Cardiac organoid with stable chamber structure

Abstract

Provided is a method for producing a cardiac organoid with a stable chamber. A cardiac organoid produced using the method is capable of maintaining the stable presence of at least one internal chamber within a period of at least 90 days. Also provided are a cardiac organoid prepared using the method and use of the cardiac organoid.

Description

Heart organoid with stable chamber structure TECHNICAL FIELD

[0001] The present application relates to the field of regenerative medicine and cardiac tissue engineering, in particular to a heart organoid with stable internal chamber, its induction culture method and its application. BACKGROUND

[0002] The human heart is able to play a vital role in regulating nutrient circulation and waste removal without interruption throughout the time span of a lifetime (about 80 years on average). The complexity and importance of the heart make it a great challenge to study the therapeutic mechanism, therapeutic effect or toxic side effect of drug molecules on it through animal models (non-patent literature 1). In the past two decades, although the development of organoid technology has provided a preliminary idea to solve this problem, due to the uniqueness and complexity of the heart in structure (composed of myocardial cells and endocardium, epicardium and other cell types, with stable multi-chamber structure) and function (producing regular spontaneous action potential and systolic and diastolic movement), the construction of in vitro model based on heart organoid still has abnormally high technical difficulty.

[0003] Early studies constructed models that essentially had no chamber structure and at most could only be considered as myocardial cell-based cultures (non-patent literature 2). Other reported chamber models were mostly obtained by coating myocardial cells and other cultured cells on chamber structures built using scaffolds, molds, and protein matrices (non-patent literatures 2, 3, 4). Such artificially built structures do not mimic the natural occurrence of the heart, and their application has great limitations, especially in terms of reliability as models for studying and verifying the therapeutic mechanisms, therapeutic effects, and toxic side effects of drug molecules. In 2021, Mendjan and colleagues reported the construction of the world's first human heart organoid with an internal structure similar to that of a complete heart chamber by regulating the WNT-BMP signaling pathway, which was named "cardioid" (non-patent literature 2). On this basis, Mendjan and colleagues also developed a special protocol for generating different cardiac structures, such as chambered OFT, chambered atrial organoids, and chambered ventricular organoids, and then fused them to form a cardioid with multiple chambers (non-patent literature 5). In addition, Aguirre and colleagues reported in vitro self-organizing chamber models with special cell types, such as the sinoatrial node (see, for example, Volmert, B. et al. Nat Commun 14, 8245 (2023) and Lewis-Israeli, Y. R. et al. Nat Commun 12, 5142 (2021)). However, the organoids with the above chamber structures all present significant limitations in actual use, such as unstable chamber structure, which can only be maintained for at most about a week under in vitro culture conditions, and continued culture will observe the gradual collapse of the internal chamber, which is far from enough for the time span to fully simulate the development and physiological function of the human heart for cardiac modeling (non-patent literatures 2, 5, 6). In addition, problems such as poor self-organizing ability, unclear myocardial and endothelial layer demarcation, and single dependence on the WNT-BMP-HAND1 axis are also observed, which leads to the inability to conduct detailed and continuous analysis using these organoids.

[0004] Wnt / p-catenin signaling pathway and FGF signaling pathway are both known to affect heart development. It has been reported that activation of Wnt pathway (e.g. Wnt3a) at the pluripotent stem cell to mesoderm stage significantly improves the differentiation rate of cardiomyocytes in EB differentiation or 2D differentiation methods in vitro (non-patent literature 15, 16), and inhibition of Wnt pathway after mesoderm formation can improve the cardiac differentiation efficiency of pluripotent stem cells (non-patent literature 17, 18). It has also been reported that FGF can regulate stem cell pluripotency and heart development. For example, when FGF2 is combined with BMP2, it can promote the differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) into cardiomyocytes (non-patent literature 19, 20); FGF2 helps stem cells differentiate into cardiac fibroblasts (non-patent literature 21) and cardioids (non-patent literature 2, 5); FGF2 regulates Wnt signaling by activating the PI3-K / GSK3 pathway to maintain pluripotency (non-patent literature 22, 23). The above-mentioned complex interactions between signaling pathways pose a great challenge to clearly elucidate the key factors affecting heart formation and development.

[0005] So far, it is not clear how to solve the problem of long-term stability of in vitro cultured heart models. SUMMARY

[0006] The present inventors have found, through intensive research and development, that there is a close synergy between the fibroblast growth factor (FGF) signaling pathway and the WNT-BMP axis for the initiation of heart chamber development, chamber formation and stabilization. By activating and / or inhibiting FGF and Wnt-BMP axis at different stages of induction culture, especially by inhibiting the FGF signaling pathway at the key stage of differentiation from mesoderm to cardiac mesoderm, the natural development of the heart can be well simulated, and a heart organoid with an internal chamber can be induced to self-assemble from pluripotent stem cells without the need for additional scaffolds or molds, and the maintenance period of the internal chamber can be significantly prolonged from several days to several months or even longer, thereby providing a heart organoid with an internal chamber that can exist stably in vitro for a long time. Such organoids can serve as scientific research tools to enhance the understanding of heart development biology, and more importantly, have extremely wide application prospects in the fields of bioengineering and medicine, including but not limited to disease modeling, drug screening, screening and evaluation of drug toxicity, heart tissue engineering, regenerative medicine, etc. For example, heart organoids generated from iPSC libraries containing iPSCs derived from healthy individuals and patient individuals of specific indications, respectively, can be used to obtain more personalized test results on the efficacy and toxicity of drugs according to the techniques described herein.

[0007] It is therefore an object of the present application to provide a method of generating a cardiac organoid, the method comprising the steps of: A. activating Wnt signaling in a cell aggregate comprising pluripotent stem cells, inducing the cell aggregate to differentiate into a mesoderm cell aggregate; B. inhibiting FGF signaling in the cell aggregate during a process of stopping the activation of Wnt signaling and allowing the mesoderm cell aggregate to differentiate into a cardiac mesoderm, promoting the cell aggregate to differentiate into a cardiac mesoderm; and C. stopping the inhibition of FGF signaling, allowing the cardiac mesoderm to continue to differentiate into a cardiac organoid.

[0008] In some embodiments of the method of the application, inhibiting FGF signaling comprises contacting the mesoderm cell aggregate with an FGF receptor inhibitor. In some embodiments, the FGF receptor inhibitor is selected from any one of or any combination of an FGFR1 inhibitor, an FGFR2 inhibitor, an FGFR3 inhibitor, or an FGFR4 inhibitor. In preferred embodiments, the FGF receptor inhibitor is an FGFR1 inhibitor. In some embodiments, the FGF receptor inhibitor is an FGFR2 inhibitor. In some embodiments, the FGF receptor inhibitor is an FGFR3 inhibitor. In some embodiments, the FGF receptor inhibitor is an FGFR4 inhibitor. In preferred embodiments, the FGF receptor inhibitor is selected from any one of or any combination of PD173074, PD166866, SSR128129E, AZD4547, Pemigatinib, ASP5878, PRN1371, Infigratinib, Futibatinib, LY2874455, FIIN-2, Derazantibnib, Zoligratinib, ODM-203, FIIN-3, Lucitanib, S49076, or Ferulic acid. In preferred embodiments, the FGF receptor inhibitor is PD173074.

[0009] In some embodiments of the method of the present application, the duration of inhibition of FGF signaling is more than 0.1 hours. In some embodiments of the method of the present application, the duration of inhibition of FGF signaling is not more than the time required for differentiation of a cell aggregate of mesoderm cells into cardiac mesoderm in the absence of said inhibition of FGF signaling. This specific differentiation time varies depending on the starting cells used for generation of the organoid and the induction culture conditions, but can be easily determined by the skilled person in the art, for example from the size of the chamber formed and the time of stable existence, or from the expression of surface markers specific for mesoderm cells and cardiac mesoderm cells, or from the empirical results of reported induction protocols. As non-limiting examples, the duration of inhibition of FGF signaling in the method of the present application can be cited, for example, 0.1 hours to 48 hours, preferably, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours.

[0010] In some embodiments, the duration of inhibition of FGF signaling is the time for which the cell aggregate of mesoderm cells is contacted with the FGF receptor inhibitor. The concentration of the FGF receptor inhibitor used can be determined depending on the inhibitory activity of the inhibitor, as long as it is capable of substantially inhibiting FGF signaling of mesoderm cells. For example, the specific concentration used can be easily determined depending on the IC50 value or the Ki value of the inhibitor, using a gradient dilution method. As non-limiting examples, the concentration of the FGF inhibitor can be 0.1 to 10 μΜ, preferably 0.2 to 5 μΜ, more preferably 0.5 to 2.5 μΜ, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0 μΜ.

[0011] In some embodiments of the method of the application, step B of the method of the application further comprises inhibiting Wnt signaling during the process of differentiating the cell aggregate of mesoderm cells into cardiac mesoderm. Inhibition of Wnt signaling and FGF signaling can be initiated in any order, as long as both occur during the stage of differentiation of mesoderm into cardiac mesoderm, and inhibition of Wnt signaling is at least partially concurrent with inhibition of FGF signaling. In some embodiments, inhibition of Wnt signaling is initiated prior to initiation of inhibition of FGF signaling. In other embodiments, inhibition of Wnt signaling is initiated concurrently with initiation of inhibition of FGF signaling. In other embodiments, inhibition of Wnt signaling is initiated after initiation of inhibition of FGF signaling. One of skill in the art can determine the time of concurrent inhibition of Wnt signaling and FGF signaling based on the size and duration of the lumen formed. By way of non-limiting example, the time of concurrent inhibition of Wnt signaling and FGF signaling can be selected from the group consisting of 1-40 hours, preferably, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 hours.

[0012] In some embodiments of the method of the application, inhibiting Wnt signaling comprises contacting the cell aggregate of mesoderm cells with a Wnt inhibitor. In some embodiments, the Wnt inhibitor is selected from the group consisting of any one of IWP2, XAV939, ICG-001, IWR-1, Capmatinib, PRI-724, Salinomycin, FH535, PNU-74654, LF3, KYA1797K, KY02111, Adavivint, MSAB, Isoquercitrin, NCB-0846, IQ-1, iCQR14, CCT251545, WIKI4, JW55, Resibufogenin, M435-1279, RCM-1, JW74, Zamaporvint, M2912, Lanatoside C, Ginsenoside Rh4, Prodigiosin, Triptonide, IWP-4, or KY-05009, or any combination thereof. In preferred embodiments, the Wnt inhibitor is IWP2.

[0013] In some embodiments, the duration of inhibition of Wnt signaling is the time for which the cell aggregate of mesoderm cells is contacted with the Wnt inhibitor. The concentration of the Wnt inhibitor used can likewise be determined according to the inhibitory activity of the inhibitor, so long as it is capable of substantially inhibiting Wnt signaling of the mesoderm cells. For example, the specific concentration used can be readily determined according to the IC50or Ki value of the inhibitor using a gradient dilution method. As non-limiting examples, the concentration of the Wnt inhibitor can be 1-10 μM, preferably 2-7 μM, more preferably 3-6 μM, e.g., 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 μM.

[0014] In some embodiments of the methods of the application, step B of the methods of the application further comprises contacting the cell aggregates of mesoderm cells with a TGF-β inhibitor, a RA agonist, and / or a BMP pathway agonist. In some embodiments, the TGF-β inhibitor is selected from any one of SB431542, Dorsomorphin 2HCl, Dorsomorphin, LDN-193189, Galunisertib (LY2157299), LY2109761, SB525334, LDN-1931892HCl, SIS3 HCl, RepSox (E-616452), LY364947, Pirfenidone, DMH1, SB505124, GW788388, A-83-01, K02288, SD-208, Vactosertib (TEW-7197), Sulfasalazine, SIS3, ITD-1, Halofuginone, LDN-212854, LY 3200882, ML347, TP0427736 HCl, LDN-214117, TGFβRI-IN-3, PD 169316, 3,3-Dimethyl-1-butanol, Lycopus Extract, AUDA, BIBF-0775, Ginsenoside Rh4, R-268712, or TA-02 or any combination thereof, preferably SB431542. In some embodiments, the RA agonist is selected from any one of Tretinoin, Bexarotene, TTNPB, AM580, Adapalene, Acitretin, Tazarotene, Tamibarotene, SR 11237, Etretinate, BMS493, Palovarotene, All trans-Retinal, CD437 (AHPN), or MSU-42011 or any combination thereof, preferably Tretinoin. In some embodiments, the BMP pathway agonist is selected from any one of BMP4, Activin A, BMP2, SJ000291942, or SB 4 or any combination thereof, preferably BMP4 and / or Activin A. In some embodiments, the TGF-β inhibitor is at a concentration of about 0.1 to 20 µM, preferably 1 to 10 µM, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 µM. In some embodiments, the RA agonist is at a concentration of 10 nM to 10 µM, preferably 100 nM to 1 µM, for example about 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1 µM.In some embodiments, the concentration of the BMP pathway agonist is 0.5 to 500 ng / mL, preferably about 5 to 20 ng / mL, for example about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL. In some embodiments, the BMP pathway agonist is about 5 μΜ SB431542. In some embodiments, the RA agonist is about 500 nM retinoic acid. In some embodiments, the BMP pathway agonist is about 10 ng / mL BMP4 and 10 ng / mL Activin A.

[0015] In some embodiments of the method of the present application, the generated cardiac organoid comprises cardiomyocytes and endocardial cells that together with the cardiomyocytes define at least one chamber, which is capable of being maintained for more than 30 days. In preferred embodiments, the chamber in the cardiac organoid generated by the method of the present application is capable of being maintained for more than 60 days. In more preferred embodiments, the chamber in the cardiac organoid generated by the method of the present application is capable of being maintained for more than 90 days. Herein, "maintained" in the context of a chamber means that the size and / or structure of the chamber has not substantially changed. For example, the size of the chamber in its largest dimension has not decreased by more than 20%, preferably more than 15%, more preferably more than 10%, compared to the organoid just obtained by the method of the present application. Or, for example, the stratified structure, integrity, cellular composition, function and / or activity of the chamber wall has not substantially changed compared to the organoid just obtained by the method of the present application.

[0016] In some embodiments, the generated cardiac organoid further comprises epicardial cells disposed at least on the outer surface of the organoid.

[0017] In some embodiments, the method of the present application further comprises the step of fusing the generated cardiac organoid with epicardial cells. The epicardial cells can be obtained by any known method, for example the method described in Non-patent Literature 2.

[0018] The second aspect of the present application also provides a cardiac organoid prepared by the method disclosed herein.

[0019] The third aspect of the application also provides a cardiac organoid comprising: a cardiac tissue comprising cardiomyocytes, endocardial cells, and optionally epicardial cells, and at least one internal lumen located inside the cardiac tissue, wherein the at least one internal lumen is capable of existing for at least 30 days, preferably at least 60 days, more preferably at least 90 days under in vitro culture conditions. In some embodiments, the epicardial cells comprised by the cardiac organoid of the application are disposed on the outer surface of the organoid. In preferred embodiments, the cardiac organoid of the application comprises an endocardial layer formed substantially of endocardial cells, a cardiomyocyte layer comprising predominantly cardiomyocytes, and an epicardial layer formed substantially of epicardial cells.

[0020] In some embodiments, the cardiac organoid of the application can spontaneously beat under in vitro culture conditions. In preferred embodiments, the spontaneous beating frequency of the cardiac organoid of the application is from about 30 to about 150 beats per minute. In more preferred embodiments, the spontaneous beating frequency of the cardiac organoid of the application is from about 60 to about 90 beats per minute.

[0021] In some embodiments, the cardiac organoid of the application comprises from about 40% to about 80% cardiomyocytes, from about 5% to about 15% endocardial cells, based on the total number of cells of the organoid. In some embodiments, the cardiac organoid of the application comprises from about 40% to about 80% cardiomyocytes, from about 5% to about 15% endocardial cells, and from about 1% to about 15% epicardial cells, based on the total number of cells of the organoid. In some embodiments, the cardiac organoid of the application comprises from about 40% to about 80% cardiomyocytes, from about 5% to about 15% endocardial cells, and from about 1% to about 2% epicardial cells, based on the total number of cells of the organoid. In some embodiments, the cardiac organoid of the application comprises from about 40% to about 80% cardiomyocytes, from about 5% to about 15% endocardial cells, and from about 5% to about 15% epicardial cells, based on the total number of cells of the organoid. In preferred embodiments, the cardiac organoid of the application comprises from about 50% to about 75% cardiomyocytes, from about 7% to about 10% endocardial cells, and from about 7% to about 10% epicardial cells, based on the total number of cells of the organoid. In some embodiments, the ratio of cardiomyocytes to endocardial cells comprised by the cardiac organoid of the application is from about 8: 1 to about 15: 1, preferably from about 10: 1 to about 12: 1. In some embodiments, the endocardial cells comprised by the cardiac organoid of the application are aggregated in the interior of the organoid and define at least one internal lumen. In some embodiments, the internal lumen is closed or partially closed.

[0022] In some embodiments, the cardiac organoid of the present application is a spherical or approximately spherical three-dimensional structure. In some embodiments, the cardiac organoid of the present application has a size of about 0.5 to about 2.5 mm in its largest dimension, preferably about 1 to about 2 mm. In some embodiments, the cardiac organoid of the present application has a size of about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 mm in its largest dimension. In some embodiments, the internal cavity in the cardiac organoid of the present application has a size in its largest dimension of at least about 60% or more, preferably at least about 70% or more, more preferably at least about 80% or more, and even more preferably at least about 90% or more of the size of the cardiac organoid in its largest dimension.

[0023] The fourth aspect of the present application also provides the use of the cardiac organoid of the present application in constructing a disease model. In some embodiments, the disease is selected from a disease or indication associated with abnormal tissue structure and / or physiological function of the heart. By way of non-limiting examples, any one of myocardial infarction, heart failure, coronary artery disease, arterial stenosis, heart attack, abnormal heart rhythm, arrhythmia, heart failure, heart valve disease, congenital heart disease, cardiomyopathy, myocardiosis, pericardial disease, aortic disease, Marfan syndrome, genetic cardiomyopathy, non-genetic cardiomyopathy, cardiac hypertrophy, cardiac dysfunction caused by pressure overload, or damage to heart tissue can be mentioned.

[0024] The fifth aspect of the present application also provides the use of the cardiac organoid of the present application in screening a drug candidate, or a method of screening a drug candidate using the cardiac organoid of the present application. In some embodiments, the drug candidate is a drug candidate for preventing and / or treating a disease or indication associated with abnormal tissue structure and / or physiological function of the heart. In some embodiments, the use or method comprises contacting the cardiac organoid of the present application with a candidate molecule to be screened, and selecting those candidate molecules that are not detected to cause a substantial difference in a property of the cardiac organoid before and after the contacting. In some embodiments, the property can be selected from any one or any combination of external morphology of the cardiac organoid, size (e.g. volume, cross-sectional area, etc.) of the cardiac organoid in its largest dimension, total number of cells comprised by the cardiac organoid, proportion of a particular cell type (e.g. cardiomyocyte, endocardial cell, epicardial cell) in the total number of cells, proportion of a particular cell type to each other, spontaneous beating rate, size (e.g. volume, cross-sectional area, etc.) of the internal cavity, proportion of the size of the internal cavity in the size of the cardiac organoid in its largest dimension, and a particular physiological or biochemical function (e.g. expression level of a particular gene, enzyme or marker, etc.) of the cardiac organoid.

[0025] The sixth aspect of the present application also provides the use of the cardiac organoid of the present application in assessing toxicity of a drug candidate, or a method of assessing toxicity of a drug candidate using the cardiac organoid of the present application. In some embodiments, the toxicity comprises undesirable effects on the histological structure and / or physiological function of the heart. In some embodiments, the use or method comprises contacting the cardiac organoid of the present application with a drug candidate, and measuring the change in a property of the cardiac organoid before and after the contacting. In some embodiments, the property can be selected from any one or any combination of the external morphology of the cardiac organoid, the size (e.g. volume, cross-sectional area, etc.) of the cardiac organoid in the largest dimension, the total number of cells comprised by the cardiac organoid, the proportion of total cells that are of a particular cell type (e.g. cardiomyocytes, endocardial cells, epicardial cells), the proportion of particular cell types to each other, the spontaneous beating rate, the size (e.g. volume, cross-sectional area, etc.) of the lumen, the proportion of the size of the lumen in the size of the organoid in the largest dimension, a particular physiological or biochemical function of the cardiac organoid (e.g. expression level of a particular gene, enzyme or marker, etc.).

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[0052] FIG. 1a is a schematic diagram of a scheme for in vitro differentiation to form a cardioid with a stable lumen;

[0053] Figure lb shows real-time brightfield images of the time course of forming a heart organoid with a chamber. Scale bar is 500 pm;

[0054] Figure lc shows a typical brightfield image at day 15.5, showing pulsatile lumen structures that consistently appeared in three independent biological replicates;

[0055] Figure Id shows a heart organoid frozen section at day 60.5, showing lumen formation and expression of the cardiomyocyte marker TNNT2 (top panel, green), the ventricular marker MYL3 (red), and the atrial marker NR2F2 (bottom panel, green), scale bar is 200 pm;

[0056] Figure le shows results of scRNA-seq analysis, depicting the proportion of cardiomyocytes, atrial cells, and ventricular cells in the heart organoid;

[0057] Figure If shows a heatmap depicting expression patterns of key genes during cardiomyocyte differentiation, VST stands for variance-stabilized transformed counts;

[0058] Figure lg shows an exemplary image of endocardial lining appearing at day 20.5, with the cardiomyocyte marker TNNT2 shown in green and the endocardial layer marker CDH5 shown in red, scale bar is 200 pm;

[0059] Figure lh shows real-time images of the heart organoid taken under tdTomato or brightfield conditions;

[0060] Figure li is a schematic of the three-layer structure of the heart organoid: epicardium, myocardium, and endocardium;

[0061] Figure 2a shows confocal images depicting sarcomere structure in cardiomyocytes of the heart organoid (day 62), scale bar is 10 pm;

[0062] Figure 2b shows electron microscopy images of the heart organoid at day 90.5, with arrows pointing to key structures: S for sarcomere, ID for intercalated disc, and Z for Z-line;

[0063] Figure 2c shows a typical electrocardiogram (trace) and averaged action potential recording of the heart organoid (D100);

[0064] Figure 2d shows features of the action potential observed in the data shown in Figure 2c, RMP stands for resting membrane potential, APD for action potential duration, and APA for action potential amplitude;

[0065] Figure 2e shows the beat rate of the heart organoid at different days, BMP stands for beats per minute, all bar graphs show mean ± SD;

[0066] Figure 2f shows the results of calcium imaging analysis of the change in fluorescence intensity over time after loading the organoids with Fluo-4-AM, F / F0 represents the fluorescence intensity relative to the background level, scale bar is 500 pm;

[0067] Figure 3a shows chamber formation in the cardioids is dependent on PD173074 (PD), scale bar is 500 pm;

[0068] Figure 3b shows a typical brightfield image at day 16.5, showing the solid heart organoid structure consistently appearing in three independent biological replicates without PD addition, scale bar is 500 pm;

[0069] Figure 3c shows frozen sections of cardioids at day 16.5 with or without PD addition, scale bar is 200 pm;

[0070] Figure 3d is a heatmap depicting the expression pattern of key genes during cardiomyocyte differentiation, including cardiomyocyte markers and ion channels, with or without PD addition, VST stands for variance-stabilized count;

[0071] Figure 3e is a PCA plot showing different cell fates with or without PD addition;

[0072] Figure 3f is a Venn diagram showing upregulated or downregulated genes in cardioids at day 3.5, with PD addition as control;

[0073] Figures 3g and 3h show GO and KEGG terms upregulated or downregulated in cardioids at day 3.5, with PD addition as control;

[0074] Figure 3i shows cell distribution in the organoids with or without PD addition. Red represents cells in the PD addition group (+PD), and blue represents cells in the PD non-addition group (-PD);

[0075] Figure 3j is a UMAP plot showing cell cluster annotation based on single-cell RNA-seq data;

[0076] Figure 3k shows the proportion of each cell cluster in the total cells in the PD addition group and the non-addition group;

[0077] Figure 3l is a UMAP scatter plot showing cell cluster annotation based on the expression of different marker genes, the darker the color, the higher the expression level;

[0078] FIG. 4a shows a visualization result of data distribution for illustrating the distribution of cells in 2D and 3D culture samples, in which the cells in the 2D sample are marked in red and the cells in the 3D sample are marked in blue, which helps to visualize the distribution of cells in each sample of the data set;

[0079] FIG. 4b shows a visualization result of cell type annotation, which is a UMAP visualization of cell type annotation for 2D and 3D cultures using single-cell RNA-seq data, different colors represent different cell types;

[0080] FIG. 4c shows the expression of marker genes, which depicts the marker genes of various cell types by UMAP scatter heat map, and the color gradient represents the expression level, and the cell annotation is based on the marker gene expression in different clusters;

[0081] FIG. 4d shows the diversity of cell types, according to the annotation results, the 3D culture shows higher cell type diversity than the 2D group, and the differential genes specific to endocardial and epithelial progenitor cells of the 3D culture are enriched and analyzed;

[0082] FIG. 4e is a violin plot showing the results of gene expression analysis, comparing the expression level distribution range of key genes marking heart maturation under 2D and 3D culture conditions;

[0083] FIG. 5a shows real-time bright field images of heart-like organs with stable cavities treated with different drugs, all test drugs are added to the heart-like organs from day 0, in which aspirin is 30 mM, thalidomide is 10 pg / ml, and acitidine is 50 nM;

[0084] FIG. 5b shows the quantification results of the cavity area of the heart-like organs treated with drugs;

[0085] FIG. 5c shows the beat rate per minute of the heart-like organs treated with drugs;

[0086] FIG. 5d shows real-time bright field images of heart-like organs with stable cavities at day 36.5, which are treated with thalidomide from day 15.5;

[0087] FIG. 6a is a graph showing the temporal changes of heart-like organs with cavities formed using the induction protocol of Non-Patent Literature 2, which is captured in real-time bright field imaging, and the scale bar is 500 pm;

[0088] FIG. 6b shows a frozen section of a heart-like organ at day 6.5 prepared using the induction protocol of Non-Patent Literature 2, and the scale bar is 200 pm;

[0089] FIG. 6c shows the proportions of myocardial cells, atrial cells and ventricular cells in the heart-like organ shown in the UMAP graph;

[0090] Figure 6d is a heatmap showing the gene expression of cardiac structure genes and ion channel genes in the heart-like in day 0 to day 25;

[0091] Figure 6e shows representative brightfield images of two independent biological replicates at day 90.5, scale bar is 500 pm;

[0092] Figure 6f is a confocal image showing the formation of 2D epicardium expressing the marker WT1 at day 9.5 using a reported method;

[0093] Figure 6g is a representative brightfield image showing heart-like with a lumen at day 15.5 derived from H1 and UiPS cell lines, UiPS is urine-induced pluripotent stem cells, scale bar is 500 pm;

[0094] Figure 6h shows frozen sections of heart-like (day 16.5) derived from H1 and UiPS cell lines;

[0095] Figure 7a is a time-lapse immunostaining image of frozen sections showing that the formation of heart-like with a lumen is independent of cell apoptosis and proliferation, scale bar is 200 pm;

[0096] Figure 7b is a heatmap showing the expression pattern of genes associated with proliferation and apoptosis during cardiomyocyte differentiation;

[0097] Figure 7c shows confocal frozen section images of HAND1-KO heart-like (from two cell lines) stained with TNNT2;

[0098] Figure 7d shows confocal frozen section images of NKX2.5 KO heart-like (from two cell lines) stained with TNNT2;

[0099] Figure 8a is a time-lapse brightfield image showing the formation of heart-like with a lumen without the addition of PD173074;

[0100] Figure 8b is a time-lapse brightfield image showing the organoids induced with 1 mM PD166866;

[0101] Figure 8c is a representative brightfield image showing heart-like induced with 1 mM PD166866 at day 17.5;

[0102] Figure 8d is a volcano plot comparing the gene expression profile of the cardiac mesoderm stage (day 3.5) and the ventricle formation stage (day 10.5) with the addition of PD as a control;

[0103] Figure 8e shows the results of gene expression analysis of heart-specific cell types in the cardioids, with the horizontal axis representing days and the vertical axis representing log fold change compared to day 1.5;

[0104] Figure 8f shows the expression levels of different FGFRs at day 0, namely FGFR1, FGFR2, FGFR3 and FGFR4, respectively;

[0105] Figure 9a shows the UMAP visualization of cell type annotation results for scRNA data of 2D cultures, with cell types represented by colors;

[0106] Figure 9b shows the UMAP visualization of cell type annotation results for scRNA data of 3D cultures, with cell types represented by colors;

[0107] Figure 9c shows the UMAP scatter heat map of marker genes of different cell types in 2D cultures, with expression levels represented by color tones;

[0108] Figure 9d shows the UMAP scatter heat map of marker genes of different cell types in 3D cultures, with expression levels represented by color tones;

[0109] Figure 9e shows the results of enrichment analysis of highly expressed genes in atrial cells under 3D culture conditions, compared with 2D culture conditions;

[0110] Figure 9f shows the results of enrichment analysis of highly expressed genes in ventricular cells under 3D culture conditions, compared with 2D culture conditions;

[0111] Figure 9g is a violin plot visualizing the distribution of expression levels of key genes, comparing the expression differences of maturation marker genes in atrial cardiomyocytes between 2D cultures and 3D cultures;

[0112] Figure 9h is a violin plot visualizing the distribution of expression levels of key genes, comparing the expression differences of maturation marker genes in ventricular CMs between 2D cultures and 3D cultures;

[0113] Figure 10a shows typical bright field images of cardioids treated with various drugs at day 20.5, with aspirin at 30 mM, thalidomide at 10 pg / ml, and acitidine at 50 nM;

[0114] Figure 10b shows magnified bright field images of cardioids treated with different drugs at day 1.5;

[0115] Figure 10c shows magnified bright field images of cardioids treated with different drugs at day 3.5;

[0116] Figure 10d shows quantification of chamber area at day 36.5 for heart-like organoids treated with thalidomide starting at day 15.5;

[0117] Figure 10e shows heart rate per minute at day 36.5 for heart-like organoids treated with thalidomide starting at day 15.5;

[0118] Figure 11A shows changes in endothelial marker expression levels during organoid formation (n=2), FPKM represents fragments per kilobase of transcript per million mapped reads;

[0119] Figure 11B shows myocardial (outer) and endothelial (inner) layers in organoids at day 7.5 and day 17.5; scale bar is 200 pm;

[0120] Figure 11C is a heatmap showing gene expression of markers associated with chamber formation and heart circulation during organoid formation from day 0 to day 25;

[0121] Figure 11D shows representative brightfield images of organoids treated with or without 0.5 mM SB431542 for 20 days at day 48.5, and ETS1 knockout heart organoids at day 28.5; scale bar is 500 pm;

[0122] Figures 11E and 11F are chamber area (N=3, N=48) and beat rate (N=3, N=30), respectively, measured for heart organoids treated with or without 0.5 mM SB431542 from day 40.5 to day 48.5;

[0123] Figure 11G is a heatmap showing gene expression of markers associated with endothelial-mesenchymal transition (EndoMT) during organoid formation from day 0 to day 25

[0124] Figure 11H shows representative brightfield images of organoids treated with or without 0.5 mM SB431542 for 20 days at day 48.5;

[0125] Figure 12 is a violin plot showing expression of myocardial maturation related genes in organoids at day 25.5 and day 43.5;

[0126] Figure 13 shows development of exemplary organoids of the disclosure at day 25.5 and 43.5 obtained using Slingshot pseudo-time trajectory analysis;

[0127] Figure 14 shows average motion amplitude at 0 seconds and 1 second after isoproterenol treatment of organoids of the disclosure, scale bar is 170 pm;

[0128] Figure 15 shows the beat rate per minute (left panel, N=3, N=7) and average displacement of contraction activity over time (right panel) of organoids after isoproterenol treatment, respectively;

[0129] Figures 16A and 16B show representative brightfield images (A) and chamber area (B, N=3, N=9) of organoids of the present disclosure before and at 24 and 48 hours after treatment with 5 mM doxorubicin, respectively;

[0130] Figures 16C-16G show cell viability (C, N=3, N=48), LDH levels in the culture medium (D, N=3, N=48), mRNA expression levels of stress marker ANP (E), mRNA expression levels of inflammation-related factors TNFA and IL-6 (F and G) in organoids treated with doxorubicin, respectively;

[0131] Figure 17A shows cell communication in organoids at day 25.5, the upper panel represents signaling pathways with endothelial cells as sender, affecting other cell types such as fibroblasts, atrial cardiomyocytes and ventricular cardiomyocytes, the lower panel represents pathways with endothelial as receiver, the arcs represent different signaling pathways;

[0132] Figure 17B is a probability heatmap showing the interaction of key signaling pathways between different cell types;

[0133] Figures 17C and 17D show signaling pathways from endothelial cells to other cell types and from other cell types to endothelial cells, respectively, the color of the circle represents the probability of communication, the size represents the importance of the pathway;

[0134] Figure 18 is a heatmap showing the probability of interaction of LAMA signaling pathways between various cell types in an exemplary organoid of the present disclosure (A) and a 5-6 week human fetal heart (B).

[0135] Figure 19A is a time-lapse brightfield image showing the development of cardiac organoids under conditions of different concentrations of activin A (AA) and retinoic acid (RA);

[0136] Figure 19B is a representative brightfield image showing cardiac organoids induced with different concentrations of AA and RA at day 25.5;

[0137] Figure 19C is a representative brightfield image showing cardiac organoids induced with different concentrations of AA at day 8.5;

[0138] Figure 19D is a graph showing chamber area of cardiac organoids measured at day 15.5 and day 25.5 induced with different concentrations of AA and RA. DETAILED DESCRIPTION

[0139] Definitions

[0140] As used in the specification of the application, the following words and phrases are generally intended to have the meanings set forth below, except to the extent that the context in which these words and phrases are used indicate otherwise.

[0141] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise. As used herein, the terms "comprises," "comprising," "includes," "including" and the like are specifically intended to be open-ended. These terms mean that a composition or method comprising, including, or including the recited components or steps does not exclude additional components or steps.

[0142] As used herein, the term "about" refers to the usual error tolerance range of a person skilled in the art. For example, ±10% or less, ±5% or less, ±2% or less, ±1% or less, or ±0.1% or less of the described numerical value. A value or parameter described herein as "about" includes the value or parameter itself.

[0143] As used herein, the term "pluripotent stem cell" refers to a cell that has the potential to differentiate into multiple different cell types derived from the ectoderm, mesoderm, and endoderm (e.g., neural cells, cardiomyocytes, muscle cells, etc.) under suitable conditions. Examples of pluripotent stem cells include, but are not limited to, embryonic stem cells and induced pluripotent stem cells.

[0144] As used herein, the term "organoid" refers to a cell mass or aggregate that resembles an organ or a portion of an organ and possesses cell types associated with that particular organ. An organoid typically has the same or similar structural or organizational features as the particular organ and exhibits at least one physiological process or function of the particular organ.

[0145] As used herein, the term "cell aggregate" refers to a structure composed of a plurality of cells that form a three-dimensional (3D) shape rather than growing as a monolayer. In some embodiments, a cell aggregate of the application is an aggregate of multiple layers of cells. In some embodiments, a cell aggregate of the application is spheroid or approximately spheroid.

[0146] As used herein, the term "activation" when used in reference to a signaling pathway refers to the initiation, occurrence, or increase in the level of activity of a certain biological process, physiological response, or molecular event associated with the signal conducted by the pathway. In contrast, the term "inhibition" when used in reference to a signaling pathway refers to the initiation, occurrence, or decrease in the level of activity of a certain biological process, physiological response, or molecular event associated with the signal conducted by the pathway.

[0147] As used herein, the term "mesoderm" refers to one of the three primary layers of cells formed during embryonic development (ectoderm, endoderm, mesoderm), located between the ectoderm and endoderm, with the potential to form a variety of organs and tissues, including but not limited to the heart, skeleton, muscle, urinary system, reproductive system, and circulatory system. Methods and markers to identify mesoderm cells are generally known in the art, as an example, mesoderm can express one or more markers (e.g., at least 3 to 5 markers) selected from the group consisting of: TBXT + , MESP1 + , MIXL1 + , Brachyury, Foxa2, Sox17, Nodal, Eomesodermin, Tbx6, and Wnt3a, among others.

[0148] As used herein, the term "cardiac mesoderm" is an intermediate state in the process of development of various cell types of the heart (such as cardiomyocytes, endocardial cells, and epicardial cells, among others) from primitive mesoderm, representing cells derived from primitive mesoderm that have already specialized with the fate to differentiate into various cell types that constitute the heart. Methods and markers to identify cardiac mesoderm are generally known in the art, see, e.g., Van Vliet P et al., Early cardiac development: a view from stem cells to embryos. Cardiovasc Res. 2012 Dec 1;96(3):352-62; Brade T et al., Embryonic heart progenitors and cardiogenesis. Cold Spring Harb Perspect Med. 2013 Oct 1;3(10):a013847. One of the characteristics of cardiac mesoderm is the expression of markers specific to cardiac mesoderm, including, e.g., one or more of HAND1, Isl1, NKX2-5, and GATA4, among others.

[0149] As used herein, the term "cardiomyocyte" encompasses cells at any stage of cardiomyocyte ontogeny, including primarily cardiomyocyte precursor cells and mature cardiomyocytes, unless otherwise specified. As an example, a cardiomyocyte can express a marker selected from any one or more (e.g., at least 3 to 5 markers) of TNNT2, NR2F2, cardiac troponin I (cTnl), cardiac troponin T (cTnT), sarcomeric myosin heavy chain (MHC), GATA-4, Nkx2.5, N-cadherin, βl-adrenergic receptor (βl-AR), ANF, MEF-2 family transcription factors, creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor (ANF), etc. Further, in the present application, a "cardiomyocyte" can be characterized by beating. Further, a "cardiomyocyte" in the present application can also be characterized by forming a striated structure.

[0150] As used herein, the term "endocardial cell" refers to a cell at any stage of endocardium formation and maintenance, including primarily endocardial precursor cells and mature endocardial cells, unless otherwise specified. As an example, an endocardial cell can express a marker selected from any one or more (e.g., at least 3 to 5 markers) of CD31 (PECAM-1), VE-cadherin, von Willebrand factor (vWF), Connexin 43 (Cx43), Troponin I (cTnl), and CDH5, etc.

[0151] As used herein, the term "epicardial cell" refers to a cell at any stage of epicardium formation and maintenance, including primarily epicardial precursor cells and mature epicardial cells, unless otherwise specified. As an example, an epicardial cell can express a marker selected from any one or more (e.g., at least 3 to 5 markers) of WT1, CD90 (Thyl), Desmin, Vimentin, a-SMA (alpha smooth muscle actin), Fibronectin, PDGFR-a (platelet-derived growth factor receptor alpha subunit), etc.

[0152] The various specific markers described in the present application can be detected in cells by biochemical or immunochemical methods (e.g., enzyme-linked immunosorbent assay, immunohistochemical assay, etc.). Detection can also be made by measuring the expression of nucleic acids encoding these markers, for example, by molecular biological methods such as RT-PCR and hybridization methods. These methods are generally known in the art, and primers and probes used can be appropriately designed and manufactured by those skilled in the art based on information in publicly available databases such as Genbank.

[0153] As used herein, the term "chamber" refers to a space or cavity within a living organism, organ, or tissue, typically used to contain a liquid, gas, or other substance. In cardiac anatomy, chambers generally refer to the atria and ventricles of the heart, which play important roles in collecting and pumping blood.

[0154] Examples

[0155] Reagents or materials

[0156] Table 1

[0157] Cell line culture

[0158] HEK293T cells (CRL-3216) and human ESCs (H9 and H1) were obtained from the American Type Culture Collection (ATCC). HEK293T cells were cultured with Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Human ESCs and homemade UiPSCs (non-patent documents 11, 12) were cultured with mTeSR1 medium (Stemcell Technologies, #88550) in plates coated with Matrigel (Corning, #354230) and passaged every 2-4 days at 70-90% confluency using TrypLE Express Enzyme (Thermo Fisher, #12563029) or 0.5 mM EDTA-DPBS (Sigma-Aldrich, #E6758). Cells were routinely checked for mycoplasma contamination. All cell lines were cultured in a humidified atmosphere at 37°C with 5% CO2.

[0159] Generation of 3D cardiac organoids

[0160] hPSCs were dissociated using Accutase (Stemcell Technologies, #07920) and resuspended in mTeSR1 medium supplemented with 10 µM ROCK inhibitor Y-27632. Subsequently, hPSCs were plated at 20,000 cells / well into Ultra-Low-Attachment U-bottom 96-well plates (Corning #7007 or Thermo Fisher #1742929) and recorded as Day -1. Then, the plates were left undisturbed for 24 hours. On Day 0, the medium was changed to Chemically Defined Medium (CDM) containing 30 ng / mL FGF, 3 ng / mL BMP4, 5 µM CHIR99021, 5 µM LY294002, and 10 ng / mL Activin A (see Non-patent Literature 24). The composition of CDM was a mixture of 50% IMDM (Gibco, #112440053) with 50% F12 NUT-MIX (Thermo Fisher, #111765054), which was further supplemented with 15 µg / mL transferrin, 450 µM monothioglycerol, and 5 mg / mL BSA. After a 36-40 hour period of nutrition, embryonic bodies were incubated for 2 days with CDM medium containing 3 ng / mL BMP4, 5 µM IWP2, 10 µg / mL insulin, 0.5 µM PD173074, 5 µM SB431542, and 0.5 µM retinoic acid. PD173074 was removed after 24 hours. From Day 3.5 to Day 6.5, spheroids were cultured in CDM medium containing 30 ng / mL FGF, 10 ng / mL BMP4, and 10 µg / mL insulin, with fresh medium changed every day. To maintain cardioids, CDM medium containing 10 µg / mL insulin was used from Day 6.5, with medium changed every 2-4 days. Different chambers were observed from Day 6.5, enabling further investigation procedures.

[0161] Generating 2D epicardium

[0162] Cells were plated at 150,000 cells / well into MG-coated 24-well plates. On Day 0, the medium was changed to CDM containing 30 ng / mL FGF, 3 ng / mL BMP4, 10 mM CHIR99021, and 5 mM LY2940021. The composition of CDM was a mixture of 50% IMDM with 50% F12 NUT-MIX, which was also supplemented with 15 pg / mL transferrin, 450 mM monothioglycerol, and 5 mg / mL BSA. After a 36-40 hour period of nutrition, the embryoid bodies were incubated for 2 days with CDM medium containing 3 ng / mL BMP4, 5 mM IWP2, and 1 mM BMS. From day 3.5 to day 6.5, the cell spheroids were cultured in CDM medium containing 10 pg / mL insulin, 10 ng / mL BMP4, 5 mM CHIR99021, and 1 mM retinoic acid. To maintain epicardium, CDM medium containing 10 pg / mL insulin and 5 mM SB-431542 was used from day 6.5, and the medium was refreshed every 2 days.

[0163] Freezing section and immunostaining

[0164] Before sectioning, the organoids were dehydrated in 30% sucrose in PB and fixed in 4% PFA (Beyotime, #P0099). For sectioning, the organoids were embedded in O.C.T. (Sakura, #4583), sectioned into 10 pm slices at -22°C to -20°C by Leica cryostat, and subjected to immunofluorescence.

[0165] The frozen sections were post-fixed in 4% PFA, permeabilized in blocking solution (Beyotime, #P0102) with 0.2% Triton X-100 (Sigma-Aldrich, #T9284) for 15 min each. After washing with DPBS, the frozen sections were blocked with the above blocking solution, and then incubated with primary antibodies diluted in the primary antibody dilution matrix (Beyotime, #P0103) at room temperature (RT) for 4 h or at 4°C overnight according to the instructions. Then, the sections were washed with PBS / 0.1% Tween20 three times and stained with secondary antibodies diluted in the secondary antibody dilution matrix (Beyotime, #P0108) at RT for 2 h. The sections for observation were prepared by co-staining with DAPI (Abeam, #ab104139) at RT for 5 min. Images were collected from no less than 3 organoids using an inverted confocal microscope (Zeiss, LSM900 or LSM800).

[0166] It is also possible to not slice and to immunostain the whole organoid. The heart organoids are rinsed once in PBS and then fixed in 4% PFA at 4°C overnight (16-20 hours). The next day, the organoids are washed twice in PBS and then in BS / Tween20 for at least 15 minutes each. Then, the organoids are permeabilized with 0.2% Triton X-100 blocking solution for 15-30 minutes at room temperature and blocked in blocking solution for 1 hour. Primary antibodies are put in primary antibody buffer and incubated at 4°C on a shaker. Incubation is for 24 hours. After washing the labeled samples three times in PBS / 0.1% Tween20 for 10 minutes each, the samples are incubated with secondary antibodies at 4°C on a shaker for 24 hours. Then, the samples are washed once in PBS / Tween20 for 10 minutes and stained with DAPI solution (2 pg / mL) for 5 minutes. Then, the samples are washed three times in PBS / Tween20 for 10 minutes each. Then, the prepared samples are imaged.

[0167] Generation of cell lines with knockouts of DETS1, HAND1 and NKX2-5

[0168] Knockout strategy was performed using the CRISPR / Cas9 system. sgRNAs were identified using the website (http: / / crispor.tefor.net / crispor.py) and cloned into the hU6-sgScaffold site of pX330-puro. The targeting efficiency of sgRNAs was verified in 293T cell line. Cells were transfected with 2 pg pX330 sgRNA and 4 pg Donor per 1 x 10 TM 2b (Lonza BioResearch, programme B16) using 2 pg pX330 sgRNA and 4 pg Donor per 1 x 10 6 6 cells. After nucleofection, cells were incubated in mTeSR1 supplemented with 10 mM Y-27632 for 24 h, then selected with 0.1 ng / mL puromycin (Beyotime, #ST551) for 48-72 h, and then transferred into mTeSR1 containing 10 mM Y-27632. When cells grew to 50% confluency, ESCs were digested with Accutase and plated at 0.8 cells / well into 96-well plates. Monoclonal will become visible to the naked eye after 4-5 days and can be transferred for genotyping (using two different pairs of primers) after 10 days to confirm successful knockout.

[0169] Transmission electron microscopy

[0170] Samples were fixed in 150 mM HEPES buffer (pH 7.2) containing 2.5% glutaraldehyde and 2% formaldehyde for 30 min at room temperature or overnight at 4 °C. Fixed samples were washed with 0.1 M PB / Cacodylate buffer (pH 7.2-7.4). Fixed samples were post-fixed in 1% osmium tetroxide for 1 h at 4 °C and in 1% uranyl acetate for 1 h at room temperature or overnight at 4 °C. Samples were then dehydrated in gradient diluted ethanol (50%, 70%, 90%, 95%, 100% twice) and embedded in EPON 12 resin at 60 °C for 24-48 h. 70 pm sections were cut on a UC7 microtome (Leica) using a diamond knife. Sections of 10 nm thickness were collected on copper single-slot grids coated with formvar, post-stained with 2% uranyl acetate and lead citrate, and observed in a 120 kV transmission electron microscope (Thermo-scientific, Talos 120) after staining.

[0171] Contraction property measurement

[0172] Real-time imaging was performed using a high-resolution imaging system. Videos of dynamic contraction behavior were analyzed using the algorithm described in Huebsch N et al. Tissue Eng Part C Methods. 2015; 21 :467-479. This algorithm quantitatively assesses the contraction velocity of the heart catheter by tracking the movement of a specific area over time.

[0173] Calcium imaging

[0174] Calcium flux was imaged by recording the fluorescence signal of real-time Fluo-4 AM. Briefly, after washing with DPBS, the organoids were incubated with culture medium supplemented with 2 mM Fluo-4 AM (Beyotime, #S1060) and 0.1% Pluronic F-127 (Beyotime, #St501) at 37 °C for 30 min. Then, the organoids were transferred to a glass-bottomed plate (Cellvis, #P96-1.5H-N) with fresh culture medium, and equilibrated at 37 °C for 15 min. The organoids were ready for recording by using a multi-mode Spinning Disk Confocal System (Olympus, Spin SR10) to record for more than 30 s at a frame rate of 50 ms per frame. Time-lapse images were analyzed using Fiji / ImageJ, and processed using GraphPad Prism 9. Baseline fluorescence intensity (F) was calculated using the asymmetric least squares smoothing method. Fluorescence change (AF / F) was determined by the following formula:

[0175] For assessing the effect of isoproterenol stimulation on calcium handling, isoproterenol was applied at 1 mM for 30 minutes before imaging. The calcium transient response after treatment was compared with the untreated control group to assess the effect of beta adrenergic stimulation on calcium dynamics.

[0176] Heart rate (BPM) measurement

[0177] The heart rate (beats per minute, BPM) of the organoids was measured by direct observation of the contractions of the organoids under an Olympus IX73 inverted microscope. The average BPM of the organoids was obtained by manual counting in videos of spontaneous beating activity in at least three different fields of view. The BPM after isoproterenol treatment was obtained by calculating the real-time video recording captured by the Olympus IX73 microscope using Fuji / ImageJ software after 30 minutes of stimulation with 1 mM isoproterenol. The BPM after isoproterenol treatment was compared with the untreated control group to assess the effect of beta adrenergic stimulation.

[0178] Doxorubicin treatment, cell viability and cytotoxicity

[0179] Organoids cultured for 15.5-25.5 days were exposed to 5 mM doxorubicin. After 24 hours, the organoids were washed three times with PBS and replaced with fresh medium. For determination of cell viability, 10 pL CCK-8 reagent (Beyotime, #C0038) was added to each well and incubated at 37 °C for 2 hours. The absorbance at 450 nm was measured using a microplate reader (Thermo Scientific). For assessment of cytotoxicity, supernatant after 24 hours of doxorubicin exposure was collected and the absorbance at 490 nm was measured according to the manufacturer’s protocol (LabLead, #L0117) to assess cytotoxicity by LDH release.

[0180] Quantitative polymerase chain reaction (qPCR) analysis

[0181] Total RNA was extracted using RNA-easy Isolation reagent (Vazyme, #701) according to the manufacturer’s instructions. The quality and quantity of RNA were assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham). For cDNA synthesis, total RNA was reverse transcribed using Q RT SuperMix (Vazyme, #R222). Quantitative PCR (qPCR) was performed using ChamQ SYBR qPCR Master Mix (Vazyme, #Q711) to analyze the expression level of target mRNA.

[0182] Electrophysiology ​

[0183] The method described in Non-patent literature 25 was used. The organoids were dissociated into single cells using STEMdiff Cardiomyocyte Dissociation Kit (Stemcell Technologies, #05025). Action potentials (APs) were triggered at 1 Hz with 3 ms suprathreshold stimuli. Briefly, using the whole-cell patch-clamp technique, APs were recorded at room temperature (RT) by an Axopatch 700A amplifier and a Digidata 1550B digitizer (Axon Instruments, Foster City, CA, USA). The external solution contained the following components: 132 mM NaCl, 4.8 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES, and 5 mM glucose (pH adjusted to 7.4 with NaOH). The internal solution contained the following components: 110 mM KCl, 5 mM ATP-K2, 11 mM EGTA, 10 mM HEPES, 1 mM CaCl2, and 1 mM MgCl2 (pH adjusted to 7.3 with KOH). The glass electrode series resistance was typically 1.5-3 MΩ when filled with the internal solution.

[0184] RNA-seq and data analysis

[0185] Total RNA was extracted using RNA-easy Isolation Reagent (Vazyme, #701) and library was prepared using VAHTS mRNA-seq v2 Library Prep Kit for Illumina (Vazyme, NR601-01 / 02) according to the manufacturer’s instructions (1 pg RNA per sample). Sequencing was performed by Guangzhou Aijie Biotechnology Co., Ltd. (Guangzhou, China) using illumina nova seq instrument. To analyze gene expression, raw sequencing data (raw data) was filtered to obtain high-quality sequencing data (clean data). Sequence Alignment Tool STAR was used to align it to the human reference genome (reference genome source: https: / / ftp.ensembl.org / pub / release-109 / fasta / homo_sapiens / dna / and https: / / ftp.ensembl.org / pub / release-109 / gtf / homo_sapiens / ), obtaining data that can be located to the reference genome, then RSEM software was used to quantify gene expression levels, and the final normalized matrix was obtained from the quantitative results. Then, DESeq2 (v.1.26.0) was used for data normalization and differential expression analysis. Time series experiments used Wald test (Benjamini-Hochberg corrected P value < 0.05 and absolute fold change >= 1.5) and Likelihood ratio test (Benjamini-Hochberg corrected P value < 0.05) to define differentially expressed genes. Gene ontology analysis was performed using David (https: / / david.ncifcrf.gov / ).

[0186] PCA trajectory plot

[0187] First, the prcomp function of R language was used to perform PCA analysis on the gene expression matrix, and then the PCA scores were extracted. According to the PCA analysis results of different samples at each time point, the points of the same group were connected by trajectory lines to represent the change trend between samples. Finally, the PCA trajectory plot was drawn using the ggplot2 package (v.3.5.0).

[0188] Heatmap

[0189] First, based on the gene expression matrix and experimental design information, a DESeqDataSet object was created in the R language package DESeq2 (v.1.26.0) using the DESeqDataSetFromMatrix function, then variance stabilising transformation was performed using the varianceStabilisingTransformation function to obtain the processing data matrix. Finally, a heat map was drawn using the pheatmap function.

[0190] Single-cell (sc) RNA-seq and bioinformatics analysis

[0191] The organoids were dissociated into single cells using the STEMdiff Cardiomyocyte Dissociation Kit (Stemcell Technologies, #05025). Before library preparation, cells were counted and checked for viability using a hemocytometer (Thermo Fisher, Countess 3).

[0192] The scRNA data from 2D culture, 3D culture, 25.5th day 3D culture, PD173074 addition group and no addition group were processed using R language package Seurat. Cells with less than 3 expressed genes in the sample and genes expressed in less than 300 cells or more than 9000 cells in the whole sample, mitochondrial gene content greater than 25% of the total number of unique molecular identifiers (UMI), and ribosomal gene content greater than 50% of the total number of unique molecular identifiers (UMI) were excluded. After quality control, expression matrices containing 40191 cells and 27489 genes (2D and 3D), 11910 cells and 27601 genes (25.5th day 3D culture), 33348 cells and 27583 genes (PD173074 addition group and no addition group) were obtained, respectively. Next, the data was normalized using the default parameters of the logarithmic normalization method, and the top 2000 highly variable genes were identified using the FindVariableFeatures function. Z-score transformation of gene expression was performed using the ScaleData function. Subsequently, dimensionality reduction analysis was performed using the RunPCA function. Clustering was performed based on the 20 most important principal components (PCs). Then, batch removal was performed using R package Harmony v0.1.0. Different resolutions were set, and the clustering effect was observed using the clustering tree, and finally the resolution was set to 0.9, 0.8 and 0.7. UMAP (Unified Manifold Approximation and Projection) visualization images were constructed using the same number of PCs as the number of relevant clusters. The samples of 2D culture and 3D culture groups were clustered into 18 clusters, and the marker genes of different clusters were found using the FindAllMarkers function. By consulting literature and consulting experts, the marker genes of each cell type in the heart were determined, and the cell types were annotated by the expression of marker genes in different clusters.

[0193] Cell communication analysis

[0194] CellChat software package (v1.6.1) was used for intercellular communication analysis. The pre-processed scRNA-seq data of day 25.5 samples were used to create CellChat objects with identified cell types as clustering labels. The comparison objects for LAMA signaling pathway analysis were single-cell sequencing data of human embryos at 5 weeks (5W) and 6 weeks (6W) of gestation (obtained from GEO database (GSE106118)). Using the CellChat human ligand-receptor database, potential communication networks were inferred by assessing ligand-receptor interactions across cell types. The probabilities of these interactions were calculated and summarized to analyze the main signaling pathways. Visualization methods such as chord diagrams and bubble plots were applied to illustrate the communication patterns between cells, highlighting the interactions of endothelial cells with other cell types and vice versa.

[0195] Slingshot for stage mapping of organoid-derived cardiomyocytes

[0196] Samples from HE5W to HE25W stages (5 to 25 weeks post-conception) were selected from single-cell transcriptome data of human embryonic heart development (GSE106118) for comprehensive analysis. Meanwhile, single-cell sequencing data of heart organoids at day 25.5 and day 43.5 were integrated, and the TNNT2-positive cardiomyocyte subpopulation was isolated, and the Slingshot algorithm was used for pseudo-time trajectory reconstruction. Cell density plots were generated based on pseudo-time coordinates to map the developmental stages of organoid-derived cardiomyocytes.

[0197] Key expression difference analysis of cardiomyocyte maturation (D25.5 vs. D43.5)

[0198] As described previously, single-cell RNA sequencing (scRNA-seq) data of heart organoids at day 25.5 (D25.5) and day 43.5 (D43.5) were processed. Cells annotated for cardiomyocytes were sub-clustered, and violin plots were generated to visualize the expression patterns of genes related to cardiomyocyte function and maturation.

[0199] Marker gene enrichment analysis of 2D cultures vs. 3D cultures

[0200] FindMarkers function was used to find marker genes for 3D culture-specific cell types and other cell types, and significant marker genes were filtered according to Padj < 0.05, |log2FC| > 1. The filtered genes were subjected to David (https: / / david.ncifcrf.gov / ) enrichment analysis, and significant pathways with FDR less than 0.05 were selected based on enrichment results. Then, the analysis revealed the potential biological functions of 3D cultures.

[0201] Quantification and statistical analysis

[0202] All analyses were performed using GraphPad Prism® software and all raw data were collected in Microsoft Excel. All data were normally distributed. Statistical significance was assessed using standard unpaired Student's t-test (two-tailed; not significant (n.s.), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) where appropriate. For multiple comparison analyses, one-way ANOVA followed by Tukey or Dunnett's test correction was used where appropriate (n.s., not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). All data are presented as mean ± SEM, representative of at least 3 independent experiments with at least 3 technical replicates per experiment, unless otherwise stated. All micrographs are representative images of at least 6 independent experiments per condition / marker, and calcium transient plots are representative images of 6 independent experiments.

[0203] Example 1

[0204] This example describes an exemplary method that can be induced by pluripotent stem cells to form cardioid with stable lumen.

[0205] The formation of cardiac lumens relies on the Wnt-BMP signaling pathway via HAND1, but the prior art generally considers this insufficient to produce stable lumens (non-patent documents 2, 5, 6). To address the stability issue, the inventors of the present application systematically tested and analyzed the effects of fibroblast growth factor (FGF), transforming growth factor-beta (TGF-beta), bone morphogenetic protein (BMP), activin, and Wnt on various stages of heart development from pluripotency, mesoderm, cardiac mesoderm, cardiomyocyte formation, lumen formation, to ventricle maintenance, and the results are shown in Figures la, lc, 6a, 6b, 6e. As can be seen, using the methods described herein, lumens can be formed in cardioids with a 100% success rate, with a single lumen appearing on day 11.5 and being able to persist until day 90.5 (Figures la, lc, and expanded data Figure le).

[0206] The obtained cardioids were subjected to immunofluorescence staining, and the results are shown in Figure Id. As can be seen, these cardioids with stable lumens contain a large number of TNNT2-positive cells (representing cardiomyocytes) and NR2F2 cells (representing atrial parts), and have a subpopulation of cells expressing ventricular markers (non-patent documents 2, 10). scRNA-seq analysis further verified the above results. In these stable cardioids with lumens, 87.4% of the cells were cardiomyocytes (TNNT2 +), where 76.2% of the cells expressed the atrial marker NR2F2 and 11.5% of the cells expressed the ventricular marker MYL3 (Fig. 1e and 6c); Fig. 1f shows the changes in markers during the development of these organoids, specifically the progressive process from pluripotency (marked by NANOG+, POU5F1+, and SOX2+), mesoderm (marked by TBXT+, MESP1+, and MIXL1+), to the cardiac mesoderm stage (marked by NKX2.5+, HAND1+), and the formation of cardiomyocytes (TNNT2+, NKX2.5+, MYL3+, HCN4+). Before day 6.5, these cardioids mainly transitioned to the first heart field (FHF) lineage (marked by HAND1+, TBX5+, NKX2-5+, and TBX1-) before entering the heart tube stage. In addition, during chamber formation, mature-related genes were all upregulated, including ion channel genes, structural genes, and beta-adrenergic receptor genes (Fig. 1f and 6d).

[0207] The obtained organoids were also frozen sectioned and the sections were subjected to immunofluorescence staining, and the results are shown in Fig. 1g. The results show that there are myocardium marked by TNNT2 and endocardial layer marked by CDH5 in these cardioids.

[0208] Example 2

[0209] This example investigates the effect of Wnt-BMP pathway regulation on the formation of cardioids with stable chambers.

[0210] Since the formation and expansion of stable chambers in cardioids can involve biological processes such as apoptosis or proliferation, the inventors investigated this. Using the method described in Example 1, the H9 ESC cell line was induced to form cardioids. By immunostaining for cleaved Ki67 and CASP3, it was found that apoptosis and proliferation both promoted chamber formation in cardioids in the time window from day 6.5 to day 15.5 (Fig. 7a). Subsequent heat map analysis of apoptosis- and proliferation-related genes also supported this conclusion, as the expression levels of these genes decreased during the formation of cardioids with chambers, rather than increasing (Fig. 7b).

[0211] On this basis, the influence of the pathway regulation was further analyzed through the downstream effectors of Wnt-BMP signaling axis. First, two HAND1 knockout (KO) H9 cell lines (named #9 and #15, respectively) and two NKX2.5 knockout H9 cell lines (named #14 and #24, respectively) were prepared. The same method as in Example 1 was performed on the above cell lines, and the corresponding organoids were obtained. The obtained organoids were subjected to frozen section, and then subjected to immunofluorescence analysis, and the results are shown in FIGS. 7c and 7d. In the two types of cell lines, opposite results were observed. Among them, the HAND1 KO cells produced solid organoids, lacking identifiable chambers, but the staining still showed TNNT2 positive (FIG. 7c); while the NKX2.5 KO cells not only had TNNT2 positive, but also could form clear chamber-like structures (FIG. 7d).

[0212] The above results strongly suggest that HAND1 in the Wnt-BMP signaling axis is a key factor for forming organoids with stable chambers, while NKX2.5 is not. The above results also suggest that the process of forming stable chambers is not related to the differentiation of cardiomyocytes.

[0213] Example 3

[0214] This example explores the influence of FGF on the regulation of forming organoids with stable chambers.

[0215] It has been reported that FGF plays a key role in the process of heart development (Non-patent Literature 7 to 9), but it is not clear how FGF affects chamber formation. In this regard, first, the effect of PD173074, a representative FGF signaling inhibitor, on cardiomyocyte formation was tested. Using substantially the same method as in Example 1, the H9 ESC cell line was induced to differentiate into cardiomyocytes, with the exception that the medium was not added with the FGF pathway inhibitor PD173074 at day 1.5 from mesoderm to cardiac mesoderm. As a result, it was found that, under the condition of adding PD173074, pluripotent stem cells were able to form a cardiomyocyte-like organ with a clear chamber structure, and the chamber structure formed was still stably maintained at day 90 without signs of structural disruption and collapse, and the results are shown in Figs. 1b, 1c, 1d, 3a, 3b, 3c, and 8a; while under the condition of not adding PD173074, the formed organ did not have an observable chamber at day 15 (Figs. 3a, 3b), and dynamic imaging also confirmed that no chamber formation was observed throughout the induction experiment using PD173074 (Fig. 8a). By performing immunofluorescence staining on frozen sections of the organoids, the difference between the two organoids was observed from a more microscopic perspective, and it was found that, without the addition of PD173074, the vast majority of the volume of the organoid was occupied by TNNT2-positive cardiomyocytes, and only a significantly reduced microchamber remained inside (Fig. 3c). The above results indicate that inhibiting FGF is a key factor in forming a long-term stable heart chamber structure during the induction of cardiac mesoderm.

[0216] Further, no difference in the differentiation of cardiomyocytes was observed between the PD173074 addition group and the non-addition group (Fig. 3d). However, principal component analysis (PCA) experiments revealed that the cell fate of the PD173074-induced organoids was significantly changed (Fig. 3e). The volcano plot showed that the key genes related to cardiomyocyte differentiation were significantly upregulated at the cardiac mesoderm stage at day 3.5, and the upregulation amplitude in the samples of the PD173074 non-addition group was significantly greater than that in the addition group. At day 10.5, changes in cardiomyocyte fate were observed: in the PD-non-added organoids, the expression of atrial-specific genes such as NR2F2 increased, and in the PD-added organoids, the level of ventricular-specific genes such as IRX4 increased (Fig. 8d). Further analysis of the expression of genes specific to cardiac cell types confirmed the above observation (Fig. 8e). The results of single-cell RNA sequencing also showed that PD-treated organoids exhibited an increased level of atrial cardiomyocytes (Figs. 3i, 3j, 3k, and 3l).

[0217] A more extensive gene expression analysis was then performed at five time points to track changes in the up- and down-regulated gene sets, the results of which are shown in Figure 3f. Dynamic changes in gene expression can be observed from the Venn diagrams, with some genes showing temporal stability, suggesting that they can play a role in the regulatory process. At day 3.5, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that up- and down-regulated genes were significantly enriched in various biological processes, cellular components, and molecular functions. Up-regulated genes were enriched in processes such as cholesterol biosynthesis, heart development, glycolysis, metabolic pathways, and adrenergic signaling, while down-regulated genes were associated with processes such as cell proliferation and adhesion, Ras signaling, and PI3K-Akt signaling (Figures 3g, 3h). Down-regulated genes were involved in cell proliferation and adhesion, and the proteins they encoded were involved in cellular components such as the plasma membrane and chromatin (Figure 3h). The above enrichment analyses provided insights into the regulatory mechanisms leading to the formation of heart-like organoids with stable lumens.

[0218] Example 4

[0219] This example verifies the effects of other FGF pathway inhibitors on regulating lumen formation in heart organoids

[0220] For H9 ESC cells, 3D organoids were induced and cultured using essentially the same method as in Example 1, with the exception that the FGF pathway inhibitor was replaced by PD166866, a selective inhibitor of FGFRI tyrosine kinase activity, at a concentration of 1 μΜ. Changes in the induced cultures were continuously observed, and live cell imaging was used, the results of which are shown in Figures 8b, 8c, 14.

[0221] As can be seen from the figures, similar to the results of Example 1, heart-like organoids with lumens were observed in the group with added PD166866 at day 15.5.

[0222] Example 5

[0223] Based on the 2D epicardium formation method reported in Non-patent Literature 2, 2D epicardium tissues were generated from H9-tdTomato cell line, in which the tdTomato gene was randomly integrated into the cell genome (Fig. 6d). By detecting the red fluorescence of tdTomato, the location and differentiation of these cells can be tracked. The differentiated epicardial cells were cultured to day 9.5, harvested and seeded at 100 cells / well into AggreWell400 plates to allow aggregation. Then, the epicardial cell aggregates were added to the organoids obtained in Example 1 (day 15.5-25.5) and the co-culture was maintained in CDM medium supplemented with 10 pg / mL insulin and 0.5 mM SB431542, with medium refreshed every 2 days, allowing the aggregates to fuse with the organoids. By this method, we successfully generated 3D structured cardioballs, including epicardial, myocardial and endocardial layers. This indicates that our method can mimic the structure of the heart in vitro, providing a new experimental model for heart development and disease research.

[0224] Example 6

[0225] The same induction culture method as in Example 1 was performed on H1 ESC cell line and UiPS cell line (Non-patent Literatures 11, 12) derived from male individuals, and the results are shown in Figs. 6g and 6h.

[0226] As can be seen from Figs. 6g and 6h, the cardiac organoid preparation method of the present application can be applied to two different sources of pluripotent stem cells and consistent results with the H9 cell line (from female individuals) above are obtained. This indicates that the method of the present application has good robustness and can effectively induce different cell lines to generate organ-like hearts with stable cavities, without being adversely affected by potential genetic background differences between cell lines and gender, and has the potential for customized applications.

[0227] Example 7

[0228] This example explores the physiological and ultrastructural features of the organ-like hearts prepared using the exemplary method of the present application.

[0229] The heart-like organoids prepared using the method described in Example 1 exhibited strong and rhythmic contractions and relaxations that were visible to the naked eye. The rhythmic nature of the contractions and relaxations was confirmed by the results of heartbeat recordings and calcium imaging experiments. For example, Figure 2e shows that an exemplary heart-like organoid maintained a beating frequency of 60 beats per minute (BPM) from day 21.5 to day 68, and Figure 2f shows that calcium ion influx and efflux consistent with the above-described heart rate could be observed in the heart-like organoid. The results of whole-cell patch clamp experiments (Figures 2c and 2d) showed that the heart-like organoids exhibited typical atrial-like action potential profiles at the level of individual cardiomyocytes, such as action potential shape, resting membrane potential (RMP), action potential amplitude (APA), and action potential duration (APD).

[0230] To observe the ultrastructure of the organoids, they were subjected to immunofluorescence staining and confocal microscopy, the results of which are shown in Figure 2a, and to cryosectioning and transmission electron microscopy, the results of which are shown in Figure 2b. The results of the ultrastructure were also consistent with the physiological function, and the ultrastructural features characteristic of cardiomyocytes, including structurally correct and ordered sarcomeres, Z lines, and interconnectedness via intercalated discs (IDs), were observed in the organoids. Whole-cell patch clamp was performed on individual cardiomyocytes derived from the organoids of the present disclosure, and the results are shown in Figures 2c and 2d. It was observed that the derived cardiomyocytes predominantly exhibited an atrial-like action potential (AP) phenotype, including a resting membrane potential (RMP) of -60.99 ± 4.91 mV, an action potential amplitude (APA) of 119.05 ± 8.69 mV, and an AP duration (APD50) of 88.57 ± 25.65 ms (Figure).

[0231] Thus, the heart-like organoids obtained in the present application have desirable characteristics in both microstructure and physiological function, such as a beating frequency that is synchronized with the patient’s heart rate, which is of critical importance for effective integration with the cardiovascular system of the transplant recipient. Moreover, these desirable characteristics are stable over a relatively long window of observation, indicating that the heart-like organoids of the present application have great potential for application in various clinical application scenarios that require stable cardiac function.

[0232] Example 8

[0233] This example describes the formation of a structured myocardial layer and endothelial layer by the heart-like organoids disclosed herein through self-organization.

[0234] A large RNA-seq was performed on the heart organoids of different culture days in Example 1, and the results showed that endothelial markers including CDH5, PECAM1, CD34, and ESAM were gradually upregulated from day 3.5 (Figure 11A). In the immunofluorescence assay of their frozen sections, an outer myocardial layer formed by TNNT2+cardiomyocytes and an endothelial layer formed by CDH5+endothelial cells arranged on the inner surface of the chamber-like structure were observed (Figure 11B). This indicates that the heart organoids disclosed herein are capable of orderly self-organization in three-dimensional space, eventually forming a structured myocardial layer and an endothelial layer, which are crucial for maintaining the stability and structural integrity of the heart chamber. This is significantly different from the reported heart organoid model (such as Non-patent document 2), which cannot form a self-organized myocardial layer and endothelial layer even under optimized conditions supplemented with VEGF.

[0235] The RNA-seq results also showed that in the heart organoids disclosed herein, dynamic upregulation of marker genes involved in ventricular morphogenesis and early cardiac patterning can be observed, such as NPPA, NPPB, TBX5, HAND1, and HAND2 (Figure 11C). This indicates that the molecular signature exhibited by the heart organoids disclosed herein is similar to that of the early stages of human heart development.

[0236] Endothelial cells play a key role in heart development through endothelial-to-mesenchymal transition (EndoMT). EndoMT is a process regulated by TGF-β signaling, which helps form the endocardial cushion, which further develops into heart valves, and is very important in cardiac morphogenesis. In the heart organoids disclosed herein, significant upregulation of key EndoMT-related markers of CDH5+endothelial cells was observed, including TWIST1, ACTA2, SNAI2, and CDH2 (Figure 11G). This is consistent with the underlying EndoMT process.

[0237] The organoids from day 28.5 to day 48.5 were treated with a selective TGF-β inhibitor SB431542 (SB), and the results showed that the chamber area of the SB-treated group was about twice that of the blank control group (Figures 11D, 11E, and 11H), but there was no significant difference in the beating rate between the treated and control groups (Figure 11F). This indicates that TGF-β signaling may affect chamber morphology by affecting endothelial and mesenchymal cell populations, but does not directly affect contractile dynamics.

[0238] ETS1 gene was also knocked out by CRISPR / Cas9-mediated gene editing technology to generate ETS1 knockout (ETS1 KO) organoids. It has been reported that ETS1 is a key transcription factor that affects endothelial cell survival and function, and its deletion disrupts coronary artery development, leading to severe defects in vascular formation. It has also been reported that ETS1 deletion is associated with Jacobsen syndrome. It was found that ETS1 KO organoids were completely unable to form ordered lumens (Figure 11D). This suggests that endothelial defects can be related to lumen morphogenesis.

[0239] Based on this, the heart organoids of the present disclosure have the potential to serve as a good model for evaluating drugs and mechanisms targeting endothelial cells.

[0240] Example 9

[0241] This example characterizes the constituent cell types of the cardioid prepared using the exemplary method of the present application.

[0242] By the method described in Example 1, corresponding 2D and 3D cultures were prepared from H9 ESC cells, and single-cell sequencing analysis was performed to determine the cell composition of these cultures. 25274 and 14614 cells were obtained from 2D and 3D cultures, respectively. Through UMAP and Louvain analysis, 7 and 18 different cell clusters were identified from 2D and 3D cultures, respectively. Specifically, four differentiated cell types were identified in the 2D culture, namely atrial cardiomyocytes, ventricular cardiomyocytes, cardiac progenitor cells, and fibroblasts (Figures 4a, 4b, and 9a); while in the 3D culture, in addition to the above four types of cells, two other cells were identified, namely valve cells expressing PRRX2 and MSX1 markers, and epithelial progenitor cells expressing GABRP and GRHL2 markers (Figures 4a-4c and Figures 9a-9d). The proportion of the main cells clustered and the typical marker genes are shown in Table 2.

[0243] Table 2:

[0244] Valve cells and epithelial progenitor cells are unique to 3D cardioids. Based on GO and KEGG analysis, valve cells are endocardial cells involved in angiogenesis, outflow tract morphogenesis, focal adhesion, and related signaling pathways such as Rap1, MAPK, and PI3K-AKT. In contrast, epithelial progenitor cells are associated with processes such as apoptosis, motor proteins, tight junction regulation, and p53 signaling pathways (Figures 4d and 9e, 9f).

[0245] Further comparisons of gene expression in 2D and 3D cultures also revealed significant expression of relevant genes, such as ion channels (RYR2, CACBA1C, HCN4), progenitor cells (EDNRA), b-oxidation (ACADVL), and 3D cell-specific cardiomyocyte-related genes (TNNT2, MYL7, NR2F2, TBX5, GATA4, LAMB2, TTN) (Fig. 4e, 9g, 9h). These results confirmed that the heart-like generated by 3D culture has a broader cell diversity and maturity.

[0246] Example 10

[0247] This example describes the gradual maturation of the heart organoids over time.

[0248] Single-cell RNA sequencing (scRNA-seq) was performed on heart organoids at day 25.5 (D25.5) and day 43.5 (D43.5), and the results are presented as violin plots in Fig. 12. It can be seen that, compared to D25.5, the expression of genes associated with the following characteristics was upregulated in the organoids at D43.5: cardiac muscle contraction (TNNT2, MYL7, MYL6), glycolytic metabolism (PGAM1), fatty acid metabolism (HADH4, HADHB), and ion channel activity (RYR2, KCNQ1, SCN5A). This indicates that the gene expression pattern associated with cardiomyocyte maturation has changed over time.

[0249] Pseudo-time trajectory analysis was performed on the scRNA-seq data measured for D25.5 and D43.5 heart organoids, respectively, and human fetal heart scRNA-seq data obtained from a public database (Cui Y, et al., Cell Rep. 2019; 26: 1934-1950 e1935) by the Slingshot method, and the results are shown in Fig. 13. It can be seen that the cardiomyocytes of D25.5 mainly correspond to an early developmental state before 5-week human fetal (HE5W); while the cells of D43.5 show a clear progression along the pseudo-time trajectory, which is closer to the transcriptional features observed in 5- to 9-week human fetal hearts.

[0250] The above results show that the heart organoids of the present disclosure can gradually shift to the later stage of heart development during long-term culture.

[0251] Example 11

[0252] This example describes the use of the exemplary heart-like of the present application for toxicity testing of drug candidates.

[0253] The cardioids of the present application have the potential to become a platform for drug safety and toxicity testing. Thalidomide and Acitretin were used as examples. Thalidomide has been reported to cause severe birth defects, especially neonatal heart abnormalities (non-patent document 13), while Acitretin can significantly affect heart development, posing a serious risk to fetal heart health (non-patent document 5). In contrast, aspirin has not been reported to have such side effects, and thus was used as a negative control (non-patent document 2).

[0254] The corresponding 2D and 3D cultures were prepared from H9 ESC cells by the method described in Example 1, and thalidomide, acitretin or aspirin was added to the culture medium to a final concentration of 10 μg / ml, 50 nM and 30 μM, respectively, on day 0 of culture. The morphological changes of the cultures were observed, and the results are shown in Figures 5a and 10a. It can be clearly seen that, compared with the aspirin group as a control, both the thalidomide group and the acitretin group can be observed to have severe defects accompanied by morphological changes. Until day 20.5, the cardioids of the thalidomide group and the acitretin group showed significantly smaller chamber areas compared with the control group (Figure 5b). Specifically, it was observed that the thalidomide group formed transparent chambers from day 6.5 to day 15.5, but could not stably maintain the chamber structure, and eventually collapsed into a spherical shape on day 20.5. The acitretin group showed irregular shapes as early as day 6.5. More detailed morphological observation results also suggest that the time at which thalidomide and acitretin begin to affect the cardioids may be different, with the former affecting the mesoderm stage and the latter appearing to have observable effects at the pluripotency stage (Figures 10b and 10c). In addition, in both the thalidomide group and the acitretin group, the beat rate was not detected or was severely reduced (Figure 5c). This indicates that the cardioids of the present application can effectively detect compounds that have potential cardiotoxicity or teratogenicity.

[0255] It has been reported that thalidomide-induced embryonic damage mainly occurs during the early development stage, usually from day 20 to day 36 after fertilization (non-patent document 14), and that late exposure to thalidomide induces brain damage in rat fetuses. Based on this, in the same method as Example 1, thalidomide (10 μg / ml) was injected into the cardioids from day 15.5 (when chamber formation is complete) to day 36.5 to evaluate whether the response window of the heart cardioids of the present application to thalidomide teratogenicity is consistent with the natural development window in vivo. The results show that the administration of thalidomide after day 15.5 does not change the chamber area in the cardioids, nor does it affect their beat rate (Figures 5d, 10d, 10e). This indicates that the heart cardioids of the present application have the potency and specificity as a screening model to respond to drug toxicity.

[0256] Therefore, the above results show that the cardioid of the present application can be used as a drug testing model, and has the potential for further optimization.

[0257] Example 12

[0258] This example further demonstrates the application of the cardioid of the present disclosure as a drug candidate screening model using isoproterenol (ISO) and doxorubicin (DOX).

[0259] ISO is a synthetic beta adrenergic agonist, commonly used to stimulate heart rate and mimic stress-induced cardiac responses. ISO treatment mainly affects the contractile activity of the heart, with no detectable morphological changes. The change in the movement speed of the cardioid after exposure to ISO was detected by …, and the results are shown in Figures 14 and 15. A positive chronotropic effect was observed in the cardioid treated with a small amount of ISO (1 mM), with a significant increase in BPM from 61.43 ± 2.34 beats per minute to 87.26 ± 3.99 beats per minute, and a significant shortening of the duration of each contraction cycle.

[0260] DOX is an anthracycline chemotherapy drug known for its strong cardiotoxicity. DOX treatment can cause severe deterioration of the structure of the cardioid, such as a decrease in chamber size. The cardioid was observed by bright field microscopy after 24 and 48 hours of DOX exposure, and the results are shown in Figures 16A and 16B. A significant decrease in chamber size was observed with the extension of the DOX treatment time (Figure 7F), indicating that the damage to the structure of the heart by DOX can be detected using the cardioid disclosed herein. The cardiotoxicity of DOX was further evaluated by cell survival and cytotoxicity assays. The results of the CCK-8 assay showed a significant decrease in cell survival in the DOX treatment group (Figure 16C), as well as an increase in lactate dehydrogenase (LDH) levels in the culture medium, suggesting cell membrane damage and increased cytotoxicity (Figure 16D). At the same time, in the DOX treatment group, the mRNA expression of the cardiac stress marker atrial natriuretic peptide (ANP) was increased (Figure 16E), and the inflammation-related markers such as tumor necrosis factor alpha (TNFA) and interleukin-6 (IL6) were significantly upregulated, suggesting an inflammatory response of the heart to the drug (Figures 16F and 16G).

[0261] As can be seen, the cardioid platform of the present disclosure can sensitively detect changes in cardiac function and a series of undesirable potential toxicities caused by candidate drug molecules, including but not limited to cardiac / chamber structure damage, cell damage, cytotoxicity, cardiac stress response, inflammatory response, etc.; thereby allowing rapid, multi-dimensional, high-throughput screening of candidate molecules from the perspective of cardiotoxicity.

[0262] Example 13

[0263] This example describes important signaling pathways involved in the induction and differentiation of cardiac organoids, in addition to the FGF pathway, using the cardiac organoids of the present disclosure can build experimental models to study these signaling pathways.

[0264] 1. Endothelial cell-mediated signaling

[0265] During heart development, endothelial cells play a critical role by interacting with various cell types, including cardiomyocytes. These interactions involve multiple signaling pathways, such as NOTCH, BMP / VEGF, WNT, EPHA / EPHB, NRG, and Hedgehog (HH).

[0266] The above interactions were revealed by cell communication analysis in the cardiac organoids of the present disclosure, the results of which are shown in FIGS. 17A, 17B, and 17C, 17D. It was observed that signaling from endothelial cells to atrial cardiomyocytes was mainly mediated by EPHA, EPHB, NOTCH, VEGF, NRG, and WNT; only BMP-mediated signaling was detected in the communication from endothelial cells to ventricular cardiomyocytes, and signaling from atrial or ventricular cardiomyocytes to endothelial cells involved multiple pathways, except for HH. In addition, specific ligand-receptor interactions were found in these intercellular communications; for example, BMP produced by endothelial cells interacted with BMPR / ACVR receptors on ventricular cardiomyocytes (FIGS. 17C, 17D).

[0267] 2. Laminin alpha family related regulatory pathways

[0268] Laminin alpha (LAMA) is an important transcription factor that plays different roles in endothelial structure, cell communication, and cardiac morphogenesis depending on the subunit. For example, laminin alpha 4 (LAMA4) has been shown to be critical for maintaining endothelial cell growth and proliferation, and its mutation or dysregulation has been associated with various cardiovascular diseases, including cardiomyopathy and vascular dysfunction. Defects in LAMA4 expression can lead to impaired function of cardiomyocytes and endothelial cells, resulting in abnormal cardiac structure and function. Laminin LAMA4 and LAMA5 also regulate immune cell trafficking, which is associated with impaired T cell localization function and exacerbated graft rejection.

[0269] Different spatial expression patterns of LAMA family members were revealed by single-cell communication analysis. FIG. 18A shows that LAMA2 is mainly expressed in cardiomyocytes (atrial and ventricular), LAMA4 is mainly detected in endothelial cells and fibroblasts, LAMA1 is detected in endodermal cells, and LAMA5 shows a more extensive expression profile, highly enriched in epithelial progenitor cells, but also found in some cardiomyocytes and endothelial cells. Further analysis suggests that LAMA2 can promote interactions between cardiomyocytes, cardiac progenitor cells, endothelial cells, and fibroblasts, while LAMA4 seems to interact with surrounding heart-related cell types.

[0270] Single-cell RNA sequencing data shows that the signaling mediated by LAMA family members observed in the organoids of the present disclosure has significant similarity to the distribution and inferred signaling in 5-6 week human fetal hearts, particularly in endothelial and cardiomyocyte-mediated interactions (FIG. 18B). LAMA2 is highly expressed in cardiomyocytes and shows extensive interactions with other heart-related cell types. LAMA4 is enriched in endothelial cells and also detected in some cardiomyocytes, cardiac progenitor cells, and epicardial cells. LAMA1 and LAMA5 are mainly secreted by cardiac progenitor cells and epithelial cells, respectively. This also indicates that the heart-like model of the present disclosure has a similar ECM-related intercellular communication pattern as the early human heart development stage.

[0271] 3. Activin A (AA) and retinoic acid (RA) related signaling pathways

[0272] AA and RA are believed to play an important role in the induction of mesoderm and cardiac mesoderm, and have also been reported to be associated with heart disease. However, their precise mechanisms in chamber formation and structural stability have not been fully elucidated. This example tests their specific effects by three concentration combinations of AA and RA.

[0273] A4R50 group: AA 4nM and RA 50nM;

[0274] A4R500 group: AA 4nM and RA 500nM;

[0275] A50R500 group: AA 50nM and RA 500 nM.

[0276] Figures 19A, 19D show that all three conditions can effectively induce the formation of stable cavities. In the condition of low concentration of AA, the cavity size of A4R500 group is significantly larger than that of A4R500 group, indicating that higher RA concentration can promote the expansion of cavity volume in the tested range. Figures 19A, 19B and 19C show that in the condition of higher concentration of AA (A50R500 group), cavity formation can be observed at an earlier stage, but the area of the formed cavity gradually decreases from the 15.5th day, indicating that higher AA concentration can accelerate cavity formation, promote cavity expansion, but is not conducive to long-term maintenance of the cavity, while lower AA concentration is more conducive to the long-term stability of the cavity.

[0277] The present application has been described in detail. For those skilled in the art, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present application, and without unnecessary experiments. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, the present application is intended to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.

Claims

A method of generating a cardiac organoid, the method comprising: A. activating Wnt signaling in a cell aggregate comprising pluripotent stem cells, inducing the cell aggregate to differentiate into a mesoderm cell aggregate; B. inhibiting FGF signaling in the mesoderm cell aggregate, promoting the cell aggregate to differentiate into a cardiac mesoderm, during a process of stopping the activation of Wnt signaling, allowing the mesoderm cell aggregate to differentiate into a cardiac mesoderm; C. stopping the inhibition of FGF signaling, allowing the cardiac mesoderm to continue to differentiate into a cardiac organoid. The method of claim 1, wherein, The inhibition of FGF signaling comprises contacting the mesoderm cell aggregate with an FGF receptor inhibitor. The method according to claim 1 or 2, wherein The FGF receptor inhibitor is selected from any one of an FGFR1 inhibitor, an FGFR2 inhibitor, an FGFR3 inhibitor, or an FGFR4 inhibitor, or any combination thereof; Preferably, the FGF receptor inhibitor is selected from an FGFR1 inhibitor; More preferably, the FGF receptor inhibitor is selected from any one of PD173074, PD166866, SSR128129E, AZD4547, Pemigatinib, ASP5878, PRN1371, Infigratinib, Futibatinib, LY2874455, FIIN-2, Derazantibnib, Zoligratinib, ODM-203, FIIN-3, Lucitanib, S49076, or Ferulic acid, or any combination thereof. The method of any one of claims 1 to 3, wherein, The duration of the inhibition of FGF signaling is more than 0.1 hour and not more than the time required for the mesoderm cell aggregate to differentiate into a cardiac mesoderm without the inhibition of FGF signaling, preferably 0.1 hour to 48 hours. The method of any one of claims 1 to 4, wherein, The step B further comprises further inhibiting Wnt signaling during the process of allowing the mesoderm cell aggregate to differentiate into a cardiac mesoderm. The method of claim 5, wherein, The inhibition of Wnt signaling is at least partially simultaneous with the inhibition of FGF signaling; Preferably, the time of the simultaneous inhibition of Wnt signaling and FGF signaling is more than 12 hours. The method of claim 5, wherein, The inhibition of Wnt signaling comprises contacting the mesoderm cell aggregate with a Wnt inhibitor; The cardiac organoid is a three-dimensional structure comprising cardiomyocytes and non-cardiomyocytes, wherein the non-cardiomyocytes are selected from the group consisting of endothelial cells, fibroblasts, and epicardial cells. Optionally, the Wnt inhibitor is selected from any one of IWP2, XAV939, ICG-001, IWR-1, Capmatinib, PRI-724, Salinomycin, FH535, PNU-74654, LF3, KYA1797K, KY02111, Adavivint, MSAB, Isoquercitrin, NCB-0846, IQ-1, iCQR14, CCT251545, WIKI4, JW55, Resibufogenin, M435-1279, RCM-1, JW74, Zamaporvint, M2912, Lanatoside C, Ginsenoside Rh4, Prodigiosin, Triptonide, IWP-4, or KY-05009, or any combination thereof. The method of any one of claims 1 to 7, wherein, The step B further comprises contacting the cell aggregates of mesoderm cells with a TGF-β inhibitor, a RA agonist, and / or a BMP pathway agonist; Optionally, the TGF-β inhibitor is selected from any one of SB431542, Dorsomorphin 2HCl, Dorsomorphin, LDN-193189, Galunisertib (LY2157299), LY2109761, SB525334, LDN-1931892HCl, SIS3 HCl, RepSox (E-616452), LY364947, Pirfenidone, DMH1, SB505124, GW788388, A-83-01, K02288, SD-208, Vactosertib (TEW-7197), Sulfasalazine, SIS3, ITD-1, Halofuginone, LDN-212854, LY 3200882, ML347, TP0427736HCl, LDN-214117, TGFβRI-IN-3, PD 169316, 3,3-Dimethyl-1-butanol, Lycopus Extract, AUDA, BIBF-0775, Ginsenoside Rh4, R-268712, or TA-02, or any combination thereof; Optionally, the RA agonist is selected from any one of Tretinoin, Bexarotene, TTNPB, AM580, Adapalene, Acitretin, Tazarotene, Tamibarotene, SR 11237, Etretinate, BMS493, Palovarotene, All trans-Retinal, CD437 (AHPN), or MSU-42011, or any combination thereof; Optionally, the BMP pathway agonist is selected from any one of BMP4, Activin A, BMP2, SJ000291942, or SB 4, or any combination thereof. The method of any one of claims 1 to 8, further comprising the step of fusing the cardiac organoid with epicardial cells. A cardiac organoid prepared by the method of claims 1 to 9. A cardiac organoid comprising cardiac tissue comprising cardiomyocytes, endocardial cells, and optionally epicardial cells, at least one internal lumen located inside the cardiac tissue, wherein, the at least one internal lumen is capable of existing for at least 30 days or more, preferably at least 60 days or more, more preferably at least 90 days or more under in vitro culture conditions; Optionally, the cardiac organoid further comprises epicardial cells disposed on the outer surface. The cardiac organoid of claim 10 or 11, which can spontaneously beat under in vitro culture conditions; Optionally, the spontaneous beating frequency of the cardiac organoid is from 30 to 150 beats per minute. The cardiac organoid according to any one of claims 10 to 12, wherein, The cardiomyocytes constitute from about 40% to about 80% of the total number of cells constituting the organoid, the endocardial cells constitute from about 5% to about 15% of the total number of cells constituting the organoid, and the epicardial cells constitute from about 1% to about 15% of the total number of cells constituting the organoid; Optionally, the ratio of the cardiomyocytes to the endocardial cells is from about 8: 1 to about 15: 1; Optionally, the endocardial cells are aggregated inside the organoid and define at least one internal lumen; Optionally, the cardiac organoid has a size of from about 0.5 to about 2.5 mm in its largest dimension; and / or Optionally, the size of the internal lumen in its largest dimension is at least about 60% or more of the size of the cardiac organoid in its largest dimension. Use of a heart organoid according to any one of claims 10 to 13, wherein, The use is selected from: (1) constructing a disease model; (2) screening drug candidates; and / or (3) evaluating drug candidate toxicity.

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