reveals compartment-specific identities defined by marker combinations. Key compartment markers were selected to determine marker combinations specific for each compartment.

[0386] C. Application of key compartmental markers to determine clusters shows overlap of different identities and clusters. Example

[0387] Example 1: Generation of cardiac organoids

[0388] Cell lines

[0389] The human H9 (female) ES cell line was provided by WiCell Institute (USA). The WTC iPS cell line (male, skin fibroblast derived) was purchased from Coriell Institute for Medical Research (USA). The reporter cell line of Allen Institute for Cell Science was derived from the WTC cell line and received from Coriell Institute for Medical Research (USA).

[0390] All human pluripotent stem cell lines were cultured in customized in-house culture medium using the E8 culture system (Chen et al. (2011) Nat. Methods 8, 424-429). 0.5% BSA (Europa Biosciences, #EQBAH70), internally prepared FGF2 and 1.8 ng / ml TGFb1 were added to the original E8 mixture (R&D RD-240-B-010). Cells were cultured on vitronectin XF (Stem Cell Technologies, #7180) coated Eppendorf (Eppendorf SE, #0030721.110) or TPP (TPP Techno Plastic Products AG, #92012) tissue culture treated plates and passaged every 2-4 days with about 70% confluence using Try-pLE Express Enzyme (GIBCO, #12605010). The lack of mycoplasma contamination in cells was tested regularly.

[0391] Generation of ISL1, TBX5, and FOXF1 knockout cell lines

[0392] For targets on exon 3 of ISL1, exon 5 of TBX5, and exon 1 of FOXF1, CRISPR / Cas9 multi-guide sgRNA (Synthego) was used to knock out ISL1, TBX5, and FOXF1 in WTC cells (Figure M1I-K). Cells were transfected using P3Primary Cell 4D-Nucleofector X Kit S (Lonza-BioResearch, #: V4XP-3032) and Amaxa4D-Nucleofector (Lonza-BioResearch). After nucleofection, cells were incubated in E8 supplemented with 5 μM Y-27632 (Tocris, #72302) on 6-well plates previously coated with vitronectin XF (StemCell Technologies, #7180). After two days, the medium was replaced with E8 without Y-27632 every other day.

[0393] Once the cells are about 70% confluent, single cell inoculation is performed, and the remaining cells are collected for gDNA extraction. Successful editing is first evaluated at the pool level using agarose gel and Sanger sequencing. Subsequently, single colonies are picked and genotyped to confirm the knockout. Colonies are collected by means of a microscope (EVOS) and transferred to a pre-coated 96-well plate (Corning, Cat#CLS3370) with 150 μL E8 / well, and E8 is supplemented with 5 μM Y-27632 and antibiotic-antimycotic. Genome editing at the pool and colony levels is analyzed using Synthego's online tool ICE (https: / / ice.synthego.com / # / ).

[0394] Cardiac organoid generation

[0395] hPSCs (WTC or H9 line) were seeded at 30-40k cells / well in 24-well plates (TPP, #92024) in E8 medium + ROCKi (5 μM Y-27632, Tocris #1254). All differentiation media were based on CDM containing 5 mg / ml bovine serum albumin (Europa Biosciences, #EQBAH70) in 50% IMDM (Gibco, #21980065) plus 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 culture medium contained BSA, which is important for efficient cardiac organoid generation. 24 hours after seeding in 24-well plates, cells were induced with mesoderm induction medium. Mesoderm induction medium consisted of CDM containing: FGF2 (30 ng / ml, QKine, Cambridge / UK), LY294002 (5 μM, Tocris, #1130), Activin A (specific concentrations for different cardiac organoid subtypes - see below, QKine, Cambridge / UK), BMP4 (10 ng / ml, R&D Systems RD-314-BP-050) and CHIR99021 (specific concentrations for different cardiac organoid subtypes - see below, R&D Systems RD-4423 / 50). After 36-40 hours, cells were dissociated with TrypleE (Gibco, #12605010) and seeded in Corning ultra-low attachment 96-well plates (Corning, #7007) at 15-20k cells / well in Cardiac Mesoderm Patterning Media One consisting of: CDM containing ROCK inhibitors, and 1 μg / ml insulin (Roche, #11376497001) for all schemes except LV plus specific factors depending on the cardiac organoid subtype (see below). After seeding, cells were spun down at 200g in a centrifuge for 4 min. This scheme is called the 2D-3D standard scheme. Alternatively, hPSCs were seeded into Corning ultra-low attachment 96-well plates at a density of 5000 cells / well.Cells are seeded in a 200mL volume containing E8+ROCKi, and collected by centrifugation at 200g for 5 minutes. As another option, 2500 cells / well are directly seeded into induction medium+ROCKi, and collected by centrifugation at 200g for 5 minutes. For these two schemes, as described in the 2D->3D scheme, cells are induced with mesoderm induction medium. These are called 3D schemes. For these two schemes, after 24 hours (or at the 2.5th day), feeder cells with cardiac mesoderm patterning medium-one. For the next two days, the culture medium is replaced with the cardiac mesoderm patterning medium-two consisting of the following and replaced every day: CDM containing the specific factor (see below) depending on the subtype of cardiac organoids. In the following two days, the culture medium is replaced every day with the cardiomyocyte differentiation medium CDM culture medium containing BMP4 (10ng / ml), FGF2 (8ng / ml) and insulin (10μg / ml). For the cultivation of the following days, the culture medium is replaced every other day with the CDM containing insulin (10μg / ml).

[0396] Alternatively, the entire protocol can be completed entirely in 2D by seeding 80,000-170.000 cells / 24 vitronectin-coated wells and adding media on the same timeline as for cardiac organoids. This is called 2D differentiation.

[0397] Mesoderm induction medium (Day 0-1.5)

[0398] For left ventricular (LV-FHF progenitor derived) cardiac organoid differentiation, 5ng / mL of activin and 3μM of CHIR99021 were used. For right ventricle (RV), atrium and outflow tract (OFT) differentiation, 50ng / mL of activin and 4μM of CHIR99021 were used. For AVC, 10ng / mL of activin and 2μM of CHIR99021 were used. For SAN, 50ng / ml of activin and 1μM of CHIR99021 were used. These mesoderm induction conditions result in high (>70%) efficiency, homogeneity and reproducibility of different cardiac organoid subtypes. This stage is characterized by the expression of BRA, EOM, MIXL1, FOXA2 and other early mesoderm markers and the lack of pluripotency and early neural marker SOX2. This culture medium works best for the WTC line.

[0399] Alternative Mesoderm Induction Medium (Day 0-1.5)

[0400] For LV (FHF-derived) cardiac organoid differentiation, use 5 ng / mL of Activin and 1 μM of CHIR99021. For RV (aSHF-derived) and atrial (pSHF-derived) differentiation, use 50 ng / mL of Activin and 1.5 μM of CHIR99021. This medium works best for the H9 line.

[0401] Cardiac Mesoderm Patterning Medium 1 (Day 1.5-3.5)

[0402] For LV (FHF progenitors): 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). Retinoic acid concentration should be low, as higher concentrations (>100 nM) lead to FHF-derived atrial, not ventricular fate. Early specific markers for LV are IRX4 and HEY2, which are characterized in Hofbauer et al., Cell 2021, 184(12):3299-3317.e22.

[0403] For RV (aSHF progenitor): TGF-β inhibitor SB 431542 (10 μM, Tocris, #1614 / 10) and C59 (2 μM) or XAV-939 (5 ​​μM, SelleckChem, #S1180). The use of SB 431542 resulted in reaching the SHF lineage, thus leading to RV, OFT, SHF-derived atrial and AVC fates. The earliest aSHF markers at day 3.5 were TBX1 and FOXC1 / 2, and were shared by RV and OFT.

[0404] For OFT (aSHF ancestor): SB 431542 (10 μM) and XAV-939 (5 ​​μM). Same as for RV at this stage.

[0405] For atria (pSHF progenitors): SB 431542 (10 μM), XAV-939 (5 ​​μM) and retinoic acid (500 nM). The combination of SB and high retinoic acid concentrations resulted in pSHF progenitor identity at day 3.5, characterized by high HOXB1, TBX5 and FOXF1 and the absence of TBX1 or IRX4 expression.

[0406] For AVC and AVN (pSHF / AVC progenitors): SB 431542 (10 μM), XAV-939 (5 ​​μM), retinoic acid (500 nM) and BMP4 (10 ng / ml). At this stage, the combination of SB, high retinoic acid and additional BMP4 results in a pSHF / AVC progenitor identity characterized by high MSX2 and TBX2 expression in addition to standard pSHF markers.

[0407] Sinoatrial Node (SAN) - Similar as for AVC, but with high activin and low WNT activation during the induction step (Activin 50 ng / ml and CHIR99021 1 μM). Optionally, also during Patterning 1: No WNT inhibition, otherwise same as for AVC.

[0408] Cardiac Mesoderm Patterning Medium II (Day 3.5-5.5)

[0409] For 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., supra).

[0410] For RV: C59 (2 μM) or XAV-939 (5 ​​μM), BMP4 (10 ng / ml), FGF2 (8 ng / ml), insulin (10 μg / ml) and retinoic acid (50-500 nM range). The addition of retinoic acid here helps drive RV identity.

[0411] For 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 resulted in a specific OFT identity characterized by high WNT5A, MSX1, BMP4 and RSPO3 expression.

[0412] For atria, 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 SAN, same but without WNT inhibition (no XAV-939) or even WNT activation (CHIR99021). For AVN, same as for AVC, with an additional push in the AVN direction, e.g., using BMP and Sonic Hedgehog signaling ( Fig.15 ). The atria will begin to express NR2F1 / 2 and HEY1 at this stage, and the AVC expresses high levels of TBX2, MSX2, and RSPO3.

[0413] Example 2: Cardiac Organoids

[0414] Example 2.1: Chamber Specialization Solution

[0415] For the atrial specification protocol, day 7 atrial cardiac organoids 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 until day 21, atrial cardiac organoids were transferred to DMEM with low glucose (1 g / L, Sigma Aldrich, #G8644) containing dexamethasone (250 nM, SigmaAldrich, #D4902), indomethacin (50 μM, Sigma Aldrich, #I7378), T3 hormone (4 nM, SigmaAldrich, #T6397) and chemically defined lipid concentrate (1X, Invitrogen, #11905031).

[0416] For the ventricular specialization protocol, day 7 LV and RV cardiotype organoids were transferred to DMEM with high glucose (4,5 g / L, Sigma Aldrich, #G8644) containing IGF2 (25 ng / mL), CHIR99021 (1 μM, R&D Systems RD-4423 / 50) and human insulin (10 ng / mL, Sigma Aldrich, #G8644) until day 14. From day 14 to day 16, LV and RV cardiotype organoids were maintained in DMEM with high glucose (4.5 g / L) containing XAV-939 (4 μM, SelleckChem, #S1180). Finally, from day 14 to day 16, LV and RV cardiac organoids were transferred to DMEM with low glucose (1 g / L, Sigma Aldrich, #G8644) 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).

[0417] Example 2.2: 2D endothelial cell differentiation

[0418] hPSCs were seeded at 100,000 cells / 24 wells in E8 medium supplemented with 5mM ROCK-i, the wells being coated with vitronectin. The next day, for H9 cells, cells were induced with FLyAB and 1-3mM CHIR99021 and incubated for 36-40 hours. In the next two days, the culture medium was replaced with its respective cardiac mesoderm patterning medium 1 for FHF, aSHF, and pSHF. Afterwards, CDM with 200ng / ml VEGF (200ng / ml, Peprotech, #AF-100-20) and 2mM forskolin (Sigma-Aldrich, #F3917) was given for 2 days, and then the cells were cultured in CDM with 100ng / ml VEGF for 1 day.

[0419] Example 2.3: Mixing ancestors

[0420] Cardiac differentiation of different progenitor cell populations (FHF, aSHF and pSHF) was performed in 24-well plates coated with vitronectin until day 3.5 (2D differentiation). Cell populations were labeled using different colored cell lines (WTC: H2B-GFP, WTC: LMNB1-RFP). On day d3.5, progenitor cells were dissociated by adding 200ul Try-pLE Express Enzyme (GIBCO, #12605010) for 3-4min at room temperature. Dissociation was terminated by adding 1mL of CDM containing ROCKi (5mM). After centrifugation at 130g for 4min, the cells were resuspended in CDM containing ROCKi (5mM). Then, the two progenitor populations were mixed by seeding 15,000-20,000 cells per progenitor population into co-developmental patterning medium in ultra-low attachment (corning), which contained C59 (2 μM), BMP4 (10 ng / ml), FGF2 (8 ng / ml), insulin (10 μg / ml), retinoic acid (500 nM) and ROCKi (5 mM). On day 5.5, the medium was changed to cardiomyocyte-specific medium for the next two days.

[0421] Example 2.4: Preparation of multi-chambered cardiac organoids

[0422] Generate cardiac organoids as above until the 3.5th day of differentiation.At the 3.5th day, according to whether to expect double fusion, triple fusion or multiple fusion, the cardiac organoids in development are combined in two different ways (see description below).The culture medium for fusion conditions is a cardiac mesoderm patterning culture medium two based on CDM for LV, RV and atrium, which contains: BMP4 (10ng / ml), FGF2 (8ng / ml), insulin (10μg / ml), C59 (2μM) and retinoic acid (500nM).The cardiac organoids of fusion are fed with cardiomyocyte differentiation culture medium every day for the subsequent 2 days.For the remaining time, feed every other day with the CDM containing insulin (10μg / ml).

[0423] Example 2.5: Generation of multi-chambered cardiac organoids

[0424] Double Fusion

[0425] At day 3.5, the developing organoids are transferred from each well of a 96-well Corning ultra-low attachment plate to a well shared with another desired organoid subtype using a wide opening pipette tip. This can be achieved by any combination of LV, RV or atrial heart-type organoids. For this type of fusion, the heart-type organoids are placed together in a co-developmental patterned culture medium. Alternatively, at day 1.5, LV differentiation can be combined with RV or atrial progenitor differentiation at day 3.5 in a co-developmental patterned culture medium or a cardiac mesoderm patterned culture medium to obtain a multi-chambered heart-type organoid with at least one shared cavity. Importantly, if fused at these early stages, the dual-chamber / multi-chambered heart-type organoids will co-develop and share a cavity. Subsequent fusion (e.g., from day 5.5) will damage the formation of a shared cavity.

[0426] Triple Fusion

[0427] Create a mold (made of silicone) shaped to place early cardiac organoids to be fused in a certain order, as expected in a natural heart (e.g., linear order). Sterilize the mold by covering the mold with 70% ethanol for at least one hour with the UV of the laminar flow turned on. The mold is then coated with an anti-adhesion rinse solution (STEMCELL Technologies, #07010) for a few seconds and immediately washed once with PBS. After washing, the mold is stored in a refrigerator until the day of fusion or immediately used. On the 3.5th day of cardiac differentiation, the cardiac organoids are transferred to the mold using a wide-opening pipette tip. By using a mold, cardiac organoids can be arranged in a desired direction (e.g., first the atrium, then the LV and RV, as in vivo). From the 3.5th to 5.5th day, when the cardiac organoids are fused in the mold, the culture medium is not changed. Early timing improves fusion efficiency and produces better morphology. Depending on the developmental stage, fusion with more developed cardiac organoids may not be complete. On day 5.5, confluent cardiac organoids were transferred back to the 96-well plate and media changes were continued as described above.

[0428] To track which cardiac organoids in the fusion were derived from which cell population, colored cell lines (WTC: H2B-GFP, WTC: LMNB1-RFP) or dyes were used. To this end, cells were stained 1 hour before induction with SP-DiIC18 (3) (Invitrogen, #D7777) to fluoresce at 564 nm or with DiIC18 (5) (Invitrogen, #D12730) to fluoresce at 668 nm.

[0429] Example 2.6: Mold for multi-chambered cardiac organoids

[0430] The embedding mold was designed in Tinkercad and adjusted in diameter and length based on the size of the cardiac organoids on the day of fusion. The file was exported as a .stl file and loaded into the slicer software XYZ Print1.4.0. The negative was printed using clear PLA with 100% infill density and 0.1mm layer height and 215°C nozzle temperature. After printing, the negative was treated with a Heatgun (Bosch Hot Air Blower 1800W) at 550°C to carefully melt the surface of the negative, create a smooth finish and remove the rough surface typical of 3D printing.

[0431] Then use polydimethylsiloxane (PDMS) to cast the positive film. Briefly, 5 ml of curing agent and 45 ml of Monomer (both 184 Elastomer Kit, VWR) was mixed vigorously. The mixture was then spun down to remove air bubbles and used directly.

[0432] In order to reduce the extent of bubbles formed during curing, the mold is cast at low temperature (40 ° C). To this end, the negative is placed in a 10 cm culture dish and slowly covered with 30 ml of liquid PDMS mixture. The negative is then carefully removed from the polymerized PDMS, and the residual PDMS is cut off using a scalpel. The mold is then glued to the bottom of a clean 10 cm culture dish using about 5 ml of PDMS and cured at 40 ° C. In order to sterilize the mold, it is washed in 70% ethanol in a fume hood with UV turned on for about 30 min. In order to locate the cardiac organoid in the mold, the mold is rinsed once with PBS and then coated with an anti-adhesion rinse solution (StemCell Technologies, #07010) to further increase the non-stick properties of PDMS. After coating, the mold is rinsed once with PBS and is then ready for use.

[0433] Example 2.7: Analytical methods

[0434] Cryosectioning

[0435] Cardiac organoids were fixed with 4% PFA in PBS and cryoprotected with 30% sucrose in PBS before embedding. Embedding was performed using OCT cryoembedding medium (Scigen, #4586K1). Embedded tissues were frozen using a metal surface immersed in liquid nitrogen and stored in a -80°C freezer until sectioning on a Leica cryostat. Sections were collected on SuperFrost Plus slides (Thermo Fisher Scientific, #10149870) and kept at -20°C or -80°C until immunostaining.

[0436] Immunostaining

[0437] To remove OCT, fixed specimens were washed in 1X PBS for 15 min. Optionally, tissues were placed in permeabilization solution 0.5% Triton-X100 (Sigma-Aldrich, #T8787) for 5 min to increase antibody permeabilization. Then, the tissue was incubated in a blocking solution (PBS (GIBCO, #14190094) with 4% donkey serum (Bio-Rad Laboratories, #C06SB) and 0.2% TritonX-100 for at least 30 minutes. Subsequently, the specimen was incubated at room temperature for 3 hours or incubated overnight at 4°C in a blocking solution containing the primary antibody. Then, it was washed for 20 minutes in PBS with 0.1% Tween20 (Sigma-Aldrich, #P1379), and then incubated in a blocking solution containing the secondary antibody for 1 hour at room temperature. Finally, the tissue was washed in PBS with 0.1% Tween20. The slides were mounted using a fluorescent mounting medium (Dako Agilent PathologySolutions, #S3023) and covered with a cover slip ( #631-0853VWR)cover.

[0438] RNAscope and in situ hybridization chain reaction (HCR)

[0439] RNAscope was performed using RNAscope Probe-hs-TBX1-C2 (target region: 100-769) and RNAscope Probe-hs-HOXB1-C2 (target region: 528-2015) using the ACDBio (https: / / acdbio.com) manual assay kit according to the manufacturer's instructions. RNAscope Probe-hs-PPIB-C1 was used as a positive control. The probes were designed and manufactured by ACDBio.

[0440] In situ HCR fluorescence was performed using the HCR kit (v.3) purchased from Molecular Instruments (molecularinstruments.org) according to the manufacturer's instructions with the following slight modifications: 100 μg / ml salmon sperm DNA was added to the pre-amplification solution and the amplification solution including the hairpin to reduce non-specific binding. The HCR probe WNT5A (B3) was designed and manufactured by Molecular Instruments.

[0441] Image acquisition and analysis

[0442] Fixed tissue sections were imaged using a spinning disk confocal microscope (Olympus spinning disk system based on an IX3 Series (IX83) inverted microscope, equipped with a Yokogawa W1 spinning disk). Live imaging was performed using an inverted widefield microscope (Axioobserver Z1 equipped with a sCMOS camera (Hamamatsu Orca Flash 4)) for brightfield and fluorescence. Cardiac organoids in 96-well plates were also imaged using a Celigo Imaging Cytometer microscope (Nexcelom Biosciences, LLC).

[0443] Flow cytometry

[0444] Cardiac organoids (8 cardiac organoids per condition) were dissociated at 37°C using 1.5 mL CM dissociation medium (Stem Cell Technologies, #05025) for 7-10 minutes. Dissociation of CM was terminated by adding 7.5 ml of support medium. After centrifugation at 130 g for 4 min, the cells were resuspended in 600 μl of PBS with 0.5 mM EDTA (Biological Industries, #01-862-1B) and 10% FBS (PAA Laboratories, #A15-108). Cells were acquired with FACS LSR Fortessa II (BD) and analyzed with FlowJo V10 (FlowJo, LLC) software. FACS sorting was performed using Sony SH800 CellSorter (Sony Biotechnology).

[0445] RNA extraction and bulk RNA-seq preparation and analysis

[0446] RNA was isolated using a KingFisher device (KingFisher Duo Prime) using a semi-automated in-house RNA bead isolation kit. 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, samples were checked for adequate size distribution using a fragment analyzer (Advanced Analytical Technologies, Inc). The RNA-seq libraries were then submitted to the Vienna Biocenter Core Facilities (VBCF) Next-Generation-Sequencing (NGS) facility for sequencing. Reads were preprocessed using umi2index (Lexogen) to add UMI sequences to the read identifiers and trimmed using BBDuk v38.06 (ref=polyA.fa.gz, truseq.fa.gz k=13ktrim=ruseshortkmers=t mink=5qtrim=r trimq=10minlength=20). Bowtie2v2.3.4.1 was used to compare and remove the reads that were located to the abundant sequences contained in the iGenomes NCBI GRCh38 reference. The remaining reads were analyzed using the genome and gene annotation of the GRCh38 assembly obtained from Homo sapiens Ensembl Release 94. Reads were aligned to the genome using star v2.6.0c, and reads in genes were counted using feature counts (featureCounts) (sub-reads v1.6.2) using strand-specific read counts (-s 1). DESeq2 v1.18.1 was used to perform differential gene expression analysis on the raw counts, and principal component analysis was performed on variance-stabilized, converted count data.

[0447] Real-time quantitative polymerase chain reaction

[0448] The isolated RNA was reverse transcribed into cDNA using a reverse transcription kit (Invitrogen, #18080044) and a Touch Bio-Rad thermal cycler. Quantitative PCR was performed using a GoTaq qPCR master mix 2x (Promega, #A6001) and a Bio-Rad CFX384 real-time thermal cycler. Gene expression values ​​for each sample were obtained in triplicate. The logarithmic fold change of the sample relative to PBGD as a housekeeping gene and the pluripotent stem cell sample for normalization was used.

[0449] Contraction analysis

[0450] Fresh CDMI culture medium was supplemented to the heart-type organoids 1-2 hours before recording. 96-well plates were placed in an environmentally controlled stage incubator (37 ° C, 5% CO2, water-saturated air atmosphere, Okolab Inc, Burlingame, CA, USA). Wide-field phase contrast microscopy (Axioobserver Z1 (inverted), with sCMOS camera, Zeis) was used to image each hole for 30-60 seconds at 100 frames per second. Then MUSCLEMOTION was used to analyze the video; data were read into custom software for calculation of reports. Percent beats are defined by whether the heart-type organoids beat once throughout the recording. By counting the total number of beats in the video, dividing it by the length of the video in seconds, and multiplying by 60 to calculate the number of beats per minute. The degree of contraction is the amplitude given from MUSCLEMOTION divided by the size of the heart-type organoid.

[0451] Calcium transients

[0452] To generate a WTC line expressing the GCaMP6f gene, an AAVS1 integrative construct with a CAG promoter followed by the GCaMP6f sequence was selected (Mandegar et al. (2016) Cell Stem Cell 18, 541-553) and introduced as previously described (Hofbauer, et al. (2021b) Cell 184, 3299-3317.e22).

[0453] The above scheme is used to differentiate cardiac organoids into LV, RV, atrium, OFT and AVC or multi-chamber cardiac organoids. Fresh CDM-I culture medium is supplemented to cardiac organoids 1-2 hours before recording. 96-well plates are placed in an environmentally controlled stage incubator (37 ° C, 5% CO2, water-saturated air atmosphere, Okolab Inc, Burlingame, CA, USA). Wide-field microscopy (Axioobserver Z1 (inverted), which has a sCMOS camera, Zeis) is used to image each hole for 30-60 seconds at 50-100 (optimal 50) frames per second. Light-emitting diodes (LEDs) are used to excite cardiac organoids at 470 ± 10nm. Analysis of signal propagation: Peaks are identified using the entire cardiac organoid analysis pipeline. Only pixels with a maximum intensity higher than the specific threshold of the organoid are considered. The intensity of each pixel is calculated, normalized relative to 1, and each trace is smoothed using a rolling average within 3 frames. The frames in which the pixels reach 50% of the peak intensity are recorded. The first frame in which more than 30 pixels reach 50% of the maximum intensity is defined as the first frame. The last frame in which all pixels except up to 30 pixels reach 50% of the maximum intensity is defined as the last frame. The average position of the largest connected component of the pixels that first reach 50% of the peak intensity is considered to be the starting point of signal propagation. The signal propagation speed of all other pixels is then calculated by dividing the distance between the pixel and the starting point and the frame difference between the frame in which the pixel reaches 50% of the peak intensity and the starting point frame. For each pixel and across all beats, the speeds are averaged together to determine the speed of signal propagation in the organoid. Images of signal propagation are made using the same technique, and each pixel is color coded based on the frame difference. Cardiac organoids were excluded from this analysis for 4 reasons: (1) cardiac organoids did not beat, (2) there was no clear directionality, (3) there were 2 starting points, and (4) if less than 10% of the cardiac organoids were expressing the reporter protein.

[0454] Multi-Electrode Array (MEA):

[0455] Electrophysiological recordings of extracellular field potentials were performed using MEA. BioCAM DupleX (3Brain) and single-hole Accura MEA chips (3Brain) were used. The MEA chip consists of 4096 plated electrodes with a pitch of 60 um, covering an area of ​​3.8 x 3.8 mm.

[0456] The MEA chip reservoir was rinsed with 70% ethanol to sterilize, and then washed 4 times with Mili-Q water. Then, PBS was added, and the chip was left overnight in the case of PBS to enhance connectivity. Next, PBS was removed without complete drying, and the heart-type organoids were carefully placed in the center of the MEA chip using a 200ul wide-bore pipette tip (Thermofisher#2069G) for single heart-type organoids at day 9.6 and for differentiated multi-chamber heart-type organoids between days 12-15. In order to fix their position and maximize the contact area between the heart-type organoids and the chip, the membrane and homemade anchors were placed on top of the heart-type organoids. Finally, 1.5mL of CDM-I was added to the reservoir, and the MEA chip was kept overnight at 37°C in a 5% CO2 incubator to further improve connectivity.

[0457] Recordings were performed using BrainWave 4 software using the cardiac organoid presets. Recordings were performed at 37°C and the entire chip was covered with a black cover to prevent light exposure. Field potential signals from beating cardiac organoids were acquired through a 5 Hz high pass filter and a 1.1 electrode was used as a reference electrode. Waveform stability was confirmed over a period of 5 to 10 minutes to ensure consistency prior to a 5 minute recording.

[0458] Patch clamp recordings from single cardiomyocytes

[0459] Cardiac organoids were dissociated using the STEMdiff Cardiomyocyte Dissociation Kit (Stem Cell Technologies, #05025) according to the manufacturer’s protocol (incubated at 37°C for 10-20 min to completely dissociate organoids) and subsequently seeded at a low density of 15-40K cells on laminin-511E8 fragment (AMSBIO, #AMS.892 011, 0.5 μg / cm 2 )-coated 35 mm tissue culture treated culture dishes (Corning, #430165).

[0460] Cells were maintained at 37°C in a humidified incubator with 5% CO2, and whole-cell patch clamp experiments were performed on single beating cardiomyocytes 4-13 days after plating. Glass micropipettes with a resistance of 1.5-4 MΩ were pulled from glass capillaries (Harvard Apparatus, #BS464-0792) using a Sutter P-1000 Micropipette Puller (Sutter Instrument). The extracellular solution consisted of the following substances (in mM): 140 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES, with NaOH used to adjust the pH to 7.4. The intracellular pipette solution contained the following substances (in mM): 150 KCl, 5 NaCl, 2 CaCl2, 5 EGTA, 10 HEPES, and 5 MgATP, with KOH used to adjust the pH to 7.2. Adopt PATCHMASTER NEXT software (HEKAElektronik GmbH), use HEKA EPC 10USB Quadro (HEKAElektronik GmbH) to obtain data at 10kHz and perform low-pass filtering at 2.9kHz.Spontaneous electrical activity was recorded in current clamp mode and analyzed using customized MATLAB (MathWorks) software.Measure the action potential amplitude from peak to maximum diastolic potential, and calculate the APD value from the corresponding percentage of action potential peak to amplitude repolarization.The parameters of 15-20 consecutive action potentials of each cell were calculated separately, and then averaged.

[0461] Photoaction potential

[0462] Cardiac organoids were dissociated in the same manner as for the patch clamp experiments (see previous section) and seeded at 40k cells per well on cells previously coated with laminin-511E8 fragment (AMSBIO, #AMS.892011, 0.5 μg / cm 2 ) in a 96-well plate (Greiner Bio-One, #655182). The cells were maintained in a humidified incubator at 37°C with 5% CO2 for 7-11 days, and the culture medium was changed every 2-3 days.

[0463] After three repeated washing steps with Hank's balanced salt solution (HBSS, Gibco, #14175-053), the cells were incubated with 0.7 times the manufacturer's recommended amount of the voltage-sensitive dye Fluovolt. TMMembrane Potential Kit (Thermo Fisher Scientific, #F10488)) was loaded with CM monolayer. Loading was performed at room temperature for 30 minutes, and then the cells were washed three times with HBSS. The 96-well plate was then placed in an environmentally controlled stage incubator (37°C, water-saturated air atmosphere, Okolab Inc, Burlingame, CA, USA), and a light emitting diode (LED) was used to record the fluorescence signal at an excitation wavelength of 470 ± 10nm, and the emitted light was collected by a photomultiplier tube (PMT, Cairn Research Ltd. Kent, UK). The fluorescence signal was digitized at 10kHz. The subsequent recording of 20 seconds was analyzed offline using customized MATLAB (MathWorks) software. APD was measured at 30%, 50% and 90% repolarization. The APD value was calculated from the corresponding percentage of repolarization of the action potential peak to the amplitude. Parameters were calculated separately for all recorded action potentials in each hole, and then the average was taken. The number range of action potentials analyzed in each hole is usually between 5 and 20.

[0464] Example 3: Derivation of Cardiac Organoids from aSHF and pSHF Progenitors

[0465] We hypothesized that aSHFs would be exposed to a signaling environment similar to other anterior and dorsal embryonic regions (neurectoderm and head mesoderm), including WNT and TGFb signaling inhibition. By combining these signaling conditions and the 3D cardiac organoid method (Hofbauer et al., Cell 2021, 184(12): 3299-3317.e22), we aimed to develop methods to derive cardiac organoids from the aSHF lineage. The first phase of differentiation consists of mesoderm induction, followed by an aSHF patterning phase using either dual WNT and TGFb inhibition or TGF-β signaling inhibition ( Figure 1 A). After 3.5 days of 3D differentiation, we observed heterogeneous progenitor populations, with one progenitor subset expressing the aSHF markers TBX1 or FOXC2, while another expressed the FHF and pSHF markers TBX5 ( Figure 1 B, 1C, and 8A). To determine the starting point of this heterogeneity, we analyzed an earlier stage of mesoderm induction (day 1.5) and found that only the outer part of the organoids expressed the mesoderm marker EOMES. In contrast, the organoid core still expressed the pluripotency and neural marker SOX2 ( Figure 8B), suggesting that mesoderm is not homogenously induced in 3D. We hypothesized that cells in 2D would receive more equal mesoderm-inducing signals, which results in a homogenous exit from pluripotency and differentiation. When we induced mesoderm in 2D and initiated 3D differentiation only at the patterning stage (day 1.5), cells efficiently exited pluripotency ( Figure 8 B), homogeneously expressed high levels of TBX1 and FOXC2, and no expression of TBX5 ( Figure 1 C, 8A, and 8D). In addition, we did not observe expression of head mesoderm markers in aSHF progenitors at day 3.5 ( Figure 8 C). Thus, the 2D->3D differentiation approach generated a homogenous progenitor population of both FHF and aSHF progenitors.

[0466] In contrast to aSHF, pSHFs were exposed to retinoic acid (RA) signaling in vivo, which activated pSHF regulators (HOXB1+, HOXA1+, TBX5+) and repressed the aSHF expression signature (TBX1+, FOXC2+, SIX1+). Consistently, we observed that addition of RA during the aSHF patterning phase resulted in a shift toward a pSHF identity ( Figure 1 F and 8E). Manipulation of other signaling pathways (SHH, WNT, FGF, and BMP) did not affect major aSHF or pSHF markers ( Figure 8 E and 8F). Consistent with recent in vivo observations, distinct signaling levels during mesoderm induction promote aSHF and pSHF on the FHF lineage ( Figure 8 E). We further analyzed the three ancestral subtypes by RNA-seq, and strikingly, the major markers of FHF, aSHF, and pSHF were among the most differentially expressed genes ( Figure 1 D and E). Compared with pluripotency, 532 genes were specifically upregulated in aSHF cells, 408 were specifically upregulated in pSHF cells, and 1046 were specifically upregulated in FHF cells, and 1417 genes were shared among the three protocols ( Figure 1 H). Notably, key markers of aSHF (TBX1, SIX1, FOXC2) were lowly expressed or absent from FHF and pSHF, whereas pSHF markers (HOXB1, HOXA1, FOXF1) were barely detectable or absent in aSHF and FHF ancestry ( Figure 1 DG″). Mutually exclusive expression of lineage-specific markers TBX1 and HOXB1 observed by RNA hybridization ( Figure 1 F) and immunostaining of FOXF1, TBX5, and FOXC2 ( Figure 1G and 8H) further emphasize the specificity and homogeneity of the ancestral groups. All groups remained positive for the cardiac ancestral marker NKX2-5 ( Figure 8 I) and remained predominantly negative for the pluripotency and neuroectoderm marker SOX2 ( Fig.16 A). Overall, by day 3.5 of differentiation using the cardiac organoid system, we can efficiently and homogenously generate all three major cardiac progenitors.

[0467] FHF, aSHF, and pSHF progenitors generate several different cardiac cell types in the embryo, including CMs and endocardial cells. We have previously shown that FHF progenitors generate chamber-like contractile heart-type organoids that contain CMs and endocardial-like cells (Hofbauer et al., supra). Following this approach, we treated a / pSHF progenitors with BMP, FGF, insulin, and RA, and we inhibited WNT signaling (cardiac patterning 2) ( Figure 1 A), which results in the reproducible formation of contractile heart-type organoids containing a cavity ( Figure 1 I and 1J). In contrast to FHF-derived cardiac organoids, a / pSHF-derived cardiac organoids required higher RA doses at this stage. Efficient a / pSHF differentiation depends on lower seeding densities ( Figure 8 J), because high density leads to inefficient CM differentiation with organoid cores expressing neural markers ( Figure 8 J′). The balanced level of WNT signaling activation during mesoderm induction and the number of cells seeded during cardiac patterning stage 1 help optimize and fine-tune cardiac organoid formation ( Figure 8 J, Fig.16 B, B′). More than 85% of cardiac organoid cells expressed the key CM marker TNNI1 ( Figure 1 K and 1L), and lack expression of endoderm (FOXA2), ectoderm (SOX2), and fibroblast (COLA1) markers ( Figure 8 K). Finally, aSHF and pSHF cells also efficiently differentiated into PECAM1+ endothelial cells in 2D when exposed to VEGF and forskolin after the SHF patterning stage ( Figure 8 L). In conclusion, in vivo-like signaling specializes a / pSHF progenitors into CM and endothelial lineages and allows the formation of cardiac organoids.

[0468] Example 4: Formation of RV and Atrial Cardiac Organoids

[0469] FHF progenitors differentiate early into CMs that form the heart tube, whereas aSHF progenitors proliferate first and differentiate together with pSHF progenitors at a later time point. Therefore, we hypothesized that SHF-derived cardiac organoids would show similarly higher proliferation rates, delayed morphogenesis, and differentiation compared to FHF-derived cardiac organoids. Detailed time course analysis revealed delayed formation of SHF cardiac organoids ( Figure 2 AC), higher aSHF progenitor proliferation rate and Ki67 expression ( Figure 2 C and 2D). a / pSHF cardiac organoids are smaller than FHF cardiac organoids and begin to express the CM marker TNNI1 one day later than FHF-derived cardiac organoids ( Figure 2 A and 2B). Global gene expression confirmed that SHF-derived cardiac organoids showed delayed expression of sarcomere and structural CM genes over time ( Figure 2 E and 2F) and delayed cavitation initiation and formation ( Figure 2 C, white and yellow arrows). Different cardiac organoid subtypes also showed an increase in cell number over time ( Fig.17 A) and increase in cell size ( Fig.17 C). Taken together, the developmental timing of SHF- and FHF-derived cardiac organoids is consistent with the in vivo timing of SHF- and FHF-derived differentiation and morphogenesis.

[0470] We next investigated whether a / pSHF-derived cardiac organoids also follow a developmental trajectory with respect to chamber identity. In vivo, FHF, aSHF, and pSHF give rise to the left ventricle (LV), right ventricle (RV), and atrium, respectively. We tested the specialization potential of a / pSHF compared to FHF progenitors by adjusting the concentration of RA. We observed that aSHF progenitors gave rise to CMs with early RV identity (IRX1+, IRX2+, IRX3+, NPPA+), whereas pSHF progenitors differentiated into early atrial CMs (HEY1, NR2F1, NR2F2)( Figure 2 F). Comparison of global gene expression in FHF-cardiac organoids, aSHF-cardiac organoids, and pSHF-cardiac organoids at day 9.5 revealed that the top differentially expressed genes included ISL1+, IRX1+, and RFTN1+ ( Figure 2 G and 9D), which have been implicated in ventricular identity and physiology. Furthermore, comparison of FHF cardiomyocytes with pSHF cardiomyocytes revealed upregulation of TBX5, NR2F2, and NR2F1, consistent with an early atrial identity ( Figure 2 G and 9D, Fig.17 B). These results were further confirmed at the protein levels of IRX1, NR2F2, and HEY2 ( Figure 2H, 2H′, and 9A). When compared to the pluripotent state, 376 genes were specifically upregulated in RV cells, 645 were specifically upregulated in atrial cells, and 449 were specifically upregulated in LV cells, and 3508 genes were shared between the three regimens ( Fig. 9 C). hESC H9s ( Fig. 9 B. Fig.17 B) and other iPSC lines ( Fig.16 C, 17C) achieved CM sublineage specificity. Interestingly, we observed that the final size of pSHF-derived cardiac organoids was smaller ( Figure 2 A and 2B), which is consistent with the smaller size of the atrium compared to the ventricle. In summary, aSHF progenitors specialize into RV-like cardiac organoids, and pSHF progenitors form atrial-like cardiac organoids, suggesting that early priming of progenitors is important for acquiring distinct chamber identities in the developing heart.

[0471] Example 5: Specialization into OFT, AVC and Chambered Heart Organoids

[0472] In addition to the right ventricle, aSHF progenitors also differentiate into the outflow tract (OFT), which gives rise to the valves and vascular structures of the aorta and pulmonary artery. Abnormalities in these are the most common congenital heart defects. Next, we investigated at which stage signaling dictates compartmentalization of the RV and OFT, hypothesizing that this occurs after aSHF specialization. We observed that higher RA doses promoted the specialization of aSHF to the RV chamber identity ( Fig.10 B), whereas in the absence of luminal markers such as NPPA, the absence of RA signaling promotes OFT markers (WNT11, WNT5A, ISL1, BMP4, RSPO3) ( Figure 3 A-3C and 10B). We confirmed these observations at the levels of WNT5A, ISL1, HAND1, and HAND2 ( Figure 3 D, 3E and 10C). The differences between RV and OFT lineages appeared already at day 4.5 of differentiation, indicating an accelerated differentiation into the RV luminal program compared to OFT ( Fig.10 A). Gene expression profile of OFT shows that the marker WNT5A is already upregulated at day 4.5, and OFT cardiac organoids are generally more mesenchymal and show delayed differentiation and are smaller in size compared to RV cardiac organoids ( Figure 3 F and 10D). OFT cardiac organoids also have the potential to differentiate into smooth muscle cells ( Fig.16 D) Therefore, aSHF progenitors can subsequently be directed into RV or OFT cardiac organoid formation by the presence or absence of RA, respectively.

[0473] In vivo, pSHF-derived CMs make up the majority of the atria and contribute to the AVC, a critical region for the development of valves and pacemaker elements. Precursors of pSHFs are located in different regions of the primitive streak and will migrate out at different times. pSHF precursors that give rise to the AVC migrate early, while atrial pSHF precursors migrate later. Therefore, we hypothesized that the mesoderm induction conditions of these two different pSHF populations would be different. Consistently, moderate levels of activin and WNT activation resulted in higher expression of AVC-specific genes at later time points, while maintaining the pSHF signature. Furthermore, after the mesoderm induction stage, the three different cell populations (FHF, SHF, and AVC) showed an anterior-to-posterior gene expression pattern ( Figure 3 H). Another striking difference between AVC and atrium development in vivo is the high exposure of the AVC region to BMP ligands. Consistent with this, addition of BMPs during the patterning phase upregulates early AVC markers. Finally, as shown by RNA-seq ( Figure 3 I and 3J) and TBX2 protein expression ( Figure 3 K) shows the optimized combination of induction and patterning stages ( Figure 3 G) drives pSHF to specialize toward AVC identity. In addition, throughout the time course, AVC cardiac organoids were smaller than atrial cardiac organoids ( Figure 3 F and 10D). Overall, sub-specialization of pSHF progenitors into atrial or AVC cardiac organoids occurs as early as the mesoderm induction stage, which shows the plasticity of pSHF progenitors.

[0474] During the early stages of cardiogenesis, the atria and AVC have similar gene expression profiles. Subsequently, the atria, LV, and RV initiate an upregulated chamber gene expression program, whereas the AVC and OFT do not. To achieve chamber specification, we combined two well-established CM chamber specification and maturation signaling processes ( Fig.17 D). Importantly, LV and RV cardiac organoids upregulated the chamber markers MYL2, MYL7, NPPB, and NPPA ( Figure 3 M, 17E, 17G). We also observed an overall increase in TNNI expression ( Fig.18 A) Improved sarcomere (fishbone) structure ( Fig.18 B) Increased MYH7 / 6 ration ( Fig.18 C), and surprisingly observed improved contraction behavior / amplitude ( Fig.18 D). However, this approach did not stimulate the atrial chamber program and suppressed atrial identity. We therefore screened for signaling factors that specifically promote atrial chamber differentiation ( Fig.17D). We found that activation of FGF and RA and inhibition of NOTCH and BMP pathways promotes the atrial chamber gene program while downregulating AVC-specific genes ( Figure 3 We then combined this treatment with low glucose medium similar to the treatment for metabolic maturation of ventricular chambers and observed further chamber differentiation ( Figure 3 M, 17F). Overall, we have shown that we can specialize and differentiate cardiac organoid cells into the five major compartmental identities found in the embryonic heart.

[0475] Finally, to confirm the specialization into all five lineages (LV, RV, OFT, atrial, and AVC), we performed single-cell RNAseq analysis in biological replicates (Figure 19). Using unsupervised UMAP clustering, we found that the five cardiac organoid / cardiomyocyte subtype identities were clustered separately ( Fig.19A ). When we assigned well-characterized cardiac compartment markers to these clusters, they overlapped with the expected identities ( Fig.19B , 19C ). These results confirm that we have developed a cardiac organoid platform that contains the identities of all major compartmental lineages found in the human heart.

[0476] Example 6: Functional characterization of five cardiac organoid subtypes

[0477] The heart must function as it develops; therefore, it is essential to also understand early cardiac function during the formation of the different embryonic cardiac compartments. Animal experiments have shown considerable differences in spontaneous contraction (autonomy) and beating frequency between the chambers of the heart. The FHF-derived heart tube and early LV regions begin to contract first and lose autonomy as they mature. In contrast, the atrial regions (developing atria and AVC) begin beating later, maintain spontaneous contractions longer, and lose autonomy only after the heart's pacemaker elements have formed. We hypothesized that these early functional developmental differences could be investigated using fused cardiac organoids, before pacemaker formation and before in vivo human data are available.

[0478] Confluent cardiac organoids are particularly advantageous for investigating contractile dynamics using widefield microscopy. Contractile behavior of compartment-specific cardiac organoids at day 6.5 ( Fig.11 A and 11B) show that 90-100% of the LV, atria, and AVC showed automaticity of beat and greater contraction. In contrast, only 18% of the RV cardiotype organoids contracted spontaneously, just as only 8% of the OFT cardiotype organoids contracted spontaneously at a low degree of contraction ( Figure 4 A and 4C). At day 9.5, the atria and AVC maintained automaticity, whereas automaticity and contractility were reduced in the LV, RV, and OFT ( Figure 4 AC). As in vivo, loss of automaticity is associated with downregulation of HCN4, which encodes a potassium / sodium channel ( Figure 4 D). Interestingly, each cardiac organoid subtype had a distinct beating pattern; the atria and AVC beat very regularly, while the LV and RV beat in regular bursts ( Fig.11 A and 11C). Importantly, these observations were reproducible in both technical and biological replicates. To further understand signal propagation in cardiac organoids, we derived a GCaMP reporter line to track calcium transients ( Figure 4 F). Within a cardiac organoid, the origin of signal propagation varies between beats. We also observed differences between cardiac organoid subtypes, which is supported by expression differences observed in T-type and L-type calcium channels ( Figure 4 E). Overall, each cardiac organoid subtype has distinct contractile and signal propagation profiles.

[0479] Ion channel expression during early cardiac development is relatively uniform and subsequently develops species-specific chamber-specific expression profiles and action potential (AP) shapes. Different cardiac organoid subtypes initially display similar ion channel profiles at day 3.5 and then differentiate based on their chamber specificity at day 9.5 ( Figure 4 G). We used voltage-sensitive dye (FluoroVolt) imaging and patch clamping to characterize how APs of early human CM subtypes from cardiac organoids compare. We validated that APs measured with FluoroVolt were consistent in different regions of cardiac organoids and across cardiac organoid subtypes ( Fig.11 D), with slight variations in the AVC. This could be explained by the functional heterogeneity of the AVC, which has the potential to develop into different structures. By comparing AP length, we observed that the LV, RV, and atria were similar in shape, while the AVC was significantly different ( Fig.11 EG). RV and atrial cardiac organoids were then dissociated for patch clamp analysis ( Figure 4 H). As expected from in vivo, we observed that the AP duration in atrial CM was shorter than that in RV CM ( Figure 4 HI and 11H-I). In addition, the diastolic potential is around -70mV ( Figure 4 J and 4K), close to in vivo CM. In addition, we can measure cardiac field potentials on a multi-electrode array (MEA). In summary, electrochemical signaling in cardiac organoid subtypes is diverse and still immature, providing a system for investigating the developmental electrophysiology of early human cardiogenesis.

[0480] Example 7: Multi-compartment integration of cardiac organoid subtypes

[0481] Embryonic cardiac progenitors specialize in adjacent but separate compartments. After SHF-derived RV and atrial precursors migrate into the heart tube, they self-sort and maintain separate compartments, which is critical for cardiac function. Studying the molecular basis of this sorting is challenging in the embryo. We hypothesized that in vitro-derived a / pSHF and FHF progenitors would have the potential to self-sort as in vivo. Indeed, when we dissociated different subtypes of developing cardiac organoids at day 3.5 and then mixed them ( Figure 5 A), we observed sorting in cardiac organoids over a 24-hour period ( Fig.12 A). In contrast, ancestors of the same subtype do not sort after mixing ( Figure 5 B). We observed the highest extent of sorting of FHF-derived LV and aSHF-derived RV progenitors at day 7.5 ( Figure 5 B and 5C). Sorting and patterning are consistent with differential cadherin expression signatures in distinct ancestor types, similar to in vivo ( Figure 5 D, 12C, and 12D). Notably, the sorted progenitors developed into TNNT2+ CMs and retained the proper luminal identity ( Figure 5 C and 12B), which confirms that the first two stages of differentiation determine lineage identity. This also means that co-differentiation of different ancestry is possible starting from day 3.5. In conclusion, the multi-chambered cardiac organoid platform can be used to further study the sorting mechanisms that separate the major cardiac compartments.

[0482] During development, the LV, RV, and atrial chambers co-develop; however, we lack a multi-chamber model to study this critical stage and process of cardiac morphogenesis. Since cardiac progenitors are already specialized and sorted at day 3.5, we hypothesized that the co-developing cardiac organoids would also remain compartmentalized at this stage but undergo morphogenesis together. When we put together the different cardiac organoid subtypes at day 3.5 ( Figure 5 E), they effectively interacted (fused) after 24 hours ( Fig.12 F), but maintain distinct identities and compartments ( Figure 5 G and 5I). In contrast, co-development of cardiac organoids at day 5.5 did not result in efficient fusion ( Fig.12 E). Only confluent cardiac organoids from day 3.5 contract synchronously ( Figure 5 G), demonstrating that the different cardiac organoid subtypes interact functionally. In the following, we refer to these structures as multi-chambered cardiac organoids. Multi-chambered cardiac organoids can be formed in all combinations, allowing us to study the two-chambered cardiac organoids ( Fig.12I) or three-chambered heart organoids (fusion of atrial, LV, RV) ( Figure 5 FH).

[0483] Directional electrochemical signal propagation occurs early in cardiac development and has not been tracked in the human embryo. Directionality of electrochemical signal and fluid propagation is gradually established, initially in the absence of pacemakers, valves, and septum. The first electrochemical signal appears in the differentiating FHF / LV. Once the atrial region develops, it paces the other regions, which ensures unidirectional signal movement and flow from the atria to the RV and OFT via the LV. We first measured calcium signal propagation and tracked signal propagation on MEAs in a multichambered heart organoid system to investigate whether it recapitulates this process. We found that each beat originated from only one location and then propagated throughout the multichambered heart organoid ( Figure 5 G and 12G-H), which ensures unidirectional flow of signal propagation. At day 6.5, most beats originate from the LV ( Figure 5 K), which involves pacing the RV, which does not beat independently ( Figure 5 I). We validated these observations in different multi-chamber heart organoids and showed that multi-chamber heart organoids paced by LV maintained the same beating frequency as LV heart organoids ( Figure 5 J). Interestingly, at day 6.5, the pacemaker potential appeared to be limited, as the LV region was unable to pace the three-chamber organoids, and the atria were unable to pace the multi-chamber organoids ( Figure 5 I and 5L). However, as multichambered cardiac organoids developed to day 9.5, the signal was almost exclusively derived from the atrial region in all combinations ( Figure 5 K). Thus, we have demonstrated that multichambered cardiac organoids provide a unique tool for deciphering the ontogeny of electrochemical signal propagation throughout the co-developing cardiac chambers.

[0484] As the heart chambers develop together, they initially share a lumen, followed by septation and valve formation. Therefore, we further optimized the co-development of a shared lumen in multi-chambered heart organoids. Strikingly, when we combined developing FHF, aSHF, and pSHF heart organoids before lumen formation ( Figure 5 L) (day 1.5 for FHF / LV and day 3.5 for aSHF / RV and pSHF / atrium), they co-develop a common cavity and still retain their identity ( Figure 5 LO). In summary, we developed a human compartment-specific and multi-chamber platform to comprehensively dissect the earliest aspects of human cardiogenesis.

[0485] Example 8: Mutations lead to compartment-specific defects in cardiac organoids

[0486] Mutations in cardiac transcription factors (TFs) often lead to compartment-specific congenital defects in cardiac development. To genetically validate the cardiac organoid compartmental platform, we generated knockout (KO) hPSC lines for important cardiac TFs (ISL1, TBX5) known to cause compartment-specific defects in vivo. In addition, we used our system to investigate the KO effects of the TF FOXF1.

[0487] ISL1 is a prominent TF whose disruption causes severe cardiac malformations in the OFT and RV, partial defects in the atria, and lethality in mice at E10.5. To investigate phenotypes in a human cardiac organoid compartment platform, we developed ISL1-KO hPSCs ( Fig.13 A) and differentiated them into LV, RV, atrial, and OFT subtypes ( Figure 6 A). Misregulation of gene expression is already visible in ISL1 KO at day 3.5, as seen by lower levels of the key cardiac TFMEF2C, NKX2-5 and MYOCD, suggesting a slower differentiation progression ( Fig.13 B). We saw the most dramatic changes in gene expression in OFT cardiac organoids, where HAND2 and BMP4 were downregulated, and TBX5 was upregulated. In atrial cardiac organoids, the key pSHF marker HOXB1 was downregulated ( Fig.13 B). Time course analysis from days 2.5 to 9.5 revealed that, starting from day 4.5, ISL1 KO cardiotype organoids displayed the most severe morphological changes in OFT and atrial cardiotype organoids, while the effects on RV and LV cardiotype organoids were less visible ( Fig.13 E). At day 9.5, significant size differences were present in all cardiac organoid subtypes ( Figure 6 A and 13F). Morphological defects at day 9.5 in the KO were also reflected in the misregulation of different cardiac organoid subtypes of NPPA, NPPB, NR2F2, HEY1, RSPO3, WNT5A, and MYL7 ( Figure 6 C). CM differentiation is less efficient in all lineages except LV ( Figure 6 B). Specifically, OFT and atrial KO cardiac organoids displayed regions devoid of TNNT2 expression, however, despite delays in differentiation and contraction, atrial cardiac organoids maintained their identity ( Figure 6 BD and 13D). In contrast, the master regulator of OFT was misregulated, which resulted in a global gene expression shift from OFT identity to atrial identity ( Figure 6This was also confirmed by contraction analysis of ISL-KO OFT cardiac organoids, which acquired atrial-like beating behavior at day 14.5 ( Figure 6 E).

[0488] Another prominent cardiac TF is TBX5, which is a key regulator in FHF and pSHF progenitors and is responsible for promoting chamber gene expression programs. Disruption of TBX5 leads to atrial and ventricular septal defects, conduction defects, and mutations that cause Holt-Oram syndrome in humans. In TBX5 KO ( Fig.13 H), we observed that HOXB1 expression was attenuated in atrial and AVC cardiac organoids at day 3.5 ( Figure 6 G and 13G). Global gene expression analysis revealed that a series of aSHF markers (TBX1, FOXC2, FGF10) were upregulated in TBX5 KO atrial cardiac organoids ( Fig.13 G), and TBX2 / 3 were downregulated in AVC cardiac organoids at day 3.5 ( Fig.13 I). In addition, TBX5 KO cells that differentiated into the FHF lineage showed upregulation of HAND2, GATA4, and ISL1 ( Fig.13 G). In contrast, no major defects were detected in aSHF-derived cardiac organoids, consistent with the in vivo findings ( Fig.13 G). Consistent with these results, at day 9.5, we observed the most severe morphogenetic phenotypes in LV, atrial, and AVC cardiac organoids, whereas RV cardiac organoids showed only a slight decrease in size ( Figure 6 H and 6H′). Atrial and AVC cardiac organoids failed to differentiate into CMs, as seen by TNNT2 deficiency ( Figure 6 I). LV and RV cardiac organoids showed inefficient CM differentiation and downregulation of the chamber-specific marker NPPA ( Figure 6 I). In both RV and LV cardiac organoids, all TBX5 KO cardiac organoid subtypes showed significant defects in the expression of luminal markers such as NPPA / B, IRX3 / 4 / 1, HEY2 and upregulation of the non-luminal marker TBX2 ( Figure 6 J). Overall, TBX5 KO displayed specific phenotypes at different stages; LV, atrial, and AVC cardiac organoids were already affected at the progenitor stage and failed to form CMs, whereas LV and RV cardiac organoids displayed mild phenotypes at the CM specification stage when chamber-specific genes were downregulated.

[0489] The Forkhead box transcription factor (FOXF1) is a specific regulator of the pSHF lineage. Disruption of FOXF1 primarily results in atrial septation defects, but mutant mice die by E8.0 due to defects in extraembryonic mesoderm. The widespread expression of this TF in early mesoderm is consistent with its possible role in establishing cardiovascular progenitor identity in early lateral mesoderm. We analyzed all isoforms ( Figure 6 FOXF1KO cardiac organoids in N Fig.13 J), and severe morphological phenotypes were observed in atrial and AVC cardiac organoids starting from day 3.5 ( Figure 6 L). Major pSHF markers (HOXB1, OSR1) and AVC markers (TBX2, TBX3) were downregulated ( Figure 6 K and 6M), consistent with a failure of pSHF specification. LV cardiac organoid KO showed downregulation of TBX5, whereas RV cardiac organoids showed upregulation of TBX1 ( Figure 6 K). At day 9.5, we observed morphological phenotypes in LV and AVC KO cardiac organoids ( Figure 6 N and 13K). Interestingly, atrial cardiac organoids transformed to a more ventricular identity (IRX1+, IRX4+, HEY2+, NRF2F2-)( Fig.13 M and 13N), while AVC cardiac organoids failed to differentiate efficiently ( Fig.13 L). As expected, we observed no phenotype in RV, except for the downregulation of NPPA observed in all subtypes ( Fig.13 L and 13M). A less severe phenotype was seen in LV heart organoids, where genes involved in cardiac contraction (ENO1, ENO2) were downregulated ( Fig.13 M), which was confirmed by functional assays in which KO LV cardiac organoids displayed a lower beating rate ( Figure 6 O). KO atrial cardiac organoids also displayed a lower beating rate, whereas AVC cardiac organoids did not contract ( Figure 6 O). In conclusion, the cardiac organoid platform can be used to dissect stage-specific and compartment-specific genetic heart defects.

[0490] Example 9: Multi-chambered cardiac organoid platform for screening teratogen-induced cardiac defects

[0491] In addition to genetic causes, congenital heart defects are often induced by teratogens (e.g., drugs, toxins, metabolites). However, screening of compartment-specific teratogenic effects in efficient, high-throughput, and easily quantifiable human systems is lacking. To test the teratogenic suitability of confluent cardiac organoids, we first identified a non-teratogenic factor (aspirin, 30 μM high dose) as a negative control. We observed neither morphological differences nor significant gene expression differences in the cardiac organoid system ( Fig.14 A and 14B). Next, we tested thalidomide, a known teratogen in humans but not in rodents. Thalidomide binds to TBX5 and causes severe cardiac septal defects, downregulation of chamber markers, and limb malformations. We used a high-throughput platform to dissect the effects of thalidomide at different concentrations (0.1 μM, 1 μM, 10 μM compared to the human therapeutic plasma concentration of approximately 2.4 μM). We observed dose-specific effects in different compartments, with the most pronounced effect on AVC formation, which had not been detected previously ( Figure 7 A and 7A′). Meanwhile, RV and OFT cardiac organoids were less affected ( Figure 7 A and 7A′). Gene expression profiles of the treatments revealed downregulation of the compartment-specific marker NPPA and dosage-specific misregulation of compartment identity markers (NR2F2, IRX1 / 4) in all lineages except RV and OFT ( Figure 7 B) These results demonstrate and validate that the multicompartmental platform can discern early compartment-specific effects of teratogenic drugs in the human system and correlate these with defects observed in patients.

[0492] Another class of compounds known to induce congenital defects are retinoid derivatives used in leukemia, psoriasis, acne creams, etc., which lead to defects in AVC and OFT derivatives. Since RA plays a crucial role in vivo and in our cardiac organoid system, we hypothesized that the cardiac organoid compartmental platform would allow stage-specific dissection of the underlying mechanisms. We tested acitretin and isotretinoin and observed profound compartment-specific and stage-specific effects at very low doses (e.g. 5nM acitretin in atrium; 1nM isotretinoin in LV). OFT, atrium, and AVC cardiac organoids had defects in specialization, patterning, and morphogenesis ( Figure 7 C, 7D, 14C, 14C′). Surprisingly, when trans-retinol was used, we only observed severe morphological effects on OFT cardiac organoids, while all other cardiac organoid subtypes were unaffected ( Fig.14E and 14E′). In OFT cardiac organoids, retinoids caused downregulation of OFT genes (WNT5A, MSX1, ISL1) and upregulation of ventricular and chamber genes (IRX1, IRX4, NPPA), but not atrial genes ( Figure 7 E and 14F). In addition, OFT cardiac organoids treated with retinoids differentiated into CMs earlier ( Figure 7 C and 14D). Taken together, we further validated the cardiac organoid compartmental platform and used it to dissect drug-specific effects on different compartments with high sensitivity.

[0493] Different plastic residues are an overlooked but ubiquitous class of compounds in our environment with possible teratogenic effects. The in vivo effects of plastic residues are difficult to demonstrate because, until now, we do not have a validated system to reveal specialization and morphogenesis defects in the human heart. Here, we used our cardiac organoid compartmental platform to investigate the impact of different combinations of plastic residues (BPA, PFO, and nanoplastics). After treatment with plastic residues, we observed a severe delay in RV cardiac organoid specialization and inefficient RV and atrial cardiac organoid differentiation, while LV and AVC cardiac organoids were unaffected ( Figure 7 F and 14G). This highlights that we have a comprehensive system for testing the teratogenic effects of ubiquitous environmental compounds on the developing human heart.

[0494] discuss

[0495] We have developed a signal transduction-controlled cardiac organoid platform that represents all the major compartments of the embryonic heart. In this system, three different ancestral groups (aSHF, pSHF and FHF) produce five major cardiac compartments (LV, RV, OFT, atrium and AVC) respectively or in combination, which simulate the selected aspects of early human heart development. 2D to 3D differentiation methods ensure homogeneous ancestral specialization at an early stage, which reduces heterogeneity and increases robustness, which remains a challenge in the field of organoids. Therefore, the platform is efficient, reproducible, works with a variety of cell lines, can be screened in high-throughput applications and is versatile, including single compartment or multi-chamber cardiac organoids.

[0496] In vivo, during the mesoderm induction in the primitive streak and as mesoderm cells migrate from this time and occupy a determined position in the cardiogenic region at different times, the dosage and timing of signal transduction have driven the specialization of these pedigrees.Consistently, we found that specific activin / Nodal and WNT signal transduction activation levels drive specialization into different SHF, AVC and FHF ancestors.After mesoderm induction, inhibition of TGF-β signal transduction makes it possible to determine the SHF pedigree fate, which is consistent with the signal transduction environment in the anterior region of the embryo.The specific combination of mesoderm induction and patterning signals allows the identity, dynamics and late stage functions of the heart pedigree in simulation development.For example, these two SHF pedigrees all show delayed cavity formation and differentiation into CM, and compared to FHF, aSHF is more epithelial and highly proliferative.Contrast with aSHF and consistent with in vivo observations, pSHF covers a more diverse batch of induction and patterning conditions, which results in AVC or atrial phenotypes. The contractile differences and kinetics of the atrium, AVC, LV, RV, and OFT cardiac organoids also reflect early in vivo-like functions, as the pacemaker is still absent at this developmental stage. Finally, by virtue of their different induction and patterning stages, confluent cardiac organoids allow for the dissection of progenitor and compartmental sorting mechanisms as well as chamber interactions.

[0497] We found that the role of RA signaling in the specialization of different lineages is more complex in terms of dosage and timing than previously thought. Lack of RA signaling is a feature of initial aSHF specialization and later OFT differentiation. Early on, LV specialization requires relatively low levels of RA, and atrial specialization requires high levels of RA. However, timing is also important, as aSHF RV specialization requires high levels of RA signaling at later stages. The critical role of RA signaling dosage was confirmed by teratogenic screening experiments, which showed a strong effect of retinoids on differentiation speed, specialization efficiency and direction, morphogenesis, and physiology leading to compartment-specific defects.

[0498] As is well known, the interaction between the cardiac lineages during the earliest stages of cardiac development is difficult to analyze in embryos and cannot be obtained in early human embryos, and the stage includes cardiac mesoderm specialization, morphogenesis and functional differentiation. However, this aspect is to understand the impact of mutations and teratogens on early cardiac development and how they cause the defects of specialization, morphogenesis and contraction signal propagation to cause embryo failure. For example, it is not clear how different compartments are sorted and kept separated, which can now be solved using a multi-chamber heart type organoid platform. Another key and neglected aspect of cardiac development is the ontology (ontology) of contraction signal propagation through different early stages (20-30 days) of human heart development. This is particularly important for understanding the heart reasons of embryo failure, which have been attributed to wrong specialization and morphogenesis, but may also be caused by early contraction signal propagation defects between chambers.

[0499] Confluent cardiac organoids allow us to comprehensively and systematically dissect uncharacterized mutations in regulatory elements (such as enhancers), environmental factors (such as pollutants), diet, and the complex interactions between genetic and environmental factors.

Claims

1. A cardiac tissue model comprising cardiac tissue having at least one internal cavity or central chamber, 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, wherein the central chamber can be shared by at least two different cardiac tissues, and wherein the at least two different cardiac tissues have electrophysiological or calcium signaling connections and / or the ability to propagate tissue contraction and are adjacent.

2. The cardiac tissue model of claim 1, wherein The left ventricular tissue comprises at least 60% cardiac cells selected from the group consisting of 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 contains at least 60% cardiomyocytes; The sinoatrial node tissue comprises at least 60% cardiomyocytes; and / or The atrioventricular node tissue contains at least 60% of myocardial cells.

3. The cardiac tissue model of claim 1 or 2, wherein the interior cavity or central chamber 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 wherein the volume of the interior cavity or central chamber is not directed into a main blood vessel. The cardiac tissue model of any one of claims 1 to 3, having a size of 0.3 mm to 50 mm in its largest dimension.

5. The cardiac tissue model according to any one of claims 1 to 4, wherein: 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; 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; 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 the expression of one or more expression markers selected from TBX3, NR2F1 and NPPA; 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; 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 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.

6. The cardiac tissue model of any one of claims 1 to 5, wherein the size of the internal cavity or central chamber at its largest dimension is at least 30% of the size of the cardiac tissue model at its largest dimension.

7. A method for generating a cardiac tissue model according to any one of claims 1 to 6, comprising generating at least two different cardiac tissues in vitro, wherein the different cardiac tissues are selected from left ventricular progenitor first cardiac area tissue, right ventricular / outflow tract progenitor second cardiac area tissue, right ventricular progenitor second cardiac area tissue, atrial progenitor second cardiac area tissue, outflow tract progenitor second cardiac area tissue, atrioventricular canal progenitor second cardiac area tissue, sinoatrial node progenitor second cardiac area tissue and atrioventricular node tissue, and fusing the at least two cardiac tissues, The fused tissue model is cultured and allows calcium signaling connections between the different cardiac tissues, the ability to propagate tissue contractions, and / or central chamber formation.

8. The method of claim 7, wherein the different cardiac tissues have been cultured and differentiated from pluripotent cells, and wherein the fusion is on culture days 1 to 7 from the pluripotent stage; preferably, the right ventricle / outflow tract progenitor second cardiac zone tissue, the right ventricle progenitor second cardiac zone tissue, the atrial progenitor second cardiac zone tissue, the atrioventricular progenitor second cardiac zone tube tissue, the sinoatrial node progenitor second cardiac zone tissue and / or the atrioventricular node tissue are fused on culture days 2 to 5; or preferably, the left ventricle progenitor first cardiac zone tissue is fused when expressing the expression markers TBX5 and / or HAND1; preferably, the right ventricle / outflow tract progenitor second cardiac zone tissue or the right ventricular progenitor second cardiac zone tissue is fused when expressing the expression markers Preferably, the second cardiac zone tissue after the atrial progenitor is fused when expressing the expression markers TBX1, FOXC1 and / or FOXC2; preferably, the second cardiac zone tissue after the atrioventricular canal progenitor is fused when expressing the expression markers TBX3, FOXF1 and / or HOXB1; preferably, the second cardiac zone tissue after the sinoatrial node progenitor is fused when expressing the expression markers SHOX2, TBX3, HCN4, ISL1 and / or GJC1; and / or preferably, the atrioventricular node tissue is fused when expressing the expression markers TBX3, TBX5, KCNE1, HCN4 and / or GJC1.

9. The method according to claim 7 or 8, wherein one of the at least two different cardiac tissues is left ventricular progenitor first heart zone tissue, and generating the left ventricular progenitor first heart zone tissue comprises differentiating mesodermal cells into left ventricular progenitor cells in a culture medium comprising a bone morphogenetic protein, preferably BMP4, a fibroblast growth factor, preferably FGF2, insulin, a Wnt inhibitor, preferably Wnt-C59 or IWP2, and retinoic acid, wherein the retinoic acid has a concentration of 5 nM to 100 nM in the culture medium; wherein one of the at least two different cardiac tissues is right ventricle / outflow tract progenitor second cardiac zone tissue, and generating the right ventricle / outflow tract progenitor second cardiac zone tissue comprises differentiating mesodermal cells into right ventricle and / or outflow tract progenitor cells in a medium comprising a TGF-β inhibitor, preferably SB 431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939; wherein one of the at least two different cardiac tissues is a pre-outflow tract progenitor secondary cardiac zone tissue, and generating the pre-outflow tract progenitor secondary cardiac zone tissue comprises differentiating mesodermal cells into outflow tract tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, and a Wnt inhibitor, preferably Wnt-C59 or XAV-939; wherein one of the at least two different cardiac tissues is atrial progenitor secondary cardiac zone tissue, and generating atrial progenitor secondary cardiac zone tissue comprises differentiating mesodermal cells into atrial tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, preferably at a retinoic acid concentration of 300 nM to 800 nM; and / or wherein one of the at least two different cardiac tissues is atrioventricular canal progenitor second cardiac zone tissue, and generating atrioventricular canal progenitor second cardiac zone tissue comprises differentiating mesodermal cells into atrioventricular canal tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably, the retinoic acid concentration is 300 nM to 800 nM; wherein one of the at least two different cardiac tissues is a second cardiac region tissue after the sinoatrial node progenitor, and generating the second cardiac region tissue after the sinoatrial node progenitor comprises differentiating mesodermal cells into sinoatrial node tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a concentration of 300 nM to 800 nM of retinoic acid; and / or One of the at least two different cardiac tissues is atrioventricular node tissue, and generating the atrioventricular node tissue comprises differentiating mesoderm cells into atrioventricular canal tissue precursor cells in a culture medium comprising a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, a bone morphogenetic protein, preferably BMP4, and retinoic acid, preferably at a retinoic acid concentration of 300 nM to 800 nM; and wherein the secondary cardiac zone tissue after the atrioventricular canal progenitors is further differentiated into atrioventricular node tissue by further maturation in a culture medium containing an activator of sonic hedgehog signaling and / or BMP.

10. The method of any one of claims 7 to 9, wherein fusing the at least two cardiac tissues comprises culturing in a culture medium comprising 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.

11. The method of any one of claims 7 to 10, for screening or testing candidate compounds for their effects on cardiac development and / or function, the method comprising generating a cardiac tissue model according to any one of claims 7 to 10, treating the cells with the candidate compound, and comparing the development of the cardiac tissue model with the development and / or function of a cardiac tissue model not treated with the candidate compound.

12. A method for observing the effects of a suppressed, mutated or overexpressed gene during cardiac development, comprising generating a cardiac tissue model according to any one of claims 7 to 10, wherein the cells have a suppressed or mutated candidate gene or overexpress a candidate gene, and comparing the development of the cardiac tissue model with the development of a cardiac tissue model generated without the suppressed, mutated or overexpressed gene.

13. A method for screening or testing a candidate compound for its effect on cardiac function, comprising treating the cardiac tissue model according to any one of 1 to 6 with the candidate compound, and comparing the function with that of the cardiac tissue model not treated with the candidate compound.

14. A method of treating a cardiac injury in a patient comprising transplanting cells, preferably cardiomyocytes, from the cardiac tissue model of any one of claims 1 to 6 into the injury.

15. A cell culture medium comprising a) a bone morphogenetic protein, preferably BMP4, a 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) TGF-β inhibitor, preferably SB 431542, and 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 culture medium; d) the TGF-β inhibitor is preferably SB 431542 and the Wnt inhibitor is preferably Wnt-C59 or XAV-939; e) the Wnt inhibitor is preferably Wnt-C59 or XAV-939, the bone morphogenetic protein is preferably BMP4, the fibroblast growth factor is preferably FGF2, and insulin, and the culture medium lacks retinoic acid; f) a TGF-β inhibitor, preferably SB 431542, a Wnt inhibitor, preferably Wnt-C59 or XAV-939, and retinoic acid, preferably retinoic acid alone 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 concentration of retinoic acid is 300 nM to 800 nM; h) a TGF-β inhibitor, preferably SB 431542, 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; i) activin and CHIR99021; preferably, the activin concentration is 1 ng / mL to 8 ng / mL, preferably about 5 ng / mL, and / or the CHIR99021 concentration is 1 μM to 6 μM, preferably about 3 μM; or preferably, the activin concentration is 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or the CHIR99021 concentration is 2 μM to 8 μM, preferably about 4 μM; and / or preferably, the activin concentration is 6 ng / mL to 30 ng / mL, preferably about 10 ng / mL, and / or the CHIR99021 concentration is 0.4 μM to 4 μM, preferably about 2 μM; and / or preferably, the activin concentration is 30 ng / mL to 100 ng / mL, preferably about 50 ng / mL, and / or the CHIR99021 concentration is 0.1 μM to 4 μM, preferably about 1 μM; j) Wnt inhibitor, preferably Wnt-C59, bone morphogenetic protein, preferably BMP4, fibroblast growth factor, preferably FGF2, insulin and retinoic acid, preferably the concentration of retinoic acid is 300 nM to 800 nM, particularly preferably 500 nM.

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