Heart-like organ with stable chamber structure

Abstract

A method of producing a cardiac organoid having a stable chamber is provided, the cardiac organoid produced using the method being capable of maintaining a stable presence of at least one internal chamber for a time of at least 90 days. The invention also provides the heart organoid prepared by the method, and application of the heart organoid.

Description

Technical Field

[0001] This invention relates to the fields of regenerative medicine and cardiac tissue engineering, specifically to cardiac organoids with stable internal chambers, their induction and culture methods, and their applications. Background Technology

[0002] The human heart plays a vital role in regulating nutrient circulation and removing waste products continuously throughout a lifetime (an average of about 80 years). The complexity and importance of the heart make it extremely challenging to study the therapeutic mechanisms, efficacy, or toxic side effects of drug molecules using animal models (Non-Patent Literature 1). While the development of organoid technology over the past two decades has provided initial insights into this problem, the unique and complex structure of the heart (composed of cardiomyocytes and various other cell types such as the endocardium and epicardium, possessing a stable multi-chamber structure) and function (generating regular spontaneous action potentials and contractile-diastolic movements) make the construction of in vitro models based on heart organoids exceptionally technically difficult.

[0003] Early studies constructed models that largely lacked chamber structures, at best resembling cultures primarily composed of cardiomyocytes (Non-Patent Literature 2). Most other reported chamber models were obtained by encapsulating cardiomyocytes and other cultured cells within chamber structures built using scaffolds, molds, and protein matrices (Non-Patent Literature 2, 3, 4). These artificially constructed structures do not mimic the natural development of the heart, limiting their application, particularly their reliability as models for studying and validating the therapeutic mechanisms, efficacy, and toxic side effects of drug molecules. In 2021, Mendjan and colleagues reported constructing the world's first human heart organoid with an internal structure resembling complete heart chambers by modulating the WNT-BMP signaling pathway, naming it a "cardioid" (Non-Patent Literature 2). Building upon this, Mendjan et al. developed specialized protocols for generating different heart structures separately, such as lumenized outflow tracts (OFTs), lumenized atrial organoids, and lumenized ventricular organoids, then fusing them to form a heartoid with multiple chambers (Non-Patent Literature 5). Furthermore, Aguirre et al. reported in vitro spontaneous chamber models with specific cell types (such as the sinoatrial node) (see, for example, Volmert, B. et al. Nat Commun 14, 8245 (2023) and Lewis-Israeli, Y. Ret al. Nat Commun 12, 5142 (2021)). However, organoids with the aforementioned chamber structures exhibit significant limitations in practical use. For instance, the chamber structure is unstable and can only be maintained for about a week under in vitro culture conditions. Further culture leads to the gradual collapse of the internal chambers, a time span far from sufficient for cardiac modeling that adequately simulates human cardiac development and physiological function (Non-Patent Literature 2, 5, 6). In addition, poor self-organizing ability, unclear boundaries between the myocardium and endothelial layer, and a single dependence on the WNT-BMP-HAND1 axis have been observed, preventing detailed and sustained analysis using these organoids.

[0004] Both the Wnt / β-catenin signaling pathway and the FGF signaling pathway are known to influence cardiac development. It has been reported that in in vitro EB differentiation or 2D differentiation methods, activation of the Wnt pathway (e.g., Wnt3a) during the mesodermal stage of pluripotent stem cells significantly increases cardiomyocyte differentiation rate (Non-Patent Literature 15, 16), and inhibition of the Wnt pathway after mesodermal formation increases the cardiac differentiation efficiency of pluripotent stem cells (Non-Patent Literature 17, 18). FGF has also been reported to regulate stem cell pluripotency and cardiac development. For example, when FGF2 binds to 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 facilitates the differentiation of stem cells into cardiac fibroblasts (Non-Patent Literature 21) and heart-like cells (Non-Patent Literature 2, 5); FGF2 regulates Wnt signaling by activating the PI3-K / GSK3 pathway, thereby maintaining pluripotency (Non-Patent Literature 22, 23). The complex interactions between these signaling pathways present a significant challenge to clearly elucidating the key factors influencing heart formation and development.

[0005] To date, it remains unclear how to address the long-term stability issues of in vitro cultured heart models. Summary of the Invention

[0006] Through in-depth research, the inventors of this application discovered a close synergy between the fibroblast growth factor (FGF) signaling pathway and the WNT-BMP axis in the initiation, formation, and stabilization of cardiac chamber development. By activating and / or inhibiting the FGF and Wnt-BMP axis at different stages of induction culture, especially by inhibiting the FGF signaling pathway during the critical stage of differentiation from mesoderm to cardiac mesoderm, the natural development of the heart can be well simulated. Cardiac organoids with internal chambers 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 chambers can be significantly extended from several days to several months or even longer, thus providing cardiac organoids with internal chambers that can exist stably in vitro for a long period. Such organoids can serve as scientific research tools to enhance our understanding of cardiac developmental biology. More importantly, they have extremely broad application prospects in the fields of bioengineering and medicine, including but not limited to disease modeling, drug screening, screening and evaluation of drug toxicity and side effects, cardiac tissue engineering, regenerative medicine, and more. For example, cardiac organoids, generated separately from iPSC libraries containing healthy individuals and patients with specific indications, can be used, according to the techniques described herein, to obtain more personalized test results regarding the efficacy and toxicity of drugs.

[0007] Therefore, one object of the present invention is to provide a method for generating cardiac organoids, the method comprising the following steps: A) activating Wnt signaling in a cell aggregate containing pluripotent stem cells to induce the cell aggregate to differentiate into a cell aggregate of mesodermal cells; B) during the process of stopping the activation of Wnt signaling and causing the cell aggregate of mesodermal cells to differentiate into cardiac mesoderm, inhibiting FGF signaling in the cell aggregate to promote the differentiation of the cell aggregate into cardiac mesoderm; and C) stopping the inhibition of FGF signaling, allowing the cardiac mesoderm to continue differentiating into cardiac organoids.

[0008] In some embodiments of the method of the present invention, inhibiting FGF signaling includes contacting mesodermal cell aggregates with an FGF receptor inhibitor. In some embodiments, the FGF receptor inhibitor is selected from any one or any combination of FGFR1 inhibitors, FGFR2 inhibitors, FGFR3 inhibitors, or FGFR4 inhibitors. In a preferred embodiment, 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 a preferred embodiment, 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. In a preferred embodiment, the FGF receptor inhibitor is PD173074.

[0009] In some embodiments of the method of the present invention, the duration of inhibition of FGF signaling is greater than 0.1 hours. In some embodiments of the method of the present invention, the duration of inhibition of FGF signaling does not exceed the time required for the differentiation of mesodermal cell aggregates into cardiac mesoderm without the inhibition of FGF signaling. This specific differentiation time varies depending on the starting cells used to generate organoids and the induction culture conditions, but can be readily determined by those skilled in the art based on the size of the formed chambers and the duration of stable existence, for example, based on the expression of surface markers specific to mesodermal cells and cardiac mesodermal cells, or based on empirical results from reported induction protocols. As a non-limiting example, the duration of suppression of FGF signal transduction in the method of the present invention can be, for example, from 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 FGF signaling inhibition is the time during which the mesodermal cell aggregates are exposed to the FGF receptor inhibitor. The concentration of the FGF receptor inhibitor used can be determined based on the inhibitor's inhibitory activity, as long as it substantially inhibits FGF signaling in the mesodermal cells. For example, the specific concentration can be easily determined using a serial dilution method based on the inhibitor's IC50 or Ki value. As a non-limiting example, the concentration of the FGF inhibitor can be 0.1–10 μM, preferably 0.2–5 μM, more preferably 0.5–2.5 μM, 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 μM.

[0011] In some embodiments of the method of the present invention, step B further includes inhibiting Wnt signaling during the differentiation of mesodermal cell aggregates into cardiac mesoderm. The inhibition of Wnt and FGF signaling can begin in any order, as long as both occur during the differentiation from mesoderm to cardiac mesoderm, and the inhibition of Wnt signaling occurs at least partially simultaneously with the inhibition of FGF signaling. In some embodiments, Wnt signaling inhibition begins before FGF signaling inhibition begins. In other embodiments, Wnt signaling inhibition begins simultaneously with FGF signaling inhibition. In still other embodiments, Wnt signaling inhibition begins after FGF signaling inhibition begins. Those skilled in the art can determine the timing of simultaneous inhibition of Wnt and FGF signaling based on the size of the formed chamber and its stable duration. As a non-limiting example, the duration for which both Wnt and FGF signal transduction are suppressed can be selected from 1 to 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 present invention, inhibiting Wnt signaling includes contacting mesodermal cell aggregates with a Wnt inhibitor. In some embodiments, 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, GinsenosideRh4, Prodigiosin, Triptonide, IWP-4, or KY-05009, or any combination thereof. In a preferred embodiment, the Wnt inhibitor is IWP2.

[0013] In some embodiments, the duration of inhibition of Wnt signaling is the time during which mesodermal cell aggregates are exposed to the Wnt inhibitor. The concentration of the Wnt inhibitor used can also be determined based on the inhibitor's inhibitory activity, as long as it substantially inhibits Wnt signaling in mesodermal cells. For example, the specific concentration can be easily determined using a serial dilution method based on the inhibitor's IC50 or Ki value. As a non-limiting example, the concentration of the Wnt inhibitor can be 1–10 μM, preferably 2–7 μM, more preferably 3–6 μM, such as 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 10μM.

[0014] In some embodiments of the method of the present invention, step B of the method of the present invention further includes contacting the cell aggregates of mesodermal cells with TGF-β inhibitors, RA agonists and / or BMP pathway agonists. In some embodiments, the TGF-β inhibitor is selected from SB431542, Dorsomorphin 2HCl, Dorsomorphin, LDN-193189, Galunisertib (LY2157299), LY2109761, SB525334, LDN-193189 2HCl, 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, LY3200882, 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 retinoic acid, Bexarotene, TTNPB, AM580, Adapalene, Acitretin, Tazarotene, Tamibarotene, SR 11237, Etretinate, BMS493, Palovarotene, Alltrans-Retinal, CD437(AHPN), or MSU-42011, or any combination thereof, preferably retinoic acid. In some embodiments, the BMP pathway agonist is selected from any one or any combination of BMP4, Activin A, BMP2, SJ000291942, or SB4, preferably BMP4 and / or Activin A. In some embodiments, the concentration of the TGF-β inhibitor is 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 concentration of the RA agonist is 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 from 0.5 to 500 ng / mL, preferably from 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 μM 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 invention, the resulting cardiac organoids comprise cardiomyocytes and endocardial cells that, together with the cardiomyocytes, define at least one chamber, which can be maintained for more than 30 days. In a preferred embodiment, the chamber in the cardiac organoid produced by the method of the present invention can be maintained for more than 60 days. In a more preferred embodiment, the chamber in the cardiac organoid produced by the method of the present invention can be maintained for more than 90 days. In this document, “maintained” in the description of a chamber means that the size and / or structure of the chamber has not undergone substantial change. For example, compared to an organoid just obtained by the method of the present invention, the chamber is reduced in size by less than 20% in its maximum dimension, preferably less than 15%, more preferably less than 10%. Or, for example, compared to an organoid just obtained by the method of the present invention, the layered structure, integrity, cellular composition, function, and / or activity of the chamber wall have not undergone substantial change.

[0016] In some embodiments, the resulting cardiac organoids further include at least epicardial cells disposed on the outer surface of the organoids.

[0017] In some embodiments, the method of the present invention further includes the step of fusing the resulting cardiac organoids with epicardial cells. The epicardial cells can be obtained by any known method, such as the method described in Non-Patent Document 2.

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

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

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

[0021] In some embodiments, the cardiac organoids of the present invention comprise about 40% to about 80% of cardiomyocytes and about 5% to about 15% of endocardial cells, based on the total number of organoid cells. In some embodiments, the cardiac organoids of the present invention comprise about 40% to about 80% of cardiomyocytes, about 5% to about 15% of endocardial cells, and about 1% to about 15% of epicardial cells, based on the total number of organoid cells. In some embodiments, the cardiac organoids of the present invention comprise about 40% to about 80% of cardiomyocytes, about 5% to about 15% of endocardial cells, and about 1% to about 2% of epicardial cells, based on the total number of organoid cells. In some embodiments, the cardiac organoids of the present invention comprise about 40% to about 80% of cardiomyocytes, about 5% to about 15% of endocardial cells, and about 5% to about 15% of epicardial cells, based on the total number of organoid cells. In a preferred embodiment, the cardiac organoid of the present invention comprises approximately 50% to approximately 75% cardiomyocytes, approximately 7% to approximately 10% endocardial cells, and approximately 7% to approximately 10% epicardial cells, based on the total number of cells in the organoid. In some embodiments, the ratio of cardiomyocytes to endocardial cells in the cardiac organoid of the present invention is approximately 8:1 to approximately 15:1, preferably approximately 10:1 to approximately 12:1. In some embodiments, the endocardial cells in the cardiac organoid of the present invention are aggregated within the organoid and define at least one lumen. In some embodiments, the lumen is closed or partially closed.

[0022] In some embodiments, the cardiac organoids of the present invention are spherical or nearly spherical three-dimensional structures. In some embodiments, the cardiac organoids of the present invention have a size of about 0.5 to about 2.5 mm in their maximum dimension, preferably about 1 to about 2 mm. In some embodiments, the cardiac organoids of the present invention have 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, or 2.5 mm in their maximum dimension. In some embodiments, the size of the internal chambers in the cardiac organoids of the present invention in their maximum dimension is at least about 60%, preferably at least about 70%, more preferably at least about 80%, and even more preferably at least about 90%.

[0023] A fourth aspect of this application also provides the use of the cardiac organoids of the present invention in constructing disease models. In some embodiments, the disease is selected from diseases or indications related to abnormalities in the tissue structure and / or physiological function of the heart. As non-limiting examples, examples include any of the following: myocardial infarction, heart failure, coronary artery disease, arterial stenosis, heart attack, arrhythmia, arrhythmia, heart failure, valvular heart disease, congenital heart disease, cardiomyopathy, cardiomyopathy, pericardial disease, aortic disease, Marfan syndrome, hereditary cardiomyopathy, non-hereditary cardiomyopathy, cardiac hypertrophy, cardiac dysfunction due to stress overload, or damage to cardiac tissue.

[0024] The fifth aspect of this application also provides the use of the cardiac organoids of the present invention in screening drug candidates, or a method for screening drug candidates using the cardiac organoids of the present invention. In some embodiments, the drug candidates are drug candidates for the prevention and / or treatment of diseases or indications related to abnormalities in the tissue structure and / or physiological function of the heart. In some embodiments, the use or method includes contacting the cardiac organoids of the present invention with candidate molecules to be screened, and selecting candidate molecules that are not detected to cause a substantial difference in the characteristics of the cardiac organoids before and after contact. In some embodiments, the characteristics may be selected from any or any combination of the following: the external morphology of the cardiac organoids; the size of the cardiac organoids in the largest dimension (e.g., volume, cross-sectional area, etc.); the total number of cells contained in the cardiac organoids; the proportion of a specific cell type (e.g., cardiomyocytes, endocardial cells, epicardial cells) to the total number of cells; the proportion of specific cell types among themselves; spontaneous beat rate; luminal size (e.g., volume, cross-sectional area, etc.); the proportion of the luminal size in the organoids in the largest dimension; and specific physiological or biochemical functions of the cardiac organoids (e.g., expression levels of specific genes, enzymes, or biomarkers, etc.).

[0025] The sixth aspect of this application also provides the use of the cardiac organoids of the present invention in evaluating the toxicity of drug candidates, or a method for evaluating the toxicity of drug candidates using the cardiac organoids of the present invention. In some embodiments, the toxicity includes undesirable effects on the tissue structure and / or physiological function of the heart. In some embodiments, the use or method includes contacting the cardiac organoids of the present invention with a drug candidate and measuring changes in the properties of the cardiac organoids before and after contact. In some embodiments, the properties may be selected from any one or any combination of the following: the external morphology of the cardiac organoids; the size of the cardiac organoids in the largest dimension (e.g., volume, cross-sectional area, etc.); the total number of cells contained in the cardiac organoids; the proportion of a specific cell type (e.g., cardiomyocytes, endocardial cells, epicardial cells) to the total number of cells; the proportion of specific cell types among themselves; spontaneous beat rate; luminal size (e.g., volume, cross-sectional area, etc.); the proportion of the luminal size in the organoids in the largest dimension; and specific physiological or biochemical functions of the cardiac organoids (e.g., expression levels of specific genes, enzymes, or biomarkers, etc.).

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[0052] Figure 1 a is a schematic diagram of a scheme for in vitro differentiation to form a heart-like organism with stable chambers;

[0053] Figure 1b shows a real-time bright-field image of the time process of forming a chambered cardiac organoid. Scale bar: 500 μm;

[0054] Figure 1 c shows a typical bright-field image at day 15.5, showing pulsating cavity structures that are consistently present in three independent biological replicates.

[0055] Figure 1 Figure d shows a frozen section of a heart-like structure at day 60.5, showing cavity formation and the expression of cardiomyocyte markers TNNT2 (top, green), ventricular marker MYL3 (red), and atrial marker NR2F2 (bottom, green), with a scale bar of 200 μm;

[0056] Figure 1 e shows the results of the scRNA-seq analysis, depicting the proportions of cardiomyocytes, atrial cells, and ventricular cells in the heart-like structure;

[0057] Figure 1 f shows a heatmap depicting the expression patterns of key genes during cardiomyocyte differentiation, where VST represents variance-stable transition counts;

[0058] Figure 1 g shows an exemplary image of the endocardial lining at day 20.5, in which the cardiomyocyte marker TNNT2 is shown in green and the endocardial layer marker CDH5 is shown in red, with a scale bar of 200 μm;

[0059] Figure 1 h shows real-time images of a heart-like structure taken under tdTomato or bright-field conditions;

[0060] Figure 1 i is a schematic diagram of the three-layered structure of a heart-like organ: epicardium, myocardium, and endocardium;

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

[0062] Figure 2 b shows an electron microscope image of the heart-like structure at day 90.5, with arrows indicating key structures: S for sarcomere, ID for intercalated disc, and Z for Z-line;

[0063] Figure 2 c shows a typical electrocardiogram (trace) and mean action potential recording of a heart-like (D100) model;

[0064] Figure 2 d shows in Figure 2The characteristics of the action potential observed in the data shown in c are as follows: RMP represents the resting membrane potential, APD represents the duration of the action potential, and APA represents the amplitude of the action potential.

[0065] Figure 2 e shows the heart-like beat rate over different days, BMP represents heart rate per minute, and all bars show the mean ± SD;

[0066] Figure 2 f shows the results of calcium imaging analysis of organoid loading with Fluo-4-AM and monitoring the time-dependent changes in fluorescence intensity. F / F0 represents the fluorescence intensity relative to the background level, and the scale bar is 500 μm.

[0067] Figure 3 a shows that chamber formation in a heart-like organ depends on PD173074 (PD), scale bar 500 μm;

[0068] Figure 3 b shows a typical bright-field image at day 16.5, showing a solid heart organoid structure that appears consistently in three independent biological replicates without PD, at a scale bar of 500 μm.

[0069] Figure 3 c shows a cardiac-like frozen section (with or without PD) at day 16.5, with a scale bar of 200 μm;

[0070] Figure 3 d is a heatmap depicting the expression patterns of key genes during cardiomyocyte differentiation with or without the addition of PD. The key genes include cardiomyocyte markers and ion channels. VST represents variance-stable transition counts.

[0071] Figure 3 e is a PCA plot showing different cell fates under conditions with and without PD added;

[0072] Figure 3 f shows the Venn plot of upregulated or downregulated genes in the heart-like structure at day 3.5, with PD added as a control;

[0073] Figure 3 g and 3h show the upregulated or downregulated GO and KEGG terms in the heart-like organ at day 3.5, with the case of added PD as a control;

[0074] Figure 3 i shows the cell distribution in organoids of the PD-added and non-PD-added groups. Red represents cells in the PD-added group (+PD), and blue represents cells in the PD-unadded group (-PD).

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

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

[0077] Figure 3 l shows a UMAP scatter heatmap of cell clustering annotation based on the expression of different biomarkers; the darker the color, the higher the expression level.

[0078] Figure 4 Figure a shows a visualization of the data distribution, illustrating the distribution of cells in 2D and 3D culture samples. Cells in 2D samples are marked in red, and cells in 3D samples are marked in blue. This helps to visualize the cell distribution in each sample of the dataset.

[0079] Figure 4 b shows the visualization results of cell type annotation, a UMAP visualization that provides cell type annotation for 2D and 3D cultures using single-cell RNA-seq data, with different colors representing different cell types;

[0080] Figure 4 c shows the expression of marker genes. Marker genes of various cell types are depicted by UMAP scattering heatmaps. The color gradient represents the expression level. Cell annotation is performed based on the expression of marker genes in different clusters.

[0081] Figure 4 d shows the cell type diversity. According to the annotation results, the 3D culture showed higher cell type diversity than the 2D group. The differentially expressed genes specific to endocardial and epithelial progenitor cells in the 3D culture were enriched and analyzed.

[0082] Figure 4 e is a violin diagram showing the results of gene expression analysis, comparing the distribution range of expression levels of key genes marking cardiac maturation under 2D and 3D culture conditions;

[0083] Figure 5 a shows real-time bright-field images of a heart-like organ with stable chambers treated with different drugs. All test drugs were added to the heart-like organ starting from day 0, including aspirin at 30 μM, thalidomide at 10 μg / ml, and atratin at 50 nM.

[0084] Figure 5 b shows the quantification results of the chamber area of ​​the heart-like organ treated with the drug;

[0085] Figure 5c shows the number of heartbeats per minute treated with the drug;

[0086] Figure 5 Image d shows a real-time bright-field image of a heart-like organ with stable chambers at day 36.5, which had been treated with thalidomide since day 15.5.

[0087] Figure 6 a is a graph showing the temporal changes of a chambered cardiac organoid formed using the induction scheme of Non-Patent Document 2, captured in real-time bright-field imaging, with a scale bar of 500 μm.

[0088] Figure 6 b shows a heart-like frozen section prepared using the induction protocol of Non-Patent Document 2 at day 6.5, with a scale bar of 200 μm;

[0089] Figure 6 c shows the proportions of cardiomyocytes, atrial cells, and ventricular cells in the heart-like structure as displayed in the UMAP diagram;

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

[0091] Figure 6 e shows typical bright-field images of two independent biological replicates at day 90.5, with a scale bar of 500 μm;

[0092] Figure 6 f is a confocal image of the epicardium formed on day 9.5 using the reported method;

[0093] Figure 6 g shows a typical bright-field image of a chambered heart-like organ derived from H1 and UiPS cell lines at day 15.5, where UiPS represents urinary induced pluripotent stem cells, with a scale bar of 500 μm.

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

[0095] Figure 7 Image a is a time-lapsed immunostaining image of a frozen section, showing that the formation of cardiac organoids with chambers is not related to apoptosis and proliferation. Scale bar: 200 μm.

[0096] Figure 7 b is a heatmap showing the expression patterns of genes related to proliferation and apoptosis during cardiomyocyte differentiation;

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

[0098] Figure 7 Image d shows a confocal frozen section image of NKX2.5 KO-type heart cells (from two cell lines) stained with TNNT2;

[0099] Figure 8 a is a bright-field image that varies over time, showing the formation of a chambered heart-like structure without the addition of PD173074;

[0100] Figure 8 b is a bright-field image showing the time-dependent changes in organoids induced by 1 μM PD166866;

[0101] Figure 8 c shows a typical bright-field image of the heart-like organism induced by 1 μM PD166866 on day 17.5;

[0102] Figure 8 d is a volcano plot comparing gene expression profiles during the cardiac mesodermal stage (day 3.5) and the ventricular formation stage (day 10.5), with PD added as a control;

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

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

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

[0106] Figure 9 b shows a UMAP visualization of cell type annotation results for scRNA data from 3D cultures, with cell types represented by color.

[0107] Figure 9 c shows UMAP scattering heatmaps of marker genes in different cell types in 2D cultures, with hues indicating expression levels;

[0108] Figure 9 d shows UMAP scattering heatmaps of marker genes in different cell types in 3D cultures, with hues indicating expression levels;

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

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

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

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

[0113] Figure 10 a shows typical bright-field images of a heart-like organ treated with various drugs on day 20.5: aspirin 30 μM, thalidomide 10 μg / ml, and atracine 50 nM.

[0114] Figure 10 b shows magnified bright-field images of the heart-like organ treated with different drugs on day 1.5;

[0115] Figure 10 c shows magnified bright-field images of the heart-like organ treated with different drugs on day 3.5;

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

[0117] Figure 10 e shows the heart rate per minute of the heart-like cells treated with thalidomide starting on day 15.5 at day 36.5;

[0118] Figure 11 A shows the changes in the expression levels of endothelial markers during organoid formation (n=2), where FPKM represents the number of fragments / kilobase pairs of transcripts / million mapping reads;

[0119] Figure 11 B shows the cardiomyocyte (outer) and endothelial (inner) layers in the organoids at days 7.5 and 17.5; scale bar is 200 μm;

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

[0121] Figure 11 D shows typical bright-field images of organoids treated with 0.5 μM SB431542 for 20 days at day 48.5, and bright-field images of ETS1 knockout cardiac organoids at day 28.5; scale bar is 500 μm.

[0122] Figure 11 E and 11F are the chamber area (N=3, N=48) and stroke rate (N=3, N=30) of cardiac organoids treated with or without 0.5 μM SB431542, measured on days 40.5 to 48.5, respectively.

[0123] Figure 11 G represents the gene expression of biomarkers associated with endothelial-mesenchymal transition (EndoMT) during organoid formation from day 0 to day 25.

[0124] Figure 11 H represents a typical bright-field image of organoids treated with 0.5 μM SB431542 for 20 days on day 48.5, with or without treatment.

[0125] Figure 12 A violin diagram showing the expression of myocardial maturation-related genes in organoids at days 25.5 and 43.5;

[0126] Figure 13 The development of exemplary organoids of this disclosure at days 25.5 and 43.5, obtained using Slingshot pseudo-time trajectory analysis, is shown.

[0127] Figure 14 To show the average motion amplitude of the organoids of this disclosure at 0 seconds and 1 second after treatment with isoproterenol, the scale bar is 170 μm;

[0128] Figure 15 The figures show the number of beats per minute (left, N=3, N=7) and the average displacement of contractile activity over time in organoids after treatment with isoproterenol (right).

[0129] Figure 16 A and 16B show typical bright-field images (A) and chamber areas (B, N=3, N=9) of organoids of this disclosure before treatment with 5 μM doxorubicin, at 24 hours and 48 hours after treatment, respectively;

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

[0131] Figure 17A The diagram illustrates cell communication in organoids at day 25.5. The top diagram shows the signaling pathway that uses endothelial cells as transmitters and affects other cell types (such as fibroblasts, atrial cardiomyocytes, and ventricular cardiomyocytes), while the bottom diagram shows the pathway that uses endothelial cells as receptors. The arcs represent different signaling pathways.

[0132] Figure 17B A probability heatmap illustrating key signaling pathway interactions between different cell types;

[0133] Figure 17C and 17D The signaling pathways from endothelial cells to other cell types and from other cell types to endothelial cells are shown respectively. The color of the circle indicates the communication probability, and the size indicates the importance of the pathway.

[0134] Figure 18 is a heatmap showing the probability of LAMA signaling pathway interactions between various cell types in the exemplary organoid (A) of this disclosure and the heart of a 5- to 6-week-old human fetus (B).

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

[0136] Figure 19 B shows typical bright-field images of cardiac organoids induced with different concentrations of AA and RA on day 25.5;

[0137] Figure 19 C shows typical bright-field images of cardiac organoids induced by different concentrations of AA on day 8.5;

[0138] Figure 19 D represents the chamber area of ​​the cardiac organoids measured on days 15.5 and 25.5 after induction with different concentrations of AA and RA. Detailed Implementation

[0139] definition

[0140] As used in the specification of this invention, the following words and phrases are generally considered to have the meanings set forth below, unless otherwise specified in the context in which they are used.

[0141] As used herein, the singular forms “a,” “an,” and “the” refer to both the singular and the plural unless the context clearly indicates otherwise. As used herein, the terms “comprising,” “including,” and “having” mean that a composition or method includes the described components or steps, but does not exclude other components or steps.

[0142] As used herein, the term "about" refers to a common range of error for a corresponding value that is readily known to those skilled in the art. For example, less than ±10%, ±5%, ±2%, ±1%, or ±0.1% relative to the described value. Values ​​or parameters described herein using the term "about" include 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 various cell types (such as nerve cells, cardiomyocytes, muscle cells, etc.) derived from the ectoderm, mesoderm, and endoderm 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 cluster or aggregate of cells that resembles or is part of an organ and possesses a cell type associated with that particular organ. Organoids typically have the same or similar structural or tissue features as that particular organ and exhibit at least one physiological process or function of that particular organ.

[0145] As used herein, the term "cell aggregate" refers to a structure composed of multiple cells forming a three-dimensional (3D) shape rather than growing in a single layer. In some embodiments, the cell aggregates of the present invention are multilayered cell aggregates. In some embodiments, the cell aggregates of the present invention are spherical or nearly spherical.

[0146] As used herein, the term "activation" in describing a signaling pathway refers to the initiation, occurrence, or increased activity level of a biological process, physiological response, or molecular event associated with the signal transduced by that pathway. Conversely, the term "inhibition" in describing a signaling pathway refers to the initiation, occurrence, or decreased activity level of a biological process, physiological response, or molecular event associated with the signal transduced by that pathway.

[0147] As used herein, the term "mesoderm" refers to one of the three main cell layers (ectoderm, endoderm, and mesoderm) that forms during embryonic development, located between the ectoderm and endoderm, and has the potential to form a variety of organs and tissues (including, but not limited to, the heart, bones, muscles, urinary system, reproductive system, and circulatory system). Methods and markers for identifying mesodermal cells are well known in the art; as an example, the mesoderm may express one or more markers selected from the following (e.g., at least three to five markers): TBXT + MESP1 + MIXL1 + Brachyury, Foxa2, Sox17, Nodal, Eomesodermin, Tbx6, and Wnt3a, etc.

[0148] As used herein, the term “cardiac mesoderm” refers to an intermediate state in the development of the primitive mesoderm into the various cell types of the heart (such as cardiomyocytes, endocardial cells, and epicardial cells), representing cells derived from the primitive mesoderm that have specialized to have the fate of differentiating into the various cell types that constitute the heart. Methods and markers for identifying the cardiac mesoderm are well known in the art; see, for example, 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 characteristic of the cardiac mesoderm is the expression of cardiac mesoderm-specific markers, including one or more of, for example, HAND1, Isl1, NKX2-5, and GATA4.

[0149] As used herein, the term "cardiomyocyte" encompasses cells at any stage of cardiomyocyte individual development, primarily including progenitor cells and mature cardiomyocytes, unless otherwise stated. As an example, cardiomyocytes may express one or more markers selected from the following (e.g., at least 3 to 5 markers): TNNT2, NR2F2, cardiac troponin I (cTnI), cardiac troponin T (cTnT), myosin heavy chain (MHC), GATA-4, Nkx2.5, N-cadherin, β1-adrenergic receptor (β1-AR), ANF, MEF-2 family transcription factors, creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor (ANF), etc. Furthermore, in this invention, "cardiomyocytes" may be characterized by beating. Additionally, the "cardiomyocytes" of this invention may also be characterized by forming striped structures.

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

[0151] As used herein, the term “epidermal cells” refers to cells at any stage of epicardial formation and maintenance, primarily including epicardial progenitor cells and mature epicardial cells, unless otherwise stated. As an example, epicardial cells may express one or more markers selected from the following (e.g., at least 3 to 5 markers): WT1, CD90 (Thy1), Desmin, Vimentin, α-SMA (α-smooth muscle actin), Fibronectin, PDGFR-α (platelet-derived growth factor receptor α subunit), etc.

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

[0153] As used in this article, the term "chamber" refers to a cavity or space within an organism, organ, or tissue, typically used to contain liquids, gases, or other substances. In cardiac anatomy, chambers usually refer to the atria and ventricles of the heart, which play a vital role in collecting and pumping blood.

[0154] Example

[0155] reagents or materials

[0156] Table 1

[0157]

[0158]

[0159]

[0160] Cell line culture

[0161] HEK293T cells (CRL-3216) and human ESCs (H9 and H1) were obtained from the American Type Culture Collection (ATCC). HEK293T cells were cultured in Dulbecco modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Human ESCs and self-made UiPSCs (Non-Patent Literature 11, 12) were cultured in Matrigel (Corning, #354230)-coated plates in mTeSR1 medium (Stemcell Technologies, #88550) and passaged every 2–4 days at 70%–90% confluence using TrypLE Express Enzyme (Thermo Fisher, #12563029) or 0.5 mM EDTA-DPBS (Sigma-Aldrich, #E6758). Mycoplasma contamination was detected in the cells according to standard procedures. All cell lines were cultured at 37°C in a humid atmosphere containing 5% CO2.

[0162] 3D heart organoids

[0163] hPSCs were isolated using Accutase (Stemcell Technologies, #07920) and resuspended in mTeSR1 medium supplemented with 10 μM ROCK inhibitor Y-27632. Subsequently, hPSCs were seeded at 20,000 cells / well in Ultra-Low-Attachment U-bottom 96-well plates (Corning #7007 or Thermo Fisher #1742929), recorded as Day-1. The plates were then incubated for 24 hours. On Day 0, the medium was replaced with Chemically Defined Medium (CDM) containing 30 ng / mL LFGF, 3 ng / mL BMP4, 5 μM CHIR99021, 5 μM LY294002, and 10 ng / mL activin A (see Non-Patent Literature 24). CDM consisted of a mixture of 50% IMDM (Gibco, #112440053) and 50% F12 NUT-MIX (Thermo Fisher, #111765054), supplemented with 15 μg / mL transferrin, 450 μM monothioglycerol, and 5 mg / mL BSA. After a 36–40 hour nutrient period, embryonic bodies were incubated for 2 days in 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 the medium changed daily. To maintain the cardioids, from day 6.5 onwards, CDM medium containing 10 μg / mL insulin was used, with the medium changed every 2–4 days. Different compartments could be observed from day 6.5, allowing for further investigation procedures.

[0164] 2D epicardium

[0165] Cells were seeded at 150,000 cells / well into MG-coated 24-well plates. On Day 0, the medium was replaced with CDM containing 30 ng / mL FGF, 3 ng / mL BMP4, 10 μM CHIR99021, and 5 μM LY2940021. The CDM was a mixture of 50% IMDM and 50% F12 NUT-MIX, supplemented with 15 μg / mL transferrin, 450 μM monothioglycerol, and 5 mg / mL BSA. After a 36–40 hour nutrient period, embryoids were incubated for 2 days in CDM medium containing 3 ng / mL BMP4, 5 μM MIWP2, and 1 μM BMS. From Day 3.5 to Day 6.5, cell spheroids were cultured in CDM medium containing 10 μg / mL insulin, 10 ng / mL BMP4, 5 μM CHIR99021, and 1 μM retinoic acid. To maintain the epicardium, CDM medium containing 10 μg / mL insulin and 5 μM SB-431542 was used from day 6.5, and the medium was replaced every 2 days.

[0166] Frozen sections and immunostaining

[0167] Before sectioning, the organoids were dehydrated in 30% sucrose PB and fixed in 4% PFA (Beyotime, #P0099). For sectioning, the organoids were embedded in OCT (Sakura, #4583) and cut into 10μm sections using a Leica cryostat at -22℃ to -20℃ for immunofluorescence.

[0168] Frozen sections were post-fixed in 4% PFA and permeated for 15 minutes each in blocking solution (Beyotime, #P0102) of 0.2% Triton X-100 (Sigma-Aldrich, #T9284). After washing with DPBS, frozen sections were blocked with the above blocking solution and then incubated at room temperature (RT) for 4 hours or overnight at 4°C with primary antibody diluted in primary antibody dilution matrix (Beyotime, #P0103) according to the manufacturer's instructions. Sections were then washed three times with PBS / 0.1% Tween 20 and stained with secondary antibody diluted in secondary antibody dilution matrix (Beyotime, #P0108) at RT for 2 hours. Sections for observation were prepared by co-staining with DAPI (Abcam, #ab104139) at RT for 5 minutes. Images were acquired from at least three organoids using an inverted confocal microscope (Zeiss, LSM900 or LSM800).

[0169] Alternatively, without sectioning, the entire organoid can be immunostained. Heart organoids are collected, rinsed once in PBS, and then fixed overnight (16-20 hours) in 4% PFA at 4°C. The next day, after washing the organoids twice in PBS, they are washed with BS / Tween 20 for at least 15 minutes each time. Then, at room temperature, they are permeabilized with 0.2% Triton X-100 blocking solution for 15-30 minutes and blocked in blocking solution for 1 hour. The primary antibody is placed in primary antibody buffer and incubated on a shaker at 4°C for 24 hours. After washing the labeled sample three times with PBS / 0.1% Tween 20 (10 minutes each time), the sample is incubated with the secondary antibody on a shaker at 4°C for 24 hours. Then, it is washed once with PBS / Tween 20 for 10 minutes and stained with DAPI solution (2 μg / mL) for 5 minutes. Then, it is washed three times with PBS / Tween 20 for 10 minutes each time. Finally, the prepared sample is imaged.

[0170] Generate cell lines with DETS1, HAND1, and NKX2-5 knocked out

[0171] The knockout strategy was implemented using the CRISPR / Cas9 system. The sgRNA was identified using the website (http: / / crispor.tefor.net / crispor.py) and cloned into the hU6-sgScaffold site of pX330-puro. The targeting efficiency of the sgRNA was validated in the 293T cell line. Nucleofector was used. TM 2b (Lonza BioResearch, program B16), per 1x10 6 Cells were transfected with 2 μg pX330 sgRNA and 4 μg Donor per cell. After nuclear infection, cells were incubated in mTeSR1 containing 10 mM Y-27632 for 24 h, then selected with 0.1 ng / mL puromycin (Beyotime, #ST551) for 48–72 h, and then transfected into mTeSR1 containing 10 mM Y-27632. When cells reached 50% confluence, ESCs were digested with Accutase and seeded at 0.8 cells / well in 96-well plates. Single clones became visible to the naked eye after 4–5 days and could be transferred for genotyping (using two different primer pairs) after 10 days to confirm successful knockout.

[0172] Transmission electron microscope

[0173] Fixed the samples in 150 mM HEPES buffer (pH 7.2) containing 2.5% glutaraldehyde and 2% formaldehyde for 30 minutes at room temperature, or overnight at 4°C. Wash the fixed samples with 0.1 M PB / Cacodylate buffer (pH 7.2–7.4). Fix the samples in 1% osmium tetroxide at 4°C for 1 hour, and then in 1% uranyl acetate at room temperature for 1 hour or overnight at 4°C. Dehydrate the samples in serially diluted ethanol (50%, 70%, 90%, 95%, and 100% twice) and embed them in EPON12 resin at 60°C for 24–48 hours. Cut the samples into 70 μm sections using a diamond microtome (Leica) on a UC7 microtome. After staining with basic dimethylamine blue and compound red, 10 nm thick sections were collected onto a copper single-slot grid coated with polymethyl methacrylate (formvar), stained with 2% uranyl acetate and lead citrate, and observed under a 120 kV transmission electron microscope (Thermo-scientific, Talos 120).

[0174] Shrinkage characteristics measurement

[0175] Real-time imaging was performed using a high-resolution imaging system. The algorithm described in Huebsch N et al., *Tissue Eng PartCMethods.*, 2015; 21:467-479, was used to analyze the video of dynamic contraction behavior. This algorithm quantitatively assesses the contraction velocity of the cardiac catheter by tracking the motion of a specific region over time.

[0176] Calcium imaging

[0177] Calcium flux was imaged by recording real-time fluorescence signals from Fluo-4 AM. Briefly, after washing with DPBS, organoids were incubated at 37°C for 30 min in medium supplemented with 2 μM Fluo-4 AM (Beyotime, #S1060) and 0.1% Pluronic F-127 (Beyotime, #St501). Then, the organoids were transferred to glass-bottom plates (Cellvis, #P96-1.5HN) with fresh medium and equilibrated at 37°C for 15 min. Organoids were then prepared for recording using a multi-mode Spinning Disk Confocal System (Olympus, Spin SR10) at a frame rate of 50 ms per frame for at least 30 s. Time-series images were analyzed using Fiji / ImageJ and processed using GraphPad Prism 9. Baseline fluorescence intensity (F) was calculated using asymmetric least-squares smoothing. Fluorescence change (ΔF / F) was determined using the following formula:

[0178]

[0179] To assess the effect of isoproterenol stimulation on calcium processing, patients were treated with 1 μM isoproterenol for 30 minutes prior to imaging. The calcium transient response after treatment was compared with that of the untreated control group to assess the effect of β-adrenergic stimulation on calcium dynamics.

[0180] Heart rate (BPM) measurement

[0181] Heart rate (beats per minute, BPM) was measured by directly observing organoid contraction under an Olympus IX73 inverted microscope. The mean BPM of the organoids was obtained by manual counting in at least three different fields of view from videos of spontaneous pulsation. BPM after isoproterenol treatment was calculated using real-time video recordings captured by the Olympus IX73 microscope after 30 minutes of stimulation with 1 μM isoproterenol, using Fuji / ImageJ software. The BPM after isoproterenol treatment was compared with the untreated control group to assess the effect of β-adrenergic stimulation.

[0182] Doxorubicin treatment, cell viability and cytotoxicity

[0183] Organoids cultured for 15.5–25.5 days were exposed to 5 μM doxorubicin. After 24 hours, the organoids were washed three times with PBS and replaced with fresh culture medium. To determine cell viability, 10 μL of CCK-8 reagent (Beyotime, #C0038) was added to each well and incubated at 37°C for 2 hours. Absorbance at 450 nm was measured using a microplate reader (Thermo Scientific). To assess cytotoxicity, the supernatant was collected 24 hours after doxorubicin exposure, and absorbance at 490 nm was measured according to the manufacturer's protocol (LabLead, #L0117). Cytotoxicity was assessed by LDH release.

[0184] Quantitative polymerase chain reaction (qPCR) analysis

[0185] Total RNA was extracted using the RNA-easy Isolation reagent (Vazyme, #701) according to the manufacturer's instructions. RNA quality and quantity were assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham). For cDNA synthesis, [the following was used]. Total RNA was reverse transcribed from IIQ RT SuperMix (Vazyme, #R222). Quantitative PCR (qPCR) was performed using ChamQSYBR qPCR Master Mix (Vazyme, #Q711) to analyze the expression levels of target mRNAs.

[0186] Electrophysiology

[0187] The method described in Non-Patent Literature 25 was used. Organoids were dissociated into single cells using the STEMdiff Cardiac Cell Dissociation Kit (Stemcell Technologies, #05025). Action potentials (APs) were triggered at 1 Hz using a 3 ms overthreshold stimulus. Briefly, APs were recorded at room temperature (RT) using whole-cell patch-clamp technology via 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 MEGTA, 10 mM HEPES, 1 mM CaCl2, and 1 mM MgCl2 (pH adjusted to 7.3 with KOH). When filled with internal solution, the series resistance of the glass electrode is typically 1.5–3 MΩ.

[0188] RNA-seq and data analysis

[0189] Following the manufacturer's instructions, total RNA was extracted using the RNA-easy Isolation Reagent (Vazyme, #701), and libraries were prepared using the VAHTS mRNA-seq v2 Library Prep Kit for Illumina (Vazyme, NR601-01 / 02) (1 μg RNA per sample). Sequencing was performed using an Illumina OVA seq instrument by Guangzhou Aiji Biotechnology Co., Ltd. (Guangzhou, China). For gene expression analysis, the raw sequencing data (raw data) was filtered to obtain high-quality sequencing data (clean data). The Sequence Alignment Tool STAR was used to align this data with 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 / ) to obtain data that could be mapped to the reference genome. Gene expression levels were then quantified using RSEM software, and the final normalized matrix was obtained from the quantification results. Data normalization and differential expression analysis were then performed using DESeq2 (v.1.26.0). For time-series experiments, the Wald test (Benjamini-Hochberg corrected p-value < 0.05 and absolute fold change >= 1.5) and the likelihood ratio test (Benjamini-Hochberg corrected p-value < 0.05) were used to define differentially expressed genes. Gene ontology analysis was performed using David (https: / / david.ncifcrf.gov / ).

[0190] PCA trajectory diagram

[0191] First, PCA analysis was performed on the gene expression matrix using the prcomp function in R, and then the PCA scores were extracted. Based on the PCA analysis results of different samples at each time point, points in the same group were connected by trajectory lines to represent the changing trends between samples. Finally, the PCA trajectory plots were drawn using the ggplot2 toolkit (v.3.5.0).

[0192] Heatmap

[0193] First, based on the gene expression matrix and experimental design information, a DESeqDataSet object is created using the DESeqDataSetFromMatrix function in the R package DESeq2 (v.1.26.0). Then, the varianceStabilisingTransformation function is used to perform a variance-stabilizing transformation to obtain the processed data matrix. Finally, the pheatmap function is used to draw a heatmap.

[0194] Single-cell (sc) RNA-seq and bioinformatics analysis

[0195] Organoids were dissociated into single cells using the STEMdiff cardiomyocyte dissociation kit (Stemcell Technologies, #05025). Cells were counted and viability was checked using a hematology analyzer (Thermo Fisher, Countess 3) before library preparation.

[0196] scRNA data from 2D cultures, 3D cultures, 3D cultures at day 25.5, PD173074-added groups, and no-added groups were processed using the R package Seurat. Cells expressing fewer than 3 genes in a sample, or genes expressed in fewer than 300 cells or more than 9000 cells in the entire sample, cells with mitochondrial gene content greater than 25% of the total number of unique molecular identifiers (UMIs), and cells with ribosomal gene content greater than 50% of the total number of unique molecular identifiers (UMIs) were excluded. After quality control, expression matrices were obtained containing 40191 cells and 27489 genes (2D and 3D), 11910 cells and 27601 genes (3D cultures at day 25.5), and 33348 cells and 27583 genes (PD173074-added and no-added groups). Next, the data were 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. Dimensionality reduction analysis was then performed using the RunPCA function. Clustering was performed based on the 20 most important principal components (PCs). Batch removal was then performed using the R package Harmony v0.1.0. Different resolutions were set, and the clustering effect was observed using a clustering tree, ultimately setting the resolutions to 0.9, 0.8, and 0.7. Unified Manifold Approximation and Projection (UMAP) visualizations were constructed using the same number of PCs as the relevant clusters. Samples from both 2D and 3D culture groups were clustered into 18 clusters, and marker genes for different clusters were identified using the FindAllMarkers function. By reviewing literature and consulting experts, marker genes for each cell type in the heart were identified, and cell types were annotated by the expression of these marker genes in different clusters.

[0197] Cell communication analysis

[0198] Intercellular communication analysis was performed using the CellChat software package (v1.6.1). CellChat objects were created using preprocessed scRNA-seq data from samples taken at day 25.5, with identified cell types used as cluster tags. Single-cell sequencing data from human embryos at 5 weeks (5 weeks) and 6 weeks (6 weeks) of gestation (obtained from the GEO database (GSE106118)) were used as a comparison for LAMA signaling pathway analysis. Potential communication networks were inferred by assessing ligand-receptor interactions across cell types using the CellChat human ligand-receptor database. The probabilities of these interactions were calculated and summarized to analyze major signaling pathways. Visualization methods such as chord diagrams and bubble diagrams were applied to illustrate intercellular communication patterns, highlighting interactions between endothelial cells and other cell types, and vice versa.

[0199] Slingshot for plotting stages of organoid-derived cardiomyocytes

[0200] Samples from the HE5W to HE25W stage (5 to 25 weeks post-conception) were selected from single-cell transcriptome data of human embryonic heart development (GSE106118) for comprehensive analysis. Simultaneously, single-cell sequencing data from cardiac organoids at days 25.5 and 43.5 were integrated to isolate TNNT2-positive cardiomyocyte subsets, and pseudo-temporal trajectory reconstruction was performed using the Slingshot algorithm. Cell density maps were generated based on the pseudo-temporal coordinates to map the developmental stages of organoid-derived cardiomyocytes.

[0201] Differential expression analysis of key markers in cardiomyocyte maturation (D25.5 vs. D43.5)

[0202] As described above, single-cell RNA sequencing (scRNA-seq) data from cardiac organoids at day 25.5 (D25.5) and day 43.5 (D43.5) were processed. Annotated cardiomyocytes were sub-histologized, and violin plots were generated to visualize expression patterns of genes related to cardiomyocyte function and maturation.

[0203] Marker gene enrichment analysis of 2D and 3D cultures

[0204] The FindMarkers function was used to identify marker genes specific to 3D cultures and other cell types, and significant marker genes were screened based on Padj < 0.05 and |log2FC| > 1. The screened genes were then subjected to David enrichment analysis (https: / / david.ncifcrf.gov / ), and significant pathways with an FDR < 0.05 were identified based on the enrichment results. Further analysis was then conducted to reveal the potential biological functions of 3D cultures.

[0205] Quantitative and statistical analysis

[0206] All analyses were performed using GraphPad software, and all raw data were collected in Microsoft Excel. All data were normally distributed. Where appropriate, standard unpaired Student's t-test (two-tailed; no significance (ns), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) was used to assess statistical significance. For multiple comparison analyses, one-way ANOVA (ns, no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) was used, adjusted for Tukey or Dunnett tests, where appropriate. Unless otherwise stated, all data are expressed as mean ± SEM, representing at least three independent experiments with at least three technical replicates per experiment. All photomicrographs are typical images from at least six independent experiments for each condition / marker, and calcium transient images are typical images from six independent experiments.

[0207] Example 1

[0208] This embodiment describes an exemplary method for inducing the formation of a heart-like organ with stable chambers from pluripotent stem cells.

[0209] The formation of cardiac chambers depends on the Wnt-BMP signaling pathway via HAND1, but existing technologies generally consider this insufficient to generate stable chambers (Non-Patent Literature 2, 5, 6). To address the stability issue, the inventors of this application systematically tested and analyzed the effects of fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), bone morphogenetic protein (BMP), activin, and Wnt on various stages of cardiac development, including pluripotency, mesoderm, cardiac mesoderm, cardiomyocyte formation, chamber formation, and ventricular maintenance. The results show... Figure 1 a, 1c, 6a, 6b, 6e. It is evident that the method described herein can achieve 100% success in forming chambers in organoids, with individual chambers appearing on day 11.5 and persisting until day 90.5. Figure 1 a, 1c and extended data Figure 1 e).

[0210] Immunofluorescence staining was performed on the obtained organoids, and the results were shown in... Figure 1d. It is evident that these stable organoids contain a large number of TNNT2-positive cells (representing cardiomyocytes) and NR2F2 cells (representing the atrial portion), and also possess a subset of cells expressing ventricular markers (Non-Patent Literature 2, 10). scRNA-seq analysis further validated these results. In these stable organoids with chambers, 87.4% of the cells are cardiomyocytes (TNNT2-positive cells). + Of these, 76.2% expressed the atrial marker NR2F2, and 11.5% expressed the ventricular marker MYL3. Figure 1 e and 6c); Figure 1 f shows the changes in markers during the development of these organoids, specifically the gradual progression from pluripotency (marked by NANOG+, POU5F1+, and SOX2+), mesoderm (marked by TBXT+, MESP1+, and MIXL1+), to the cardiac mesoderm stage (marked by NKX2.5+ and HAND1+), and cardiomyocyte formation (TNNT2+, NKX2.5+, MYL3+, and HCN4+). Before day 6.5, these heartoids primarily transition to the first heart region (FHF) lineage (marked by HAND1+, TBX5+, NKX2-5+, and TBX1-) before entering the cardiac tube stage. Furthermore, during chamber formation, maturation-related genes are upregulated, including ion channel genes, structural genes, and β-adrenergic receptor genes (…). Figure 1 f and 6d).

[0211] The obtained organoids were also frozen sections, and the sections were then subjected to immunofluorescence staining. The results were shown in... Figure 1 g. The results showed that these heart-like structures contained TNNT2-labeled myocardium and CDH5-labeled endocardial layer.

[0212] Example 2

[0213] This embodiment investigates the effect of Wnt-BMP pathway regulation on the formation of a heart-like organ with stable chambers.

[0214] Since the formation and expansion of stable chambers in heart-like structures may involve biological processes such as apoptosis or proliferation, the inventors investigated this. Using the method described in Example 1, H9 ESC cell lines were induced to form heart-like structures. Immunostaining of cleaved Ki67 and CASP3 cells revealed that apoptosis and proliferation both promoted chamber formation in the heart-like structures within a time window from day 6.5 to day 15.5. Figure 7 a). Subsequent heatmap analysis of apoptosis and proliferation-related genes also supported this conclusion, as the expression levels of these genes decreased rather than increased during the formation of chambered heart-like structures. Figure 7 b).

[0215] Based on this, the influence of downstream effectors of the Wnt-BMP signaling axis on the regulation of this pathway was further analyzed. 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 used to obtain the corresponding organoids from these cell lines. The obtained organoids were frozen sections and then subjected to immunofluorescence analysis. The results are shown in... Figure 7 c and 7d. Opposite results were observed in the two cell lines. HAND1 KO cells produced solid organoids lacking discernible chambers, but still showed TNNT2 positivity (…). Figure 7 c); while NKX2.5 KO cells not only showed TNNT2 positivity but were also able to form well-defined compartmentalized structures ( Figure 7 d).

[0216] These results strongly suggest that HAND1 in the Wnt-BMP signal axis is a key factor in the formation of heart-like structures with stable chambers, while NKX2.5 is not. These results also suggest that the process of stable chamber formation is unrelated to cardiomyocyte differentiation.

[0217] Example 3

[0218] This embodiment investigates the effect of FGF on the regulation of the formation of a heart-like structure with stable chambers.

[0219] FGF has been reported to play a crucial role in heart development (Non-Patent Literature 7-9), but its effect on chamber formation remains unclear. Therefore, the effect of PD173074 (a representative FGF signaling inhibitor) on heart-like cell formation was first tested. H9 ESC cell lines were induced and cultured using essentially the same method as in Example 1, except that on day 1.5 of differentiation from mesoderm to cardiac mesoderm, a culture medium without the FGF pathway inhibitor PD173074 was used. The results showed that, under conditions with PD173074, pluripotent stem cells were able to form heart-like cells with distinct chamber structures, and these chamber structures remained stable on day 90 without signs of structural damage or disintegration. Figure 1 b, 1c, 1d, 3a, 3b, 3c, and 8a; while without the addition of PD173074, the resulting organoids did not have observable cavities on day 15. Figure 3 (a, 3b) Dynamic imaging also confirmed that no chamber formation was observed throughout the entire induction experiment using PD173074 without its addition. Figure 8a). Immunofluorescence staining of frozen sections of organoids was used to observe the differences between the two organoids from a more microscopic perspective. The results showed that without the addition of PD173074, the majority of the volume of the organoids was occupied by TNNT2-positive cardiomyocytes, with only significantly reduced microcavities remaining inside. Figure 3 c). The above results indicate that inhibiting FGF is a key factor in the formation of long-term stable cardiac chamber structures during the induction of cardiac mesoderm.

[0220] Furthermore, no difference in cardiomyocyte differentiation was observed between the PD173074 addition group and the non-addition group. Figure 3 d). However, principal component analysis revealed that PD173074 induced significant changes in organoid cell fate. Figure 3 e). Volcano plots showed that key genes associated with cardiomyocyte differentiation were significantly upregulated during the cardiac mesodermal stage at day 3.5, with the upregulation magnitude being significantly greater in the PD173074 unadded group than in the PD-added group. Changes in cardiomyocyte fate were observed at day 10.5: in organoids without PD, expression of atrial-specific genes (such as NR2F2) increased, while in organoids with PD, levels of ventricular-specific genes (such as IRX4) increased. Figure 8 d). More detailed analysis of gene expression in heart-specific cell types further confirmed the above observations. Figure 8 e). Single-cell RNA sequencing results also showed that PD-treated organoids exhibited increased levels of atrial cardiomyocytes. Figure 3 i, 3j, 3k, and 3l).

[0221] Then, five time points were selected for a more extensive gene expression analysis to track changes in the upregulated and downregulated gene sets. The results are shown in Figure 3 f. Dynamic changes in gene expression can be observed from the Venn diagram, with some genes exhibiting temporal stability, suggesting they may play a role in regulatory processes. On day 3.5, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed significant enrichment of both upregulated and downregulated genes in various biological processes, cellular components, and molecular functions. Upregulated genes were enriched in processes such as cholesterol biosynthesis, heart development, glycolysis, metabolic pathways, and adrenergic signaling, while downregulated genes were associated with processes such as cell proliferation and adhesion, Ras signaling, and PI3K-Akt signaling pathways. Figure 3 g, 3h). Downregulated genes are involved in cell proliferation and adhesion, and their encoded proteins are involved in the formation of cellular components such as the cell membrane and chromatin. Figure 3h). The enrichment analysis described above provides insights into the regulatory mechanisms leading to heart-like structures with stable chambers.

[0222] Example 4

[0223] This embodiment verifies the efficacy of other FGF pathway inhibitors in regulating compartment formation in cardiac organoids.

[0224] For H9 ESC cells, 3D organoids were induced and cultured using essentially the same method as in Example 1, except that the FGF pathway inhibitor was replaced with PD166866 (a selective inhibitor of FGFR1 tyrosine kinase activity) at a concentration of 1 μM instead of 0.5 μM PD173074. Changes in the induced culture were continuously observed using live-cell imaging techniques, and the results are shown in [the original text is missing]. Figure 8 b, 8c, 14.

[0225] As can be seen from the figure, similar to the results of Example 1, a heart-like structure with chambers was observed to form on day 15.5 in the group with added PD166866.

[0226] Example 5

[0227] Based on the 2D epicardial formation method reported in Non-Patent Literature 2, 2D epicardial tissue was generated from the H9-tdTomato cell line, and the tdTomato gene was randomly integrated into the cell genome. Figure 6 d) The location and differentiation of these cells could be tracked by detecting the red fluorescence of tdTomato. Differentiated epicardial cells were cultured to day 9.5, harvested, and seeded at 100 cells / well into AggreWell 400 plates to allow aggregation. The epicardial cell aggregates were then added to the organoids obtained in Example 1 (days 15.5–25.5), and co-cultured in CDM medium supplemented with 10 μg / mL insulin and 0.5 μM SB431542, with the medium being changed every 2 days to allow the aggregates to fuse with the organoids. Using this method, we successfully generated a three-dimensional cardiac sphere, including the epicardium, myocardium, and endocardium. This demonstrates that our method can simulate the structure of the heart in vitro, providing a new experimental model for cardiac development and disease research.

[0228] Example 6

[0229] The same induction and culture method as in Example 1 was applied to H1 ESC cell lines and UiPS cell lines (non-patent literature 11, 12) derived from male individuals, and the results showed that... Figure 6 g and 6h.

[0230] Depend on Figure 6As can be seen from g and 6h, the method for preparing cardiac organoids of the present invention can be applied to pluripotent stem cells from two different sources and obtained results consistent with those obtained from the H9 cell line (from a female individual) mentioned above. This indicates that the method of this application has good robustness and can effectively induce different cell lines to produce cardiac organoids with stable chambers, unaffected by potential genetic background differences between cell lines and sexes, and has the potential for customized applications.

[0231] Example 7

[0232] This embodiment explores the physiological and ultrastructural features of a heart-like organism prepared using the exemplary methods of this application.

[0233] The heart-like organ prepared using the method described in Example 1 exhibits strong, visually observable contractile and diastolic movements. These movements are verified to be rhythmic and consistent with a normal heart rate through heartbeat recording and calcium imaging experiments. For example, Figure 2 e shows that the exemplary heart-like organ maintained a beat rate of 60 beats / min (BPM) from day 21.5 to day 68. Figure 2 f shows that calcium ion influx and efflux consistent with the aforementioned heart rate can be observed in the heart-like structure. Results of whole-cell patch-clamp experiments ( Figure 2 c) and 2d) show that these cardiac-like organs exhibit typical atrial-like action potential patterns at the level of individual cardiomyocytes, such as action potential shape, resting membrane potential (RMP), action potential amplitude (APA), and action potential duration (APD).

[0234] To observe the ultrastructure of these organoids, they were subjected to immunofluorescence staining and confocal microscopy imaging. The results are shown in... Figure 2 a) Frozen sections were prepared and transmission electron microscopy (TEM) images were taken. The results are shown in Figure 2 b. The ultrastructural results are also consistent with physiological function, with characteristic ultrastructural features of cardiomyocytes observed in these organoids, including properly ordered sarcomeres, Z-lines, and interconnectedness achieved through intercalated discs (IDs). Whole-cell patch-clamp analysis of single cardiomyocytes derived from organoids of this disclosure yielded the following results. Figure 2 c and 2d, it can be observed that the derived cardiomyocytes mainly exhibit an atrial-like action potential (AP) phenotype, including a resting membrane potential (RMP) of -60.99±4.91mV, an action potential amplitude (APA) of 119.05±8.69mV, and an AP duration (APD50) of 88.57±25.65ms (Figure).

[0235] Therefore, the heart-like organ obtained in this application possesses ideal characteristics in both microstructure and physiological function. For example, the pulsation frequency synchronized with the patient's heart rate is crucial for effective integration with the cardiovascular system of the transplant recipient. Furthermore, these ideal characteristics remain stable over a relatively long observation window, indicating that the heart-like organ of this application has great application potential in various clinical scenarios requiring stable cardiac function.

[0236] Example 8

[0237] This embodiment describes the self-organization of the cardiac organoids disclosed herein to form a structured myocardial layer and endothelial layer.

[0238] Extensive RNA sequencing was performed on cardiac organoids cultured for different days in Example 1. The results showed that endothelial markers, including CDH5, PECAM1, CD34, and ESAM, were gradually upregulated starting from day 3.5. Figure 11 A). Immunofluorescence assays of its frozen sections revealed an outer myocardial layer formed by TNNT2+ cardiomyocytes and an endothelial layer formed by CDH5+ endothelial cells arranged on the inner surface of the ventricular structures. Figure 11 (B) This demonstrates that the cardiac organoids disclosed in this paper can undergo orderly self-organization in three-dimensional space, ultimately forming structured myocardial and endothelial layers, which are crucial for maintaining the stability and structural integrity of the cardiac chambers. This is significantly different from previously reported cardiac organoid models (such as Non-Patent Literature 2), which failed to form self-organized myocardial and endothelial layers even under optimized conditions supplemented with VEGF.

[0239] RNA sequencing results also showed that dynamic upregulation of marker genes involved in ventricular morphogenesis and early cardiac patterning, such as NPPA, NPPB, TBX5, HAND1, and HAND2, could be observed in the cardiac organoids disclosed in this paper. Figure 11 C). This indicates that the molecular tag features exhibited by the cardiac organoids disclosed in this paper are similar to those of the early stages of human heart development.

[0240] Endothelial cells play a crucial role in heart development through endothelial-mesenchymal transition (EndoMT). EndoMT, a process regulated by TGF-β signaling, contributes to the formation of endocardial cushions, which further develop into heart valves, and is essential for heart morphogenesis. In the cardiac organoids disclosed in this paper, key EndoMT-related markers of CDH5+ endothelial cells were significantly upregulated, including TWIST1, ACTA2, SNAI2, and CDH2 (…). Figure 11 G). This is consistent with the potential EndoMT process.

[0241] Organoids treated with the selective TGF-β inhibitor SB431542 (SB) from day 28.5 to day 48.5 showed that the chamber area in the SB-treated group increased to approximately twice that of the blank control group. Figure 11 D, 11E, and 11H), but there was no significant difference in pulsation rate between the treatment group and the control group ( Figure 11 F). This suggests that TGF-β signaling may affect compartment morphology by influencing endothelial and mesenchymal cell populations, but not directly affect contractile dynamics.

[0242] Furthermore, the ETS1 gene was knocked out using CRISPR / Cas9-mediated gene editing technology to generate ETS1 knockout (ETS1KO) organoids. ETS1 has been reported as a key transcription factor affecting endothelial cell survival and function; its absence disrupts coronary artery development, leading to severe defects in angiogenesis. ETS1 deficiency has also been reported to be associated with Jacobson's syndrome. The results showed that the ETS1 KO organoids were completely unable to form ordered compartments. Figure 11 D). This suggests that endothelial defects may be related to the formation of chamber morphology.

[0243] Based on this, the cardiac organoids disclosed herein have the potential to serve as a good model for evaluating drugs and mechanisms that target endothelial cells.

[0244] Example 9

[0245] This embodiment characterizes the constituent cell types of a heart-like organism prepared using the exemplary method of this application.

[0246] Following 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 cellular composition of these cultures. 25,274 and 14,614 cells were obtained from the 2D and 3D cultures, respectively. UMAP and Louvain analyses identified 7 and 18 distinct cell clusters from the 2D and 3D cultures, respectively. Specifically, four differentiated cell types were identified in the 2D culture: atrial cardiomyocytes, ventricular cardiomyocytes, cardiac progenitor cells, and fibroblasts. Figure 4 a, 4b, and 9a); and in the 3D culture, in addition to the above four cell types, two other cell types were identified: valve cells expressing PRRX2 and MSX1 markers, and epithelial progenitor cells expressing GABRP and GRHL2 markers. Figure 4 a~4c and Figure 9 (a-9d). The proportion of the main cells in the cluster and the typical marker genes are shown in Table 2.

[0247] Table 2:

[0248] Cell clustering markers 3D Culture 2D culture atrial myocardial cells NR2F2, NPPA 48.2 14.34 Ventricular myocardial cells BMP2, IRX4 21.2 72.90 Cardiac progenitor cells TOP2A 8.46 1.54 Fibroblasts FN1, COL1A1 3.05 11.22 valve cells <![CDATA[PRRX2 + 、MSX1 + ]]> 6.48 0 Epithelial progenitor cells <![CDATA[GABRP + 、GRHL2 + ]]> 11.1 0 Endocardial cells CDH5, PECAM1 0.315 0 Other cells 0.011 0

[0249] Valvular cells and epithelial progenitor cells are unique to 3D-type hearts. Based on GO and KEGG analyses, valvular cells are endocardial cells involved in angiogenesis, outflow tract morphogenesis, focal adhesion, and related signaling pathways (such as Rap1, MAPK, and PI3K-AKT). Conversely, epithelial progenitor cells are involved in processes such as apoptosis, motor proteins, tight junction regulation, and the p53 signaling pathway. Figure 4 d and 9e, 9f).

[0250] Further comparisons of gene expression in 2D and 3D cultures revealed significant expression of related genes, such as ion channels (RYR2, CACBA1C, HCN4), progenitor cells (EDNRA), β-oxidation (ACADVL), and cardiomyocyte-specific genes specific to 3D cells (TNNT2, MYL7, NR2F2, TBX5, GATA4, LAMB2, TTN). Figure 4 (e, 9g, 9h). These results confirm that the heart-like cells generated by 3D culture have broader cellular diversity and maturity.

[0251] Example 10

[0252] This embodiment describes the gradual maturation of cardiac organoids over time.

[0253] Single-cell RNA sequencing (scRNA-seq) was performed on heart organoids at day 25.5 (D25.5) and day 43.5 (D43.5). The results were as follows: Figure 12 The violin diagram is presented. 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: myocardial contraction (TNNT2, MYL7, MYL6), glycolysis (PGAM1), fatty acid metabolism (HADH4, HADHB), and ion channel activity (RYR2, KCNQ1, SCN5A). This indicates that the gene expression patterns related to cardiomyocyte maturation have changed over time.

[0254] Using the Slingshot method, pseudo-temporal trajectory analysis was performed on scRNA-seq data from cardiac organoids at D25.5 and D43.5, as well as scRNA-seq data from human fetal hearts obtained from a public database (Cui Y, et al., Cell Rep. 2019; 26:1934-1950e1935). The results showed that... Figure 13It is evident that the cardiomyocytes of D25.5 mainly correspond to the early developmental stage before 5 weeks of human fetal development (HE5W); while the cells of D43.5 show a clear progression along a pseudo-timeline, more closely resembling the transcriptional features observed in the human fetal heart from 5 to 9 weeks.

[0255] The above results indicate that the cardiac organoids disclosed herein can gradually transform into the later stages of cardiac development during long-term culture.

[0256] Example 11

[0257] This embodiment describes the use of an exemplary heart-like structure from this application for toxicity testing of drug candidates.

[0258] The heart-like structure described in this application has the potential to serve as a platform for drug safety and toxicity testing. Thalidomide and acitretin are examples. Thalidomide has been reported to cause serious birth defects, particularly neonatal cardiac abnormalities (Non-Patent Literature 13), while acitretin can significantly affect heart development, posing a serious risk to fetal heart health (Non-Patent Literature 5). In contrast, aspirin has not been reported to have such side effects and is therefore used as a negative control (Non-Patent Literature 2).

[0259] Following the method described in Example 1, corresponding 2D and 3D cultures were prepared from H9 ESC cells. On day 0 of culture, thalidomide, atracin, or aspirin were added to the culture medium to final concentrations of 10 μg / ml, 50 nM, and 30 μM, respectively. Morphological changes in the cultures were observed, and the results were shown in... Figure 5 a and 10a. It is clearly visible that, compared to the control aspirin group, both the thalidomide and atratin groups exhibited severe defects in their organoids, accompanied by morphological changes. By day 20.5, both the thalidomide and atratin groups showed significantly smaller chamber areas compared to the control group. Figure 5 b). Specifically, the thalidomide group was observed to form transparent chambers between days 6.5 and 15.5, but these chambers could not be stably maintained, eventually collapsing into a spherical shape on day 20.5. The atratin group, on the other hand, exhibited irregular shapes as early as day 6.5. More detailed morphological observations also suggest that thalidomide and atratin may begin to affect the heart-like structure at different times; the former occurs at the mesodermal stage, while the latter appears to show observable effects at the pluripotency stage. Figure 10 (b and 10c). Furthermore, in the thalidomide and atratin groups, no pulse rate was detected, or the detected pulse rate was significantly reduced. Figure 5 c). This demonstrates that the heart-like organ of this application can effectively detect compound molecules with potential cardiotoxicity or teratogenicity.

[0260] It has been reported that thalidomide-induced embryonic damage mainly occurs in the early developmental stages, typically from day 20 to day 36 post-fertilization (Non-Patent Literature 14), while late exposure to thalidomide induces brain damage in rat fetuses. Based on this, in the same manner as in Example 1, thalidomide (10 μg / ml) was injected into the heart-like organoid from day 15.5 (after ventricular formation was complete) until day 36.5 to assess whether the teratogenic window of the cardiac organoid of the present invention to thalidomide coincided with the window of natural development in vivo. The results showed that administration of thalidomide after day 15.5 did not change the chamber area in the heart-like organoid, nor did it affect its stroke rate. Figure 5 (d, 10d, 10e). This indicates that the cardiac organoids of the present invention have the efficacy and specificity as a screening model in response to drug toxicity.

[0261] Therefore, the above results indicate that the heart-like organ of this application can be used as a drug testing model and has the potential for further optimization.

[0262] Example 12

[0263] This embodiment further illustrates the application of the heart-like structure of this disclosure as a drug candidate screening model using isoproterenol (ISO) and doxorubicin (DOX).

[0264] ISO is a synthetic β-adrenergic agonist commonly used to stimulate heart rate and simulate stress-induced cardiac responses. ISO treatment primarily affects cardiac contractile activity, lacking detectable morphological changes. Changes in the velocity of a cardiac-like animal after ISO exposure were detected by… and the results are shown in… Figure 14 and 15 Positive chronotropic effects were observed in the heart-like cells treated with a small amount of ISO (1 μM), with BPM significantly increasing from 61.43 ± 2.34 beats / min to 87.26 ± 3.99 beats / min and the duration of each systolic cycle significantly shortening.

[0265] DOX is an anthracycline chemotherapy drug known to have strong cardiotoxicity. DOX treatment can lead to severe deterioration of the cardiac-like structure, such as a reduction in chamber size. The cardiac-like structures were observed using bright-field microscopy 24 and 48 hours after DOX exposure. The results are shown in... Figure 16 A and 16B. It was observed that the chamber size decreased significantly with increasing DOX processing time. Figure 16 A and 16B) indicate that the heart-like structure disclosed herein can be used to detect DOX-induced damage to cardiac structures. Further evaluation of DOX cardiotoxicity was conducted using cell viability and cytotoxicity assays. CCK-8 assay results showed a significant decrease in cell viability in the DOX-treated group (…). Figure 16C), and elevated levels of lactate dehydrogenase (LDH) in the culture medium, suggest increased cell membrane damage and cytotoxicity. Figure 16 D). Simultaneously, in the DOX-treated group, the mRNA expression of the cardiac stress marker atrial natriuretic peptide (ANP) was increased ( Figure 16 E), inflammation-related markers such as tumor necrosis factor-α (TNFA) and interleukin-6 (IL-6) were significantly upregulated, suggesting an inflammatory response of the heart to the drug. Figure 16 F and 16G).

[0266] Therefore, the cardio-like platform disclosed herein can sensitively detect changes in cardiac function and a range of undesirable potential toxicities caused by candidate drug molecules, including but not limited to cardiac / chamber structural damage, cell damage, cytotoxicity, cardiac stress response, and inflammatory response; thereby allowing for rapid, multidimensional, and high-throughput screening of candidate molecules from a cardiotoxicity perspective.

[0267] Example 13

[0268] This embodiment describes important signaling pathways involved in the induction and differentiation of cardiac organoids, in addition to the FGF pathway. The cardiac organoids disclosed herein can be used to build experimental models for studying these signaling pathways.

[0269] 1. Endothelial cell-mediated signaling

[0270] During heart development, endothelial cells play a crucial 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).

[0271] Cell communication analysis was used to reveal whether the aforementioned interactions exist in the cardiac organoids of this disclosure, and the results showed that... Figure 17A , 17B In 17C and 17D, signal transduction from endothelial cells to atrial cardiomyocytes was observed to be primarily mediated by EPHA, EPHB, NOTCH, VEGF, NRG, and WNT; only BMP-mediated signal transduction was detected in endothelial-to-ventricular cardiomyocyte communication, while signal transduction from atrial or ventricular cardiomyocytes to endothelial cells involved multiple pathways other than HH. Furthermore, 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. Figure 17C , 17D ).

[0272] 2. Laminin α family-related regulatory pathways

[0273] Laminin α (LAMA) is an important transcription factor that plays different roles in endothelial structure, cell communication, and cardiac morphogenesis, depending on its subunits. For example, laminin α4 (LAMA4) has been shown to be crucial for maintaining endothelial cell growth and proliferation, and its mutations or dysregulation are associated with various cardiovascular diseases, including cardiomyopathy and vascular dysfunction. LAMA4 expression defects can lead to impaired cardiomyocyte and endothelial cell function, resulting in abnormal cardiac structure and function. Laminins LAMA4 and LAMA5 also regulate immune cell transport and are associated with impaired T cell localization and increased graft rejection.

[0274] Single-cell communication analysis revealed different spatial expression patterns of LAMA family members. Figure 18A The results showed that LAMA2 was primarily expressed in cardiomyocytes (atria and ventricles), LAMA4 was mainly detected in endothelial cells and fibroblasts, LAMA1 was detected in endoderm cells, and LAMA5 exhibited a broader expression profile, being highly enriched in epithelial progenitor cells but also found in some cardiomyocytes and endothelial cells. Further analysis suggested that LAMA2 may promote interactions among cardiomyocytes, cardiac progenitor cells, endothelial cells, and fibroblasts, while LAMA4 appeared to interact with peripheral cardiac-related cell types.

[0275] Single-cell RNA sequencing data showed that the LAMA family members-mediated signaling observed in the organoids disclosed herein exhibited significant similarities to the distribution and inferred signaling in the heart of a 5- to 6-week-old human fetus, particularly in endothelial and cardiomyocyte-mediated interactions. Figure 18B LAMA2 is highly expressed in cardiomyocytes and exhibits extensive interactions with other cardiac-associated cell types. LAMA4 is enriched in endothelial cells and was also detected in some cardiomyocytes, cardiac progenitor cells, and epicardial cells. LAMA1 and LAMA5 are primarily secreted by cardiac progenitor cells and epithelial cells, respectively. This also indicates that the heart-like model of this disclosure possesses an ECM-associated intercellular communication pattern similar to early stages of human heart development.

[0276] 3. Activator A (AA) and retinoic acid (RA) related signaling pathways

[0277] AA and RA are thought to play important roles in the induction of mesodermal and cardiac mesodermal processes and have also been reported to be associated with heart disease. However, their precise mechanisms in compartment formation and structural stability have not been fully elucidated. This example tests the specific effects of three combinations of AA and RA concentrations.

[0278] A4R50 group: AA4nM and RA 50nM;

[0279] A4R500 group: AA 4nM and RA500nM;

[0280] A50R500 group: AA 50nM and RA500 nM.

[0281] Figure 19 A and 19D showed that all three conditions effectively induced the formation of stable chambers. Under low AA concentration conditions, the chamber size of the A4R500 group was significantly larger than that of the A4R500 group, indicating that within the test range, higher RA concentrations can promote chamber volume expansion. Figure 19 A, 19B, and 19C show that under higher concentrations of AA (A50R500 group), chamber formation can be observed at an earlier stage, but the area of ​​the formed chamber gradually decreases from day 15.5. This indicates that higher AA concentrations can accelerate chamber formation and promote chamber expansion, but are not conducive to long-term chamber maintenance, while lower AA concentrations are more conducive to the long-term stability of the chamber.

[0282] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, it is intended to include any changes, uses, or improvements to the invention, including modifications made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for producing heart organoids, the method comprising: A. Activate Wnt signaling in cell aggregates containing pluripotent stem cells to induce the cell aggregates to differentiate into cell aggregates of mesodermal cells; B. During the process of stopping the activation of Wnt signaling and causing the cell aggregates of the mesodermal cells to differentiate into cardiac mesoderm, inhibiting FGF signaling in the cell aggregates and promoting the differentiation of the cell aggregates into cardiac mesoderm; and C. Stop inhibiting FGF signal transduction, allowing the cardiac mesoderm to continue differentiating to form cardiac organoids.

2. The method according to claim 1, wherein, The inhibition of FGF signaling includes contacting the cell aggregates of the mesodermal cells with an FGF receptor inhibitor.

3. The method according to claim 1 or 2, wherein, The FGF receptor inhibitor is selected from any one of FGFR1 inhibitors, FGFR2 inhibitors, FGFR3 inhibitors, or FGFR4 inhibitors, or any combination thereof. Preferably, the FGF receptor inhibitor is selected from FGFR1 inhibitors; 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.

4. The method according to any one of claims 1 to 3, wherein, The duration of the inhibition of FGF signaling is more than 0.1 hours and does not exceed the time required for the cell aggregates of the mesodermal cells to differentiate into cardiac mesoderm under the condition of not inhibiting the FGF signaling, preferably 0.1 hours to 48 hours.

5. The method according to any one of claims 1 to 4, wherein, Step B further includes inhibiting Wnt signaling during the differentiation of the mesodermal cell aggregates into cardiac mesoderm.

6. The method according to claim 5, wherein, The suppression of Wnt signal transduction occurs at least partially simultaneously with the suppression of FGF signal transduction; Preferably, the duration of simultaneous suppression of Wnt signal transduction and FGF signal transduction is more than 12 hours.

7. The method according to claim 5, wherein, The inhibition of Wnt signaling includes contacting the cell aggregates of the mesodermal cells with a Wnt inhibitor; 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.

8. The method according to any one of claims 1 to 7, wherein, Step B further includes contacting the mesodermal cell aggregates with TGF-β inhibitors, RA agonists and / or BMP pathway agonists. Optionally, the TGF-β inhibitor is selected from SB431542, Dorsomorphin 2HCl, Dorsomorphin, LDN-193189, Galunisertib (LY2157299), LY2109761, SB525334, LDN-193189 2HCl, 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 or any combination of retinoic acid, bexarotene, TTNPB, AM580, adapalene, acitretin, tazarotene, tamibarotene, SR 11237, estretinate, BMS493, palovarotene, all trans-Retinal, CD437 (AHPN), or MSU-42011; Optionally, the BMP pathway agonist is selected from any one or any combination of BMP4, Activin A, BMP2, SJ000291942, or SB4.

9. The method according to any one of claims 1 to 8, further comprising the step of fusing the cardiac organoid with epicardial cells.

10. A heart organoid prepared by the method of claims 1 to 9.

11. A heart-like organoid comprising Heart tissue comprising cardiomyocytes, endocardial cells, and optionally epicardial cells. At least one cavity located within the heart tissue, in, The at least one inner cavity can exist under in vitro culture conditions for at least 30 days, preferably at least 60 days, and more preferably at least 90 days; Optionally, the cardiac organoid further comprises epicardial cells disposed on its outer surface.

12. The heart organoid according to claim 10 or 11, which can spontaneously beat under in vitro culture conditions; Optionally, the spontaneous pulsation frequency of the said cardiac organoid is between 30 and 150 beats per minute.

13. The heart organoid according to any one of claims 10 to 12, wherein, The cardiomyocytes comprise about 40% to about 80% of the total number of cells constituting the organoid, the endocardial cells comprise about 5% to about 15% of the total number of cells constituting the organoid, and the epicardial cells comprise 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 approximately 8:1 to approximately 15:1; Optionally, the endocardial cells are aggregated inside the organoid and define at least one lumen; Optionally, the cardiac organoid has a size of about 0.5 to about 2.5 mm in its maximum dimension; and / or Optionally, the size of the lumen in its maximum dimension is at least about 60% of the size of the heart organoid in its maximum dimension.

14. Use of the cardiac organoid according to any one of claims 10 to 13, wherein, The uses are selected from: (1) Constructing disease models; (2) Screening drug candidates; and / or (3) Assess the toxicity of drug candidates.

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