A cardiac organoid and an in vitro preparation method thereof

Abstract

The present invention relates to a cardiac organoid and an in vitro preparation method thereof, and belongs to the field of stem cell technology. The present invention provides a cardiac organoid, wherein the cardiac organoid is obtained by inducing and culturing an adherent pluripotent stem cell cluster; the adherent pluripotent stem cell cluster is obtained by adherent culturing a suspended pluripotent stem cell cluster; and the suspended pluripotent stem cell cluster is obtained by suspension culturing a pluripotent stem cell cluster. The in vitro preparation process of the cardiac organoid is to induce differentiation of the pluripotent stem cell spheres in stages under adherent culture after the pluripotent stem cells are formed into spheres, so that cell subtypes such as epicardial cells can be produced without additional addition or additional signal pathway stimulation, and the cells are more homogeneous. The spontaneously generated multi-lineage coexisting cardiac organoid can better simulate the biological events derived from multiple cell types and multiple cell subtypes that occur during the early development of the embryonic heart, and has great application prospects in exploring the pathogenic factors and potential therapeutic targets of congenital heart disease.

Description

Technical Field

[0001] The present invention relates to a heart organoid and an in vitro preparation method thereof, and belongs to the technical field of stem cells. Background Art

[0002] The heart is a human organ. Its primary function is to power blood flow and distribute it to all parts of the body. Heart disease is a general term for diseases caused by structural damage or dysfunction of the heart. Cardiac organoids, 3D cell cultures spontaneously assembled from pluripotent stem cells in vitro, exhibit remarkable self-organization properties, sharing a high degree of homology with in vivo tissues in terms of morphology, structure, and gene expression. They provide a novel research tool for investigating the pathogenic factors and potential therapeutic targets of heart disease. Ideally, the self-assembly process of cardiac organoids can mimic multiple biological events of heart development in vitro, including the formation of early cardiac mesodermal progenitor cells, the further specialization of these early cardiac mesodermal progenitor cells into first heart field (FHF) and second heart field (SHF) progenitor cells, and the emergence of various cardiac-specific non-cardiac cells in the early developing heart, including endocardial cells, epicardial cells, endothelial cells, fibroblasts, and interstitial cells.

[0003] However, existing heart organoids all have defects to some extent. For example, the cardiomyocyte cell types are not comprehensive, lacking necessary cardiomyocytes such as FHF progenitor cells (see the literature “L. Drakhlis et al., Human heart-forming organoids recapitulate early heart and foregut development, Nat. Biotechnol., vol. 39, no. 6, pp. 737-746, 2021.”) or SHF progenitor cells (see the literature “P. Hofbauer et al., Cardioids reveal self-organizing principles of human cardiogenesis, Cell, vol. 184, no. 12, pp. 3299-3317.e22, 2021.”), and cannot effectively simulate the specialization process of FHF progenitor cells or SHF progenitor cells during cardiac development; the lack of non-cardiomyocytes cannot well reproduce the autonomous and orderly occurrence of cardiac-specific non-cardiomyocytes during cardiac development, and requires the additional addition of cell subtypes such as epicardial cells (see the literature “P. Hofbauer et al., Cardioids reveal self-organizing principles of humancardiogenesis, Cell, vol. 184, no. 12, pp. 3299-3317.e22, 2021.), or additional signaling pathway stimulation is required to produce cell subtypes such as epicardial cells (see the literature “YR Lewis-Israeli et al., Self-assembling human heart organoids for the modeling of cardiacdevelopment and congenital heart disease, Nat. Commun., vol. 12, no. 1,2021.” and “AB Meier et al., “Epicardioid single-cell genomics uncover sprinciples of human epicardium biology in heart development and disease, Nat.Biotechnol., 2023."), which complicates the cardiac organoid construction process and increases its cost. In vitro preparation requires a suspension culture system, which cannot effectively maintain homogeneity among cardiac organoids, resulting in irregular distribution of different cell subtypes (see "YRLewis-Israeli et al., Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease, Nat. Commun., vol. 12, no. 1, 2021." and "S.-G. Lee et al., Generation of human iPSCs-derived heart organoids structurally and functionally similar to heart, Biomaterials, vol. 290, p. 121860, Nov. 2022."). Therefore, it is urgent to find a cardiac organoid that can autonomously and orderly generate a variety of cardiac-specific cells (including cardiomyocytes and non-cardiomyocytes such as endocardial cells, epicardial cells, fibroblasts, and interstitial cells) during differentiation, and that maintains good homogeneity. Summary of the Invention

[0004] To address the above-mentioned drawbacks, the present invention provides a cardiac organoid, wherein the cardiac organoid is obtained by induced culture of an adherent pluripotent stem cell cluster; the adherent pluripotent stem cell cluster is obtained by adherent culture of a suspension pluripotent stem cell cluster; and the suspension pluripotent stem cell cluster is obtained by suspension culture of pluripotent stem cells (PSC).

[0005] In one embodiment of the present invention, the cardiac organoids are obtained by inducing and culturing cardiovascular progenitor cell clusters; the cardiovascular progenitor cell clusters are obtained by inducing and culturing cardiac mesodermal progenitor cell clusters; the cardiac mesodermal progenitor cell clusters are obtained by inducing and culturing adherent pluripotent stem cell clusters; the adherent pluripotent stem cell clusters are obtained by adherent culture of suspension pluripotent stem cells; and the suspension pluripotent stem cell clusters are obtained by suspension culture of pluripotent stem cells.

[0006] In one embodiment of the present invention, the cardiac organoids can reproduce the rhythmic beating of the heart; the cardiac organoids can reproduce the key developmental events during the early development of the embryonic heart during the differentiation process; the cardiac organoids can autonomously and orderly produce cardiac-specific cells that appear during the early development of the embryonic heart during the differentiation process; the cardiac organoids can demonstrate the distribution patterns of different cell subtypes produced during the differentiation process.

[0007] In one embodiment of the present invention, the key developmental events include the specialization process of first heart field (FHF) progenitor cells and second heart field (SHF) progenitor cells and the endothelial-mesenchymal transition (EMT) process; the cardiac-specific cells include cardiomyocytes and non-cardiomyocytes; the cardiomyocytes include working cardiomyocytes and autonomous cardiomyocytes; the non-cardiomyocytes include endocardial cells, epicardial cells, fibroblasts and interstitial cells.

[0008] In one embodiment of the present invention, the working myocardial cells include atrial myocytes and ventricular myocytes; the autonomous myocardial cells include pacemaker cells (P cells) and Purkinje cells.

[0009] In one embodiment of the present invention, the pluripotent stem cells include embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPSCs).

[0010] In one embodiment of the present invention, the method for preparing the cardiac organoids comprises: performing suspension culture on pluripotent stem cells (PSCs) to obtain a suspension pluripotent stem cell cluster; performing adherent culture on the suspension pluripotent stem cell cluster to obtain an adherent pluripotent stem cell cluster; and performing induction culture on the adherent pluripotent stem cell cluster to obtain a cardiac organoid.

[0011] In one embodiment of the present invention, the method for preparing cardiac organoids includes: performing suspension culture on pluripotent stem cells (PSC) to obtain a suspension pluripotent stem cell cluster; performing adherent culture on the suspension pluripotent stem cell cluster to obtain an adherent pluripotent stem cell cluster; sequentially performing mesoderm induction culture on the adherent pluripotent stem cell cluster using a first cardiac organoid differentiation medium and a first basal medium to obtain a cardiac mesodermal progenitor cell cluster; performing cardiovascular induction culture on the cardiac mesodermal progenitor cell cluster using a second cardiac organoid differentiation medium to obtain a cardiovascular progenitor cell cluster; and sequentially performing organoid induction culture on the cardiovascular progenitor cell cluster using the first basal medium and the second basal medium to obtain a cardiac organoid.

[0012] In one embodiment of the present invention, the method for preparing cardiac organoids includes: using a stem cell maintenance medium to perform suspension culture on pluripotent stem cells (PSC) to obtain a suspension pluripotent stem cell cluster; using a stem cell maintenance medium to perform adherent culture on the suspension pluripotent stem cell cluster to obtain an adherent pluripotent stem cell cluster; sequentially using a first cardiac organoid differentiation medium and a first basal medium to perform mesoderm induction culture on the adherent pluripotent stem cell cluster to obtain a cardiac mesodermal progenitor cell cluster; using a second cardiac organoid differentiation medium to perform cardiovascular induction culture on the cardiac mesodermal progenitor cell cluster to obtain a cardiovascular progenitor cell cluster; sequentially using the first basal medium and the second basal medium to perform organoid induction culture on the cardiovascular progenitor cell cluster to obtain a cardiac organoid.

[0013] In one embodiment of the present invention, the suspension culture time is 1 day to 2 days; the adherent culture time is 1 day to 2 days; the mesoderm induction culture time is 2 days to 3 days; the cardiovascular induction culture time is 3 days to 5 days; and the organoid induction culture time is 4 days to 10 days.

[0014] In one embodiment of the present invention, the first cardiac organoid differentiation culture medium is a first basal culture medium additionally supplemented with a WNT pathway activator, bone morphogenetic protein 4 (BMP4) and / or activin A (Active A).

[0015] In one embodiment of the present invention, in the first cardiac organoid differentiation culture medium, the WNT pathway activator comprises a GSK-3α / β inhibitor (GSK-3, i.e., ATP-competitive glycogen synthase kinase 3), WNT3a (secreted protein) and / or R-Spondins (secreted protein).

[0016] In one embodiment of the present invention, in the first cardiac organoid differentiation culture medium, the GSK-3α / β inhibitors include raglucibin (CHIR99021), BIO (6-bromoindirubin-3'-oxime, a specific inhibitor of bisindole and glycogen synthase kinase 3), lithium chloride (LiCl, Lithium Chloride), SB216763, SB415286 (2-(1H-reduced)-3-cyano-4-(dimethylamino)cholic acid), AZD1080 (2-hydroxy-3-[5-(morpholin-4-methyl)pyridin-2-yl]-1H-indole-5-carbonitrile) and / or AR-A014418; the R-Spondins include R-Spondin 1 (R-spondin 1) and / or R-Spondin 2 (R-spondin 2).

[0017] In one embodiment of the present invention, in the first cardiac organoid differentiation culture medium, the GSK-3α / β inhibitor is raglucillinib (CHIR99021).

[0018] In one embodiment of the present invention, the concentration of the WNT pathway activator in the first cardiac organoid differentiation medium is 3 μM~12 μM; the concentration of the bone morphogenetic protein 4 (BMP4) in the first cardiac organoid differentiation medium is 1 ng / mL~3 ng / mL; the concentration of activin A (Active A) in the first cardiac organoid differentiation medium is 1 ng / mL~3 ng / mL.

[0019] In one embodiment of the present invention, the second cardiac organoid differentiation medium is the first basal medium additionally supplemented with a WNT pathway inhibitor.

[0020] In one embodiment of the present invention, in the second cardiac organoid differentiation culture medium, the WNT pathway inhibitor component comprises IWR1, IWP2, tankyrase1 / 2 inhibitor XAV939 and / or PORCN inhibitor WNT-C59.

[0021] In one embodiment of the present invention, in the second cardiac organoid differentiation culture medium, the WNT pathway inhibitor is the PORCN inhibitor WNT-C59.

[0022] In one embodiment of the present invention, the concentration of the WNT pathway inhibitor in the second cardiac organoid differentiation culture medium is 1 μM to 5 μM.

[0023] In one embodiment of the present invention, the first basal culture medium is a cell culture medium additionally supplemented with B27 supplement without added insulin.

[0024] In one embodiment of the present invention, the concentration of the B27 supplement without added insulin in the cell culture medium is 1% to 5% by volume.

[0025] In one embodiment of the present invention, the second basal culture medium is a cell culture medium additionally supplemented with B27 supplement added with insulin.

[0026] In one embodiment of the present invention, the concentration of the insulin-added B27 supplement in the cell culture medium is 1% to 5% by volume.

[0027] In one embodiment of the present invention, the cell culture medium is RPMI1640 culture medium.

[0028] In one embodiment of the present invention, the stem cell maintenance medium is Essential 8TM culture medium or TeSR™-E8™ medium.

[0029] In one embodiment of the present invention, the suspension culture is performed in a low-adsorption culture container; and the adherent culture is performed in a culture container coated with matrigel.

[0030] In one embodiment of the present invention, the low-adsorption culture container includes a low-adsorption culture well plate, a low-adsorption culture dish and / or a low-adsorption culture bottle.

[0031] In one embodiment of the present invention, the low-adsorption culture well plate is a low-adsorption U-shaped bottom 96-well deep-well plate.

[0032] In one embodiment of the present invention, the Matrigel-coated culture container includes a Matrigel-coated culture well plate, a Matrigel-coated culture dish and / or a Matrigel-coated culture flask.

[0033] In one embodiment of the present invention, the matrigel-coated culture well plate is a matrigel-coated U-bottom 96-well deep-well plate.

[0034] The present invention also provides a method for constructing the above-mentioned cardiac organoids, comprising: performing suspension culture on pluripotent stem cells (PSCs) to obtain a suspension pluripotent stem cell cluster; performing adherent culture on the suspension pluripotent stem cell cluster to obtain an adherent pluripotent stem cell cluster; and performing induction culture on the adherent pluripotent stem cell cluster to obtain a cardiac organoid.

[0035] In one embodiment of the present invention, the pluripotent stem cells include embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPSCs).

[0036] In one embodiment of the present invention, the method includes: performing suspension culture on pluripotent stem cells (PSC) to obtain a suspension pluripotent stem cell cluster; performing adherent culture on the suspension pluripotent stem cell cluster to obtain an adherent pluripotent stem cell cluster; sequentially performing mesoderm induction culture on the adherent pluripotent stem cell cluster using a first cardiac organoid differentiation medium and a first basal medium to obtain a cardiac mesodermal progenitor cell cluster; performing cardiovascular induction culture on the cardiac mesodermal progenitor cell cluster using a second cardiac organoid differentiation medium to obtain a cardiovascular progenitor cell cluster; and sequentially performing organoid induction culture on the cardiovascular progenitor cell cluster using the first basal medium and the second basal medium to obtain a cardiac organoid.

[0037] In one embodiment of the present invention, the method includes: using a stem cell maintenance medium to perform suspension culture on pluripotent stem cells (PSC) to obtain a suspension pluripotent stem cell cluster; using a stem cell maintenance medium to perform adherent culture on the suspension pluripotent stem cell cluster to obtain an adherent pluripotent stem cell cluster; sequentially using a first cardiac organoid differentiation medium and a first basal medium to perform mesoderm induction culture on the adherent pluripotent stem cell cluster to obtain a cardiac mesodermal progenitor cell cluster; using a second cardiac organoid differentiation medium to perform cardiovascular induction culture on the cardiac mesodermal progenitor cell cluster to obtain a cardiovascular progenitor cell cluster; sequentially using the first basal medium and the second basal medium to perform organoid induction culture on the cardiovascular progenitor cell cluster to obtain a cardiac organoid.

[0038] In one embodiment of the present invention, the suspension culture time is 1 day to 2 days; the adherent culture time is 1 day to 2 days; the mesoderm induction culture time is 2 days to 3 days; the cardiovascular induction culture time is 3 days to 5 days; and the organoid induction culture time is 4 days to 10 days.

[0039] In one embodiment of the present invention, the first cardiac organoid differentiation culture medium is a first basal culture medium additionally supplemented with a WNT pathway activator, bone morphogenetic protein 4 (BMP4) and / or activin A (Active A).

[0040] In one embodiment of the present invention, in the first cardiac organoid differentiation culture medium, the WNT pathway activator comprises a GSK-3α / β inhibitor (GSK-3, i.e., ATP-competitive glycogen synthase kinase 3), WNT3a (secreted protein) and / or R-Spondins (secreted protein).

[0041] In one embodiment of the present invention, in the first cardiac organoid differentiation culture medium, the GSK-3α / β inhibitors include raglucibin (CHIR99021), BIO (6-bromoindirubin-3'-oxime, a specific inhibitor of bisindole and glycogen synthase kinase 3), lithium chloride (LiCl, Lithium Chloride), SB216763, SB415286 (2-(1H-reduced)-3-cyano-4-(dimethylamino)cholic acid), AZD1080 (2-hydroxy-3-[5-(morpholin-4-methyl)pyridin-2-yl]-1H-indole-5-carbonitrile) and / or AR-A014418; the R-Spondins include R-Spondin 1 (R-spondin 1) and / or R-Spondin 2 (R-spondin 2).

[0042] In one embodiment of the present invention, in the first cardiac organoid differentiation culture medium, the GSK-3α / β inhibitor is raglucillinib (CHIR99021).

[0043] In one embodiment of the present invention, the concentration of the WNT pathway activator in the first cardiac organoid differentiation medium is 3 μM~12 μM; the concentration of the bone morphogenetic protein 4 (BMP4) in the first cardiac organoid differentiation medium is 1 ng / mL~3 ng / mL; the concentration of activin A (Active A) in the first cardiac organoid differentiation medium is 1 ng / mL~3 ng / mL.

[0044] In one embodiment of the present invention, the second cardiac organoid differentiation medium is the first basal medium additionally supplemented with a WNT pathway inhibitor.

[0045] In one embodiment of the present invention, in the second cardiac organoid differentiation culture medium, the WNT pathway inhibitor component comprises IWR1, IWP2, tankyrase1 / 2 inhibitor XAV939 and / or PORCN inhibitor WNT-C59.

[0046] In one embodiment of the present invention, in the second cardiac organoid differentiation culture medium, the WNT pathway inhibitor is the PORCN inhibitor WNT-C59.

[0047] In one embodiment of the present invention, the concentration of the WNT pathway inhibitor in the second cardiac organoid differentiation culture medium is 1 μM to 5 μM.

[0048] In one embodiment of the present invention, the first basal culture medium is a cell culture medium additionally supplemented with B27 supplement without added insulin.

[0049] In one embodiment of the present invention, the concentration of the B27 supplement without added insulin in the cell culture medium is 1% to 5% by volume.

[0050] In one embodiment of the present invention, the second basal culture medium is a cell culture medium additionally supplemented with B27 supplement added with insulin.

[0051] In one embodiment of the present invention, the concentration of the insulin-added B27 supplement in the cell culture medium is 1% to 5% by volume.

[0052] In one embodiment of the present invention, the cell culture medium is RPMI1640 culture medium.

[0053] In one embodiment of the present invention, the stem cell maintenance medium is Essential 8TM culture medium or TeSR™-E8™ medium.

[0054] In one embodiment of the present invention, the suspension culture is performed in a low-adsorption culture container; and the adherent culture is performed in a culture container coated with matrigel.

[0055] In one embodiment of the present invention, the low-adsorption culture container includes a low-adsorption culture well plate, a low-adsorption culture dish and / or a low-adsorption culture bottle.

[0056] In one embodiment of the present invention, the low-adsorption culture well plate is a low-adsorption U-shaped bottom 96-well deep-well plate.

[0057] In one embodiment of the present invention, the Matrigel-coated culture container includes a Matrigel-coated culture well plate, a Matrigel-coated culture dish and / or a Matrigel-coated culture flask.

[0058] In one embodiment of the present invention, the matrigel-coated culture well plate is a matrigel-coated U-bottom 96-well deep-well plate.

[0059] The present invention also provides a cardiac organoid differentiation medium, which is a first basal medium additionally supplemented with a WNT pathway activator, bone morphogenetic protein 4 (BMP4) and / or activin A (Active A); or, the cardiac organoid differentiation medium is a first basal medium additionally supplemented with a WNT pathway inhibitor.

[0060] In one embodiment of the present invention, in the cardiac organoid differentiation culture medium, the WNT pathway activator comprises a GSK-3α / β inhibitor (GSK-3, i.e., ATP-competitive glycogen synthase kinase 3), WNT3a (secreted protein) and / or R-Spondins (secreted protein); the WNT pathway inhibitor comprises IWR1, IWP2, tankyrase1 / 2 inhibitor XAV939 and / or PORCN inhibitor WNT-C59.

[0061] In one embodiment of the present invention, in the cardiac organoid differentiation medium, the GSK-3α / β inhibitors include raglucibin (CHIR99021), BIO (6-bromoindirubin-3'-oxime, a specific inhibitor of bisindole and glycogen synthase kinase 3), lithium chloride (LiCl, Lithium Chloride), SB216763, SB415286 (2-(1H-reduced)-3-cyano-4-(dimethylamino)cholic acid), AZD1080 (2-hydroxy-3-[5-(morpholin-4-methyl)pyridin-2-yl]-1H-indole-5-carbonitrile) and / or AR-A014418; the R-Spondins include R-Spondin 1 and / or R-Spondin 2.

[0062] In one embodiment of the present invention, in the cardiac organoid differentiation culture medium, the GSK-3α / β inhibitor is raglucillin (CHIR99021); and the WNT pathway inhibitor is the PORCN inhibitor WNT-C59.

[0063] In one embodiment of the present invention, the concentration of the WNT pathway activator in the cardiac organoid differentiation medium is 3μM~12μM; the concentration of the bone morphogenetic protein 4 (BMP4) in the cardiac organoid differentiation medium is 1ng / mL~3ng / mL; the concentration of activin A (Active A) in the cardiac organoid differentiation medium is 1ng / mL~3ng / mL; the concentration of the WNT pathway inhibitor in the cardiac organoid differentiation medium is 1μM~5μM.

[0064] In one embodiment of the present invention, the first basal culture medium is a cell culture medium additionally supplemented with B27 supplement without added insulin.

[0065] In one embodiment of the present invention, the concentration of the B27 supplement without added insulin in the cell culture medium is 1% to 5% by volume.

[0066] In one embodiment of the present invention, the cell culture medium is RPMI1640 culture medium.

[0067] The present invention also provides a method for screening drugs for preventing and / or treating heart disease, the method comprising: first performing disease modeling on the above-mentioned heart organoids to obtain an in vitro model of heart disease, and then using the in vitro model of heart disease to screen drugs for preventing and / or treating heart disease.

[0068] In one embodiment of the present invention, the heart disease comprises congenital heart disease.

[0069] In one embodiment of the present invention, the heart disease includes myocardial hypertrophy, myocardial injury and / or cardiac fibrosis.

[0070] The present invention also provides use of the cardiac organoid, the method, or the cardiac organoid differentiation culture medium in screening drugs for preventing and / or treating heart disease.

[0071] In one embodiment of the present invention, the heart disease comprises congenital heart disease.

[0072] In one embodiment of the present invention, the heart disease includes myocardial hypertrophy, myocardial injury and / or cardiac fibrosis.

[0073] The technical solution of the present invention has the following advantages:

[0074] The present invention provides a cardiac organoid, which is derived from adherent pluripotent stem cell clusters induced and cultured; the adherent pluripotent stem cell clusters derived from adherent pluripotent stem cell clusters cultured in suspension; and the suspension pluripotent stem cell clusters derived from suspension pluripotent stem cells (PSCs). The in vitro preparation of this cardiac organoid involves inducing differentiation of the pluripotent stem cell spheres in adherent culture in stages after the pluripotent stem cells have formed into spheres. This allows the organoid to generate cell subtypes such as epicardial cells without the need for additional additions or signaling pathway stimulation, resulting in a more homogeneous structure. Studies have shown that this cardiac organoid can reproduce the rhythmic beating of the heart; during differentiation, it can recreate key developmental events during early embryonic heart development, such as the specialization of first heart field (FHF) and second heart field (SHF) progenitor cells, as well as the endothelial-mesenchymal transition (EMT); during differentiation, it can autonomously and orderly produce a variety of cardiac-specific cells that appear during early embryonic heart development (including cardiomyocytes such as working cardiomyocytes and autonomous cardiomyocytes, as well as non-cardiomyocytes such as endocardial cells, epicardial cells, fibroblasts, and interstitial cells); and the distribution of different cell subtypes produced during differentiation is more regular and homogeneous. Therefore, this spontaneously generated multi-lineage coexisting cardiac organoid can better simulate the biological events derived from the multiple cell types and multiple cell subtypes that occur during early embryonic heart development, and has great application prospects in exploring the pathogenic factors and potential therapeutic targets of congenital heart disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 : Flow chart of the preparation of cardiac organoids in Example 1 and Comparative Example 1.

[0076] Figure 2: Changes in beating ratio of cardiac organoids as differentiation progresses.

[0077] Figure 3 : Bright field schematic diagrams of cardiac organoids prepared in Example 1 and Comparative Example 1. Scale bar: 200 μm.

[0078] Figure 4 : Expression of pluripotency genes during cardiac organoid formation.

[0079] Figure 5 : Expression of mesodermal genes during cardiac organoid formation.

[0080] Figure 6 : Expression of myocardial marker genes during cardiac organoid formation.

[0081] Figure 7 : Expression of epicardial marker genes during cardiac organoid formation.

[0082] Figure 8 : Distribution patterns of cardiomyocytes in cardiac organoids. Figure 8 In the figure, A shows the distribution pattern of cardiomyocytes in the cardiac organoid prepared in Comparative Example 1; B shows the distribution pattern of cardiomyocytes in the cardiac organoid prepared in Example 1. Scale bar: 100 μm.

[0083] Figure 9 Immunofluorescence staining of cardiac organoids obtained after 16 days of culture. Scale bars: 200 μm for the whole image and 60 μm for the magnified image.

[0084] Figure 10 : qRT-PCR detection results of the first and second cardiac field markers in cardiac organoids at multiple time points.

[0085] Figure 11 : Immunofluorescence staining results of epicardial cells, interstitial cells, and fibroblasts in the cardiac organoids prepared in Example 1. Scale bar: 100 μm.

[0086] Figure 12 : Schematic diagram of the process for verifying the regulatory role of the TGFβ pathway in cardiac organoids.

[0087] Figure 13 : Immunofluorescence images of WT1 and aSMA protein expression in cardiac organoids after treatment with TGFβ1 and A8301.

[0088] Figure 14 : Changes in the proportion of WT1-positive cells in organoids after treatment with TGFβ1 and A8301. DETAILED DESCRIPTION

[0089] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0090] If no specific experimental steps or conditions are specified in the following examples, the experiments were carried out according to the conventional experimental steps or conditions described in the literature in the field. If no manufacturer is specified for the reagents or instruments used, they are all commercially available conventional reagents.

[0091] The H9 embryonic stem cell line involved in the following examples was purchased from WiCell; the Matrigel matrix glue involved in the following examples was purchased from Corning, model 356231, Essential 8 TM Culture medium was purchased from Thermo Fisher Scientific, model A1517001, B27(-) was purchased from Thermo Fisher Scientific, model A1895601 (B27(-) refers to B27 supplement without added insulin), B27(+) was purchased from Thermo Fisher Scientific, model 17504044 (B27(+) refers to B27 supplement with added insulin), CHIR99021 was purchased from Hanxiang Biotechnology, model BCP26126, bone morphogenetic protein 4 (BMP4) was purchased from StemImmune LLC, model HST-B4-0100, and activin A was purchased from StemImmune. LLC, model HST-A-0100, and the PORCN inhibitor Wnt-C59 was purchased from APExBIO, model A8685. The 96-well deep-well plate (U-shaped bottom) and low-adsorption 96-well deep-well plate (U-shaped bottom) involved in the following examples were purchased from Jiete Bio and Corning, models TCP002096 and 7007, respectively.

[0092] Example 1: A method for preparing cardiac organoids in vitro

[0093] This embodiment provides a cardiac organoid, which is derived from adherent pluripotent stem cell clusters induced by culture; the adherent pluripotent stem cell clusters derived from adherent pluripotent stem cell clusters cultured by culture; and the suspension pluripotent stem cell clusters derived from suspension pluripotent stem cells (PSCs). The cardiac organoid preparation process is as follows:

[0094] Step S1: H9 embryonic stem cells were seeded at a density of 10,000 cells / well in a well containing 100 μL Essential 8 TMAfter the culture medium was added to a low-adsorption 96-well deep-well plate (U-shaped bottom), the low-adsorption 96-well deep-well plate was placed in a 37°C cell culture incubator with 5% (v / v) CO2 to culture the human pluripotent stem cells in suspension (recorded as day -2);

[0095] Step S2: After 24 hours of suspension culture (recorded as Day -1), the suspended human pluripotent stem cells in the low-adsorption 96-well deep-well plate were observed to spontaneously aggregate to form suspended pluripotent stem cell clusters. At this time, the suspended pluripotent stem cell clusters in the low-adsorption 96-well deep-well plate were transferred well by well along with 30 μL of culture medium to 120 μL of Essential 8 TM The cells were transferred to a 96-well deep-well plate coated with Matrigel matrix gel containing culture medium. After the transfer, each suspended pluripotent stem cell cluster was placed in the center of the well of the 96-well deep-well plate by tapping and shaking. After the tapping and shaking, the Matrigel matrix gel-coated 96-well deep-well plate was placed in a 37°C cell culture incubator with 5% (v / v) CO2 to culture the suspended pluripotent stem cell clusters to adhere to the wall.

[0096] Step S3: After 24 hours of adherent culture (recorded as day 0), the pluripotent stem cell clusters in the Matrigel-coated 96-well deep-well plate can be observed to be attached to the wall. At this time, 65 μL of culture medium was aspirated from each well, and the first cardiac organoid differentiation medium was added to the Matrigel-coated 96-well deep-well plate at a volume of 165 μL / well. After the addition, the Matrigel-coated 96-well deep-well plate was placed in a 37°C cell culture medium with 5% (v / v) CO2. The adherent pluripotent stem cell clusters were subjected to mesoderm induction culture in a culture incubator. After 24 hours of induction culture (recorded as day 1), 160 μL of culture medium was aspirated from each well, and 165 μL / well of the first basal culture medium was added to a Matrigel-coated 96-well deep-well plate. After the addition, the Matrigel-coated 96-well deep-well plate was placed in a 37°C cell culture incubator with 5% (v / v) CO2 to continue mesoderm induction culture of the adherent pluripotent stem cell clusters.

[0097] Step S4: After 24 hours of induction culture (recorded as day 2), cardiogenic mesodermal progenitor cell clusters can be observed in the Matrigel-coated 96-well deep-well plate. At this time, 160 μL of culture medium is aspirated from each well, and the second cardiac organoid differentiation medium is added to the Matrigel-coated 96-well deep-well plate at a volume of 165 μL / well. After the addition is completed, the Matrigel-coated 96-well deep-well plate is placed in a 37°C cell culture incubator with 5% (v / v) CO2 to culture the cardiogenic mesodermal progenitor cell clusters for cardiovascular induction.

[0098] Step S5: After 48 hours of induction culture (recorded as day 4), cardiovascular progenitor cell clusters can be observed in the Matrigel-coated 96-well deep-well plate. At this time, 155 μL of culture medium is aspirated from each well, and the first basal culture medium is added to the Matrigel-coated 96-well deep-well plate at a volume of 165 μL / well; after the addition is completed, the Matrigel-coated 96-well deep-well plate is placed in a 37°C cell culture incubator with 5% (v / v) CO2 to induce organoid culture of the cardiovascular progenitor cell clusters; after 48 hours of induction culture (recorded as day 6), 155 μL of culture medium is aspirated from each well, and the second basal culture medium is added to the Matrigel-coated 96-well deep-well plate at a volume of 165 μL / well. After the addition is completed, the Matrigel-coated 96-well deep-well plate is placed in a 37°C cell culture incubator with 5% (v / v) CO2 to continue the organoid induction culture of the cardiovascular progenitor cell cluster; during the organoid induction culture using the second basal culture medium, fresh second basal culture medium is replenished every two days (when replenishing fresh second basal culture medium, 155 μL of culture medium is still aspirated and discarded from each well, and the second basal culture medium is added to the Matrigel-coated 96-well deep-well plate at an addition volume of 165 μL / well); after 9 days of induction culture (recorded as the 15th day), cardiac organoids can be observed in the Matrigel-coated 96-well deep-well plate (the preparation process of cardiac organoids is shown in the preparation process of the second basal culture medium). Figure 1 );

[0099] The preparation process of the Matrigel-coated 96-well deep-well plate (U-shaped bottom) is as follows: Dilute the Matrigel to a concentration of 2% (v / v) using DMEM / F12 medium to obtain a Matrigel diluent; Add the Matrigel diluent to a 96-well deep-well plate (U-shaped bottom) at a volume of 70 μL / well, and then place the 96-well deep-well plate in a 37°C cell culture incubator with 5% (v / v) CO2 for 30 minutes to obtain a Matrigel-coated 96-well deep-well plate;

[0100] The first cardiac organoid differentiation medium is the first basal medium supplemented with 6 μM CHIR99021, 1.875 ng / mL bone morphogenetic protein 4 (BMP4), and 1.5 ng / mL activin A.

[0101] The second cardiac organoid differentiation medium was the first basal medium supplemented with 3 μM of the PORCN inhibitor WNT-C59;

[0102] The first basal culture medium was RPMI1640 medium containing 2% (v / v) B27(-);

[0103] The second basal culture medium was RPMI1640 medium containing 2% (v / v) B27(+).

[0104] Comparative Example 1: A method for preparing cardiac organoids in vitro

[0105] This comparative example provides a heart organoid, and the heart organoid preparation process is as follows:

[0106] Based on Example 1, step S2 was replaced by the following: After 24 hours of suspension culture (recorded as day -1), it was observed that the suspended human pluripotent stem cells in the low-adsorption 96-well deep-well plate spontaneously aggregated to form a suspended pluripotent stem cell cluster. At this time, the suspended pluripotent stem cell cluster was not transferred, and the low-adsorption 96-well deep-well plate was directly placed in a 37°C cell culture incubator with 5% (v / v) CO2 to continue suspension culture of the suspended pluripotent stem cell cluster (see the preparation process of cardiac organoids for details). Figure 1 ).

[0107] Experimental Example 1: Verification of Organoid Performance

[0108] This experimental example provides a validation experiment for organoid performance. The experimental process is as follows:

[0109] 1. Experimental methods

[0110] Heart organoids (hHOs) were constructed using the methods of Example 1 and Comparative Example 1, respectively.

[0111] During the construction of cardiac organoids, the changes in the beating ratio of cardiac organoids as they differentiate were observed under a microscope every day. The observation results are shown in Figure 2 .

[0112] After the cardiac organoids were constructed, the cardiac organoids in the 96-well deep-well plate were observed under a microscope. The results are shown in the figure. Figure 3 (The heart organoids prepared in Example 1 are shown in the lower row, and the heart organoids prepared in Comparative Example 1 are shown in the upper row).

[0113] During the construction of cardiac organoids, samples were taken every 2 days for RT-qPCR detection of the expression of pluripotency genes, mesoderm genes, myocardial marker genes, and epicardial marker genes (primers are shown in Table 1). The test results are shown in Figures 4 to 7 ;

[0114] Among them, the RT-qPCR detection method is as follows: the total RNA of the sample is extracted using an RNA extraction kit (Thermo Fisher Scientific), 1 μg of RNA is reverse transcribed into cDNA using a reverse transcription kit (Beijing Quanshijin), and the cDNA is subjected to RT-qPCR analysis using a qPCR kit (Novozymes).

[0115] After the cardiac organoids were constructed, samples were taken for immunofluorescence staining. The staining results are shown in Figures 8 and 9 ; Among them, the immunofluorescence staining method is as follows:

[0116] Take the sample to be tested and immerse it in 4% (w / v, g / 100 mL) paraformaldehyde aqueous solution at room temperature (25°C) for 3 hours for fixation; after fixation, rinse it with PBS buffer 3 times, each rinse for 5 minutes; after rinsing, incubate it with PBS buffer containing 0.1% (w / v, g / 100 mL) bovine serum albumin (BSA), 0.2% (v / v) Triton X-100 and 2.5% (v / v) donkey serum at 4°C overnight (16 hours) for membrane permeabilization and blocking; after membrane permeabilization and blocking, incubate it with PBS buffer containing 0.1% (w / v, g / 100 mL) bovine serum albumin and 0.1% (v / v) Triton X-100. The primary antibody was diluted with PBS buffer containing X-100, and the organoids were incubated with the diluted primary antibody (incubated in a refrigerator at 4°C for 24 hours); after incubation, the organs were rinsed three times with PBS buffer, each rinse for 10 minutes; after rinsing, the secondary antibody was diluted with PBS buffer containing 0.1% (w / v, g / 100 mL) bovine serum albumin and 2.5% (v / v) donkey serum, and the organoids were incubated with the diluted secondary antibody (incubated overnight at 4°C); after incubation, the organs were rinsed three times with PBS buffer, each rinse for 10 minutes; after rinsing, DAPI was diluted with PBS buffer containing 0.1% (w / v, g / 100 mL) bovine serum albumin, and the organoids were incubated with the diluted DAPI (incubated at room temperature for 1 hour); after incubation, the cardiac organoids (hHOs) on day 16 were observed and photographed using a Nikon upright microscope or a Zeiss laser confocal microscope (Zeiss700b) to obtain the immunofluorescence staining results;

[0117] During incubation, the primary antibody used for cTnT protein was Mouse Anti-Cardiac Troponin T (purchased from Abcam, diluted 1000-fold before use), and the secondary antibody used for cTnT protein was Donkey anti-Mouse IgG, Alexa Fluor 555 (purchased from Invitrogen, diluted 500-fold before use). The primary antibodies used for NKX2.5 protein were Goat anti-NKX2.5 and Rabbit anti-NKX2-5 (purchased from R&D Systems and Proteintech, respectively, diluted 1000-fold before use), and the secondary antibodies used for NKX2.5 protein were Donkey anti-Goat IgG, Alexa Fluor 488 and Donkey anti-Rabbit IgG, Alexa Fluor 555 (both purchased from Invitrogen, diluted 500-fold before use). The primary antibody used for CD31 protein was Sheep Anti-Human CD31 / PECAM-1 Antigen Affinity-purified pAb (purchased from R&D Systems, Inc., Boston Children's Hospital ... Systems, diluted 1000-fold before use); the secondary antibody used for CD31 protein was Donkey anti-Sheep IgG, Alexa Fluor 488 (purchased from Invitrogen, diluted 500-fold before use); the primary antibody used for WT1 protein was Rabbit Anti-Wilms Tumor (WT1) Protein (purchased from Abcam, diluted 500-fold before use), and the secondary antibody used for WT1 protein was Donkey anti-Rabbit IgG, Alexa Fluor 555 (purchased from Invitrogen, diluted 500-fold before use); the primary antibody used for α-SMA protein was Ms Anti-Alpha SMA (purchased from Sigma-Aldrich, diluted 1000-fold before use), and the secondary antibody used for α-SMA protein was Donkey anti-Mouse IgG, Alexa Fluor 488 (purchased from Invitrogen, diluted 500-fold before use); the primary antibody used for VIM protein was Mouse monoclonal Anti-Vimentin The secondary antibody used for VIM protein was Donkey anti-Mouse IgG, Alexa Fluor 488 (purchased from Invitrogen and diluted 500-fold before use). All primary antibodies were mixed and incubated in organoids at the same time.

[0118] During the construction of cardiac organoids, samples were taken every 3 days for RT-qPCR detection of the first and second cardiac field markers (primers see Table 2 ). The test results are shown in Figure 10 ;

[0119] Among them, the RT-qPCR detection method is as follows: the total RNA of the sample is extracted using an RNA extraction kit (Thermo Fisher Scientific), 1 μg of RNA is reverse transcribed into cDNA using a reverse transcription kit (Beijing Quanshijin), and the cDNA is subjected to RT-qPCR analysis using a qPCR kit (Novozymes).

[0120] Table 1 Primers and their sequences

[0121]

[0122] Table 2 Primers and their sequences

[0123]

[0124] 2. Experimental results

[0125] Depend on Figure 2 It can be seen that the cardiac organoids in both the scheme of Example 1 and the scheme of Comparative Example 1 began to beat on the 6th day of differentiation. It can be seen that the scheme of Example 1 does not affect the efficiency of cardiac organoid formation.

[0126] Depend on Figure 3 It can be seen that the growth conditions (size changes) of the heart organoids in Example 1 and Comparative Example 1 are similar. This shows that the solution in Example 1 does not affect the growth rate of the heart organoids.

[0127] Depend on Figures 4 to 7 It can be seen that the changes in genes indicating pluripotency, mesoderm genes, and myocardium are basically the same in Example 1 and Comparative Example 1, indicating that epicardial genes are significantly upregulated in the early stages of cardiac organoid formation, which is higher than in Comparative Example 1. It can be seen that the specialization of mesoderm and myocardial cells in Example 1 and Comparative Example 1 is similar, and the early specialization of epicardial cells in Example 1 is better than that in Comparative Example 1.

[0128] Depend on Figure 8 It can be seen that the myocardial cells in the solution of Comparative Example 1 have three distribution modes, while the myocardial cells in the solution of Example 1 are mostly evenly distributed. It can be seen that the solution of Example 1 is beneficial to the even distribution of myocardial cells.

[0129] Depend on Figure 9As can be seen, in Example 1, CD31, representing endothelial cells, NKX2.5, representing cardiomyocytes, WT1, representing epicardium, and αSMA, representing mesenchymal cells, all appeared efficiently. This indicates that multiple cell subtypes coexisted in the cardiac organoids formed using Example 1.

[0130] Depend on Figure 10 It can be seen that the genes related to the first and second heart fields in both Example 1 and Comparative Example 1 were significantly upregulated during the differentiation process, and the upregulated expression of the genes in the first and second heart fields in Example 1 was higher than that in the cardiac organoids in Comparative Example 1. This shows that the cardiac organoid formation process in Example 1 reproduces the specialization events of the first and second heart fields, and the formation of cells in the first and second heart fields in Example 1 is better than that in Comparative Example 1.

[0131] Depend on Figure 11 As can be seen, the cardiac organoids obtained using the protocol in Example 1 contain all cell types involved in the epithelial-mesenchymal transition (EMT) process. Furthermore, WT1-positive cells also express aSMA and VIM, indicating a transition to mesenchymal cells. Furthermore, these cells are migrating toward the cardiomyocyte region. This demonstrates that the cardiac organoid formation process in Example 1 recapitulates the key developmental event of EMT.

[0132] Depend on Figures 12 to 14 It can be seen that in the presence of TGFβ1, the number of WT1-positive cells in the cardiac organoids provided in Example 1 decreased, while the number of αSMA-positive cells increased. In the presence of A8301, the number of WT1-positive cells increased, while the number of αSMA-positive cells decreased. It can be seen that the process of forming epicardial cells, fibroblasts, and mesenchymal cells in the cardiac organoids provided by the present invention is regulated by the TGFβ pathway, indicating that the process of cardiac organoid formation reproduces the key developmental event of EMT.

[0133] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A cardiac organoid, characterized in that The cardiac organoids are obtained by inducing and culturing cardiovascular progenitor cell clusters sequentially using a first basal medium and a second basal medium; the cardiovascular progenitor cell clusters are obtained by inducing and culturing cardiac mesodermal progenitor cell clusters using a second cardiac organoid differentiation medium; the cardiac mesodermal progenitor cell clusters are obtained by inducing and culturing adherent pluripotent stem cell clusters sequentially using a first cardiac organoid differentiation medium and a first basal medium; the adherent pluripotent stem cell clusters are obtained by adherent culture of suspension pluripotent stem cell clusters; the suspension pluripotent stem cell clusters are obtained by suspension culture of pluripotent stem cells; the adherent culture is performed in a culture vessel coated with matrigel; The first cardiac organoid differentiation medium is a first basal medium additionally supplemented with a WNT pathway activator, bone morphogenetic protein 4, and / or activin A; the second cardiac organoid differentiation medium is a first basal medium additionally supplemented with a WNT pathway inhibitor; the first basal medium is a cell culture medium additionally supplemented with a B27 supplement without insulin; the second basal medium is a cell culture medium additionally supplemented with a B27 supplement with insulin; The cardiac organoids can reproduce the key developmental events in the early development of the embryonic heart during the differentiation process; the key developmental events include the specialization process of the first cardiac field progenitor cells and the second cardiac field progenitor cells and the epithelial-mesenchymal transformation process; the cardiac organoids can reproduce the rhythmic beating of the heart; the cardiac organoids can autonomously and orderly produce cardiac-specific cells that appear in the early development of the embryonic heart during the differentiation process; the cardiac organoids show the distribution patterns of different cell subtypes produced during the differentiation process.

2. The cardiac organoid according to claim 1, wherein The cardiac-specific cells include myocardial cells and non-myocardial cells; the myocardial cells include working myocardial cells and autonomous myocardial cells; the non-myocardial cells include endocardial cells, epicardial cells, fibroblasts and interstitial cells.

3. The heart organoid according to claim 1 or 2, wherein: The preparation method of the cardiac organoids includes: performing suspension culture on pluripotent stem cells to obtain a suspension pluripotent stem cell cluster; performing adherent culture on the suspension pluripotent stem cell cluster to obtain an adherent pluripotent stem cell cluster; sequentially performing mesoderm induction culture on the adherent pluripotent stem cell cluster using a first cardiac organoid differentiation medium and a first basal medium to obtain a cardiac mesoderm progenitor cell cluster; performing cardiovascular induction culture on the cardiac mesoderm progenitor cell cluster using a second cardiac organoid differentiation medium to obtain a cardiovascular progenitor cell cluster; and sequentially performing organoid induction culture on the cardiovascular progenitor cell cluster using the first basal medium and the second basal medium to obtain a cardiac organoid.

4. The cardiac organoid according to claim 3, wherein The concentration of the WNT pathway activator in the first cardiac organoid differentiation medium is 3 μM~12 μM; the concentration of the bone morphogenetic protein 4 in the first cardiac organoid differentiation medium is 1 ng / mL~3 ng / mL; the concentration of activin A in the first cardiac organoid differentiation medium is 1 ng / mL~3 ng / mL; the concentration of the WNT pathway inhibitor in the second cardiac organoid differentiation medium is 1 μM~5 μM.

5. The cardiac organoid according to claim 1 or 2, wherein: The suspension culture is carried out in a low adsorption culture container.

6. A method for constructing the cardiac organoid according to any one of claims 1 to 5, characterized in that: The method comprises: performing suspension culture on pluripotent stem cells to obtain a suspension pluripotent stem cell cluster; performing adherent culture on the suspension pluripotent stem cell cluster to obtain an adherent pluripotent stem cell cluster; sequentially using a first cardiac organoid differentiation medium and a first basal medium to induce culture the adherent pluripotent stem cell cluster to obtain a cardiac-derived mesodermal progenitor cell cluster; performing induction culture on the cardiac-derived mesodermal progenitor cell cluster using a second cardiac organoid differentiation medium to obtain a cardiovascular progenitor cell cluster; and sequentially using the first basal medium and the second basal medium to induce culture the cardiovascular progenitor cell cluster to obtain a cardiac organoid. The first cardiac organoid differentiation medium is a first basal medium additionally supplemented with a WNT pathway activator, bone morphogenetic protein 4 and / or activin A; the second cardiac organoid differentiation medium is the first basal medium additionally supplemented with a WNT pathway inhibitor.

7. A method for screening drugs for preventing and / or treating heart disease, characterized in that: The method comprises: firstly performing disease modeling on the cardiac organoid according to any one of claims 1 to 5 to obtain an in vitro model of heart disease, and then using the in vitro model of heart disease to screen drugs for preventing and / or treating heart disease.

8. Use of the cardiac organoid according to any one of claims 1 to 5, the method according to claim 6, or the method according to claim 7 in screening drugs for preventing and / or treating heart disease.

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