Heart organoid and in-vitro preparation method thereof

Abstract

The invention relates to a heart organoid and an in-vitro preparation method thereof, and belongs to the technical field of stem cells. The invention provides a heart organoid. The heart organoid is obtained by induced culture of an adherent pluripotent stem cell mass; the adherent pluripotent stem cell mass is obtained by adherent culture of a suspended pluripotent stem cell mass; the suspension pluripotent stem cell mass is obtained by suspension culture of pluripotent stem cells. According to the in-vitro preparation process of the heart organoid, after pluripotent stem cells form balls, the pluripotent stem cell balls are subjected to staged induced differentiation under adherent culture, so that cell subtypes such as epicardial cells and the like can be generated without additional addition or additional stimulation of signal channels, and the heart organoid is more homogeneous. The self-occurring multi-lineage coexisting heart organoid can better simulate biological events derived from multiple cell types and multiple cell subtypes in the early development process of an embryonic heart, and has great application prospects in exploration of pathogenic factors and potential treatment targets of congenital heart diseases.

Description

Technical Field

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

[0002] The heart is an organ of the human body. The main function of the heart is to provide power for blood flow and to transport blood to all parts of the body. Heart disease is a general term for diseases caused by damaged heart structure or abnormal function. As a 3D cell culture spontaneously assembled from pluripotent stem cells in vitro, cardiac organoids exhibit remarkable self-organization characteristics and are highly homologous to in vivo tissues in terms of morphology, structure, gene expression, etc., providing a new research tool for exploring the pathogenic factors and potential therapeutic targets of heart disease. The self-assembly process of ideal cardiac organoids can simulate various biological events of heart development in vitro, such as the formation of early cardiac mesodermal progenitor cells, the process of early cardiac mesodermal progenitor cells further specializing into first heart field (FHF) progenitor cells and second heart field (SHF) progenitor cells, and the appearance of various cardiac-specific non-cardiac cells (including endocardial cells, epicardial cells, endothelial cells, fibroblasts, and interstitial cells) in the early heart.

[0003] However, existing heart organoids all have defects to some extent. For example, the cardiomyocyte cell types are not comprehensive, lacking necessary cardiomyocyte cells such as FHF progenitor cells (see the literature “L. Drakhlis et al., Human heart-forming organoidsrecapitulate 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., Cardioidsreveal 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 heart development; the lack of non-cardiomyocyte cells cannot well reproduce the autonomous and orderly occurrence of cardiac-specific non-cardiomyocytes during heart 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 human cardiogenesis, Cell, vol. 184, no.12, pp. 3299-3317.e22, 2021.”). 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 references “YR Lewis-Israeliet 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 uncoversprinciples of human epicardium biology in heart development and disease, Nat.Biotechnol., 2023."), which increases the complexity of the heart organoid construction process and the cost of heart organoid construction; a suspension culture system is required for in vitro preparation, and the suspension culture system cannot well guarantee the homogeneity between heart organoids, resulting in irregular distribution of different cell subtypes (see the literature "YRLewis-Israeli et al., Self-assembling human heart organoids for the modelingof 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 heart organoid that can autonomously and orderly produce a variety of heart-specific cells (including cardiomyocytes and non-cardiomyocytes such as endocardial cells, epicardial cells, fibroblasts, and interstitial cells) during differentiation, and has good homogeneity. Summary of the invention

[0004] To solve the above-mentioned defects, 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; 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 in the early development of the embryonic heart during the differentiation process; 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 distribution patterns of different cell subtypes produced by the cardiac organoids during the differentiation process.

[0007] In one embodiment of the present invention, the key developmental events include the specialization process of the first heart field (FHF) progenitor cells and the second heart field (SHF) progenitor cells and the endothelial-mesenchymal transformation (EMT); 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 cardiomyocytes include atrial cardiomyocytes and ventricular cardiomyocytes; the autonomous cardiomyocytes 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 (PSC) to obtain a suspended pluripotent stem cell cluster; performing adherent culture on the suspended 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 the 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; 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 the 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; using a first cardiac organoid differentiation medium and a first basal medium in sequence 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; using the first basal medium and the second basal medium in sequence 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 medium is a first basal medium additionally supplemented with a WNT pathway activator, bone morphogenetic protein 4 (BMP4) and / or Active A.

[0015] In one embodiment of the present invention, in the first cardiac organoid differentiation culture medium, the WNT pathway activator component includes 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 medium, the GSK-3α / β inhibitors include 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-(morpholine-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 the 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 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 adding 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 with added insulin.

[0026] In one embodiment of the present invention, the concentration of the B27 supplement with added insulin 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 carried out in a low-adsorption culture container; and the adherent culture is carried out in a culture container coated with matrix gel.

[0030] In one embodiment of the present invention, the low-adsorption culture container comprises 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 bottle.

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

[0034] The present invention also provides a method for constructing the above-mentioned cardiac organoids, the method comprising: 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; 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; performing mesoderm induction culture on the adherent pluripotent stem cell cluster using a first cardiac organoid differentiation medium and a first basal medium in sequence 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; performing organoid induction culture on the cardiovascular progenitor cell cluster using the first basal medium and the second basal medium in sequence 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; using a first cardiac organoid differentiation medium and a first basal medium in sequence 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; using the first basal medium and the second basal medium in sequence 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 medium is a first basal medium additionally supplemented with a WNT pathway activator, bone morphogenetic protein 4 (BMP4) and / or Active A.

[0040] In one embodiment of the present invention, in the first cardiac organoid differentiation culture medium, the WNT pathway activator component includes 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 medium, the GSK-3α / β inhibitors include 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-(morpholine-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 the 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 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 adding 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 with added insulin.

[0051] In one embodiment of the present invention, the concentration of the B27 supplement with added insulin 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 carried out in a low-adsorption culture container; and the adherent culture is carried out in a culture container coated with matrix gel.

[0055] In one embodiment of the present invention, the low-adsorption culture container comprises 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 bottle.

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

[0059] The present invention also provides a cardiac organoid differentiation medium, wherein the cardiac organoid differentiation medium 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 components of the WNT pathway activator include GSK-3α / β inhibitor (GSK-3, i.e., ATP-competitive glycogen synthase kinase 3), WNT3a (secreted protein) and / or R-Spondins (secreted protein); the components of the WNT pathway inhibitor include 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 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-(morpholine-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 raglucirib (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 adding 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: firstly 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 the 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: The present invention provides a cardiac organoid, wherein the cardiac organoid is obtained by inducing and culturing an adherent pluripotent stem cell group; the adherent pluripotent stem cell group is obtained by culturing a suspended pluripotent stem cell group by adherence; the suspended pluripotent stem cell group is obtained by culturing a pluripotent stem cell (PSC) by suspension. The in vitro preparation process of the cardiac organoid is to induce differentiation of the pluripotent stem cell spheres in stages under adherence 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. Studies have shown that the cardiac organoid can reproduce the rhythmic beating of the heart; during the differentiation process, the cardiac organoid can reproduce key developmental events such as the specialization process of the first heart field (FHF) progenitor cells and the second heart field (SHF) progenitor cells during the early development of the embryonic heart, as well as the endothelial-mesenchymal transformation process (EMT); during the differentiation process, the cardiac organoid can autonomously and orderly produce a variety of cardiac-specific cells that appear during the early development of the embryonic heart (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 by the cardiac organoid during the differentiation process is more regular and homogeneous. Therefore, the spontaneously generated multi-lineage coexisting cardiac organoid can better simulate the biological events derived from multiple cell types and multiple cell subtypes that appear 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 : Flow chart of preparation of cardiac organoids in Example 1 and Comparative Example 1.

[0075] Figure 2 : Changes in beating ratio of cardiac organoids as they differentiate.

[0076] Figure 3 : Bright field schematic diagram of cardiac organoids prepared in Example 1 and Comparative Example 1. Scale bar: 200 μm.

[0077] Figure 4 : Expression of pluripotency genes during cardiac organoid formation.

[0078] Figure 5 : Expression of mesodermal genes during cardiac organoid formation.

[0079] Figure 6 :Expression of myocardial marker genes during cardiac organoid formation.

[0080] Figure 7 : Expression of epicardial marker genes during cardiac organoid formation.

[0081] Figure 8 :Distribution pattern of cardiomyocytes in cardiac organoids. Figure 8 In the figure, A is the distribution law of cardiomyocytes in the cardiac organoid prepared in Comparative Example 1; B is the distribution law of cardiomyocytes in the cardiac organoid prepared in Example 1. Scale bar: 100 μm.

[0082] Fig. 9 : Immunofluorescence staining results of cardiac organoids obtained after 16 days of culture. Scale bar: 200 μm for the whole image and 60 μm for the magnified image.

[0083] Fig.10 : qRT-PCR detection results of the first and second cardiac field markers in cardiac organoids at multiple time points.

[0084] Fig.11 : Results of immunofluorescence staining of epicardial cells, interstitial cells and fibroblasts in the cardiac organoids prepared in Example 1. Scale bar: 100 μm.

[0085] Fig.12 : Schematic diagram of the process to verify the regulatory role of the TGFβ pathway in cardiac organoids.

[0086] Fig.13 : Immunofluorescence images of WT1 and aSMA protein expression in cardiac organoids after treatment with TGFβ1 and A8301.

[0087] Fig.14 : Changes in the proportion of WT1-positive cells in organoids after treatment with TGFβ1 and A8301. DETAILED DESCRIPTION

[0088] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0089] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0090] The H9 embryonic stem cell line involved in the following examples was purchased from WiCell; the Matrigel matrix gel involved in the following examples was purchased from Corning, model 356231, Essential 8 TM The 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, 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 embodiments were purchased from Clean Bio and Corning, models TCP002096 and 7007, respectively.

[0091] Example 1: A method for preparing cardiac organoids in vitro This embodiment provides a cardiac organoid, wherein the cardiac organoid is obtained by induced culture of an adherent pluripotent stem cell group; the adherent pluripotent stem cell group is obtained by adherent culture of a suspended pluripotent stem cell group; the suspended pluripotent stem cell group is obtained by suspension culture of pluripotent stem cells (PSC). The preparation process of the cardiac organoid is as follows: Step S1: H9 embryonic stem cells were seeded at a seeding 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); 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 each well of the plate containing 120 μL of Essential 8 TM The cells were placed in 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 96-well deep-well plate coated with Matrigel matrix gel was placed in a 37°C cell culture incubator with 5% (v / v) CO2 to culture the suspended pluripotent stem cell clusters adherently; 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 adhered 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 an addition 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 attached pluripotent stem cell clusters were subjected to mesoderm induction culture in a culture incubator; after 24 hours of induction culture (recorded as the first day), 160 μL of culture medium was discarded from each well, and the first basal culture medium was added to the 96-well deep-well plate coated with Matrigel matrix gel at an addition amount of 165 μL / well; after the addition, the 96-well deep-well plate coated with Matrigel matrix gel was placed in a 37°C cell culture incubator with 5% (v / v) CO2 to continue to induce mesoderm in the attached pluripotent stem cell clusters; Step S4: After 24 hours of induction culture (recorded as the second day), it can be observed that the cardiogenic mesodermal progenitor cell clusters are formed 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 an addition 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 perform cardiovascular induction culture on the cardiogenic mesodermal progenitor cell clusters; Step S5: After 48 hours of induction culture (recorded as the 4th day), 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 an addition amount of 165 μL / well; after the addition, 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 the 6th day), 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 an addition amount of 165 μL / well 96-well deep-well plate coated with Matrigel; 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 the organoid induction culture of the cardiovascular progenitor cell cluster; during the organoid induction culture using the second basal medium, fresh second basal medium was added every two days (when adding fresh second basal medium, 155 μL of medium was still aspirated from each well, and the second basal medium was added to the Matrigel-coated 96-well deep-well plate at an addition amount of 165 μL / well); after 9 days of induction culture (recorded as the 15th day), it can be observed that cardiac organoids are formed in the Matrigel-coated 96-well deep-well plate (the preparation process of cardiac organoids is shown in Figure 1 ); The preparation process of the 96-well deep-well plate (U-shaped bottom) coated with Matrigel is as follows: dilute the Matrigel to a concentration of 2% (v / v) using DMEM / F12 culture medium to obtain a Matrigel diluent; add the Matrigel diluent to the 96-well deep-well plate (U-shaped bottom) at an addition amount 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 96-well deep-well plate coated with Matrigel; The first cardiac organoid differentiation medium was the first basal medium supplemented with 6 μM CHIR99021, 1.875 ng / mL bone morphogenetic protein 4 (BMP4), and 1.5 ng / mL Active A; The second cardiac organoid differentiation medium was the first basal medium supplemented with 3 μM of the PORCN inhibitor WNT-C59; The first basal culture medium was RPMI1640 medium containing 2% (v / v) B27(-); The second basal culture medium was RPMI1640 medium containing 2% (v / v) B27(+).

[0092] Comparative Example 1: A method for preparing cardiac organoids in vitro This comparative example provides a heart organoid, and the heart organoid preparation process is as follows: On the basis of Example 1, step S2 was replaced by: 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 to culture the suspended pluripotent stem cell cluster in suspension (see the preparation process of cardiac organoids for details). Figure 1 ).

[0093] Experimental Example 1: Verification of organoid performance This experimental example provides a validation experiment of organoid performance. The experimental process is as follows: 1. Experimental methods Heart organoids (hHOs) were constructed using the methods of Example 1 and Comparative Example 1, respectively.

[0094] 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 results are shown in Figure 2 .

[0095] 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 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).

[0096] During the construction of cardiac organoids, cardiac organoids were sampled every 2 days for RT-qPCR detection of the expression of pluripotency genes, mesoderm genes, myocardial marker genes, and epicardial marker genes (primers see Table 1). The test results are shown in Figure 4~Figure 7 ; Among them, the RT-qPCR detection method is: use RNA extraction kit (Thermo Fisher) to extract total RNA of the sample, use reverse transcription kit (Beijing Quanshijin) to reverse transcribe 1 μg RNA into cDNA, and use qPCR kit (Novozyme) to perform RT-qPCR analysis on cDNA.

[0097] After the cardiac organoids were constructed, samples were taken for immunofluorescence staining. The staining results are shown in Figure 8~Figure 9 ; Among them, the immunofluorescence staining method is as follows: Take the sample to be tested, immerse it in 4% (w / v, g / 100mL) paraformaldehyde aqueous solution at room temperature (25℃) for 3h for fixation; after fixation, rinse it with PBS buffer for 3 times, each rinse for 5 minutes; after rinsing, incubate it with PBS buffer containing 0.1% (w / v, g / 100mL) bovine serum albumin (BSA), 0.2% (v / v) Triton X-100 and 2.5% (v / v) donkey serum at 4℃ overnight (16 hours) for membrane permeabilization and blocking; after membrane permeabilization and blocking, incubate it with PBS buffer containing 0.1% (w / v, g / 100mL) bovine serum albumin and 0.1% (v / v) Triton The primary antibody was diluted with PBS buffer containing X-100, and the organoids were incubated with the diluted primary antibody (incubated in a 4°C refrigerator for 24 hours); after the incubation, the organs were rinsed three times with PBS buffer, each time for 10 minutes; after the rinsing, the secondary antibody was diluted with PBS buffer containing 0.1% (w / v, g / 100mL) 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 the incubation, the organs were rinsed three times with PBS buffer, each time for 10 minutes; after the rinsing, DAPI was diluted with PBS buffer containing 0.1% (w / v, g / 100mL) bovine serum albumin, and the organoids were incubated with the diluted DAPI (incubated at room temperature for 1 hour); after the incubation, the cardiac organoids (hHOs) on the 16th day were observed and photographed using a Nikon upright microscope or a Zeiss laser confocal microscope (Zeiss700b) to obtain the immunofluorescence staining results; During the incubation, the primary antibody used for cTnT protein was Mouse Anti-Cardiac Troponin T (purchased from Abcam, used after 1000-fold dilution), and the secondary antibody used for cTnT protein was Donkey anti-Mouse IgG, Alexa Fluor 555 (purchased from Invitrogen, used after 500-fold dilution); the primary antibodies used for NKX2.5 protein were Goat anti-NKX2.5 and Rabbitanti-NKX2-5 (purchased from R&D Systems and Proteintech, respectively, used after 1000-fold dilution), and the secondary antibodies used for NKX2.5 protein were Donkey anti-Goat IgG, Alexa Fluor 488 and Donkey anti-Rabbit IgG, AlexaFluor 555 (both purchased from Invitrogen, used after 500-fold dilution); the primary antibody used for CD31 protein was Sheep Anti-Human CD31 / PECAM-1 Antigen Affinity-purified pAb (purchased from R&D Systems, used after 500-fold dilution). Systems, diluted 1000 times before use); the secondary antibody used for CD31 protein was Donkey anti-Sheep IgG, Alexa Fluor 488 (purchased from Invitrogen, diluted 500 times before use); the primary antibody used for WT1 protein was Rabbit Anti-Wilms Tumor (WT1) Protein (purchased from Abcam, diluted 500 times before use), and the secondary antibody used for WT1 protein was Donkey anti-Rabbit IgG, Alexa Fluor 555 (purchased from Invitrogen, diluted 500 times before use); the primary antibody used for α-SMA protein was Ms Anti-Alpha SMA (purchased from Sigma-Aldrich, diluted 1000 times before use), and the secondary antibody used for α-SMA protein was Donkey anti-Mouse IgG, Alexa Fluor 488 (purchased from Invitrogen, diluted 500 times before use); the primary antibody used for VIM protein was Mouse monoclonal Anti-Vimentin antibody (purchased from Abcam and used after 1000-fold dilution), the secondary antibody used for VIM protein was Donkey anti-Mouse IgG, Alexa Fluor 488 (purchased from Invitrogen and used after 500-fold dilution); all primary antibodies were mixed and incubated in organoids at the same time.

[0098] During the construction of cardiac organoids, cardiac organoids were sampled every 3 days for RT-qPCR detection of the first cardiac field marker and the second cardiac field marker (primers see Table 2 ). The detection results are shown in Fig.10 ; Among them, the RT-qPCR detection method is: use RNA extraction kit (Thermo Fisher) to extract total RNA of the sample, use reverse transcription kit (Beijing Quanshijin) to reverse transcribe 1 μg RNA into cDNA, and use qPCR kit (Novozyme) to perform RT-qPCR analysis on cDNA.

[0099] Table 1 Primers and their sequences

[0100] Table 2 Primers and their sequences

[0101] 2. Experimental results 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.

[0102] Depend on Figure 3 It can be seen that the growth conditions (size changes) of the cardiac organoids in the scheme of Example 1 and the scheme of Comparative Example 1 are similar. It can be seen that the scheme of Example 1 does not affect the growth rate of cardiac organoids.

[0103] Depend on Figure 4~Figure 7 It can be seen that the changes in the genes of pluripotency, mesoderm genes, and myocardium in the scheme of Example 1 and the scheme of Comparative Example 1 are basically the same, indicating that the genes of the epicardium are significantly upregulated in the early stage of cardiac organoid formation, which is higher than that in the scheme of Comparative Example 1. It can be seen that in the scheme of Example 1 and the scheme of Comparative Example 1, the specialization of mesoderm and myocardial cells is similar, and the early specialization of epicardial cells in the scheme of Example 1 is better than that in the scheme of Comparative Example 1.

[0104] Depend on Figure 8 It can be seen that the myocardial cells in the scheme of comparative example 1 have three distribution modes, and the myocardial cells in the scheme of embodiment 1 are mostly evenly distributed. It can be seen that the scheme of embodiment 1 is beneficial to the even distribution of myocardial cells.

[0105] Depend on Fig. 9 It can be seen that CD31 representing endothelial cells, NKX2.5 representing cardiomyocytes, WT1 representing epicardium, and αSMA representing mesenchymal cells all appeared efficiently in the scheme of Example 1. It can be seen that multiple cell subtypes exist simultaneously in the cardiac organoids formed by the scheme of Example 1.

[0106] Depend on Fig.10 It can be seen that the genes related to the first and second heart fields in the scheme of Example 1 and the scheme of Comparative Example 1 are significantly upregulated during the differentiation process, and the upregulated expression of the first and second heart field genes in the scheme of Example 1 is higher than that of the heart organoids in the scheme of Comparative Example 1. It can be seen that the heart organoid formation process of Example 1 reproduces the first and second heart field specialization events, and the formation of the first and second heart field cells in the scheme of Example 1 is better than that in Comparative Example 1.

[0107] Depend on Fig.11 It can be seen that the cell types involved in the epithelial-mesenchymal transition (EMT) process are present in the cardiac organoids obtained by the scheme of Example 1, and it can be observed that WT1-positive cells also express aSMA and VIM, indicating that they are transforming into mesenchymal cells, and these cells are migrating to the cardiomyocyte area. It can be seen that the cardiac organoid formation process of Example 1 reproduces the key developmental event of EMT.

[0108] Depend on Figure 12~Figure 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, and the number of αSMA-positive cells increased. In the presence of A8301, the number of WT1-positive cells increased, and the number of αSMA-positive cells decreased. It can be seen that the process of the cardiac organoids provided by the present invention forming epicardial cells, fibroblasts, and interstitial cells is regulated by the TGFβ pathway, indicating that the cardiac organoid formation process reproduces the key developmental event of EMT.

[0109] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A cardiac organoid, characterized in that: The cardiac organoids are obtained by induced culture of adherent pluripotent stem cell clusters; the adherent pluripotent stem cell clusters are obtained by adherent culture of suspended pluripotent stem cell clusters; and the suspended pluripotent stem cell clusters are obtained by suspension culture of pluripotent stem cells.

2. The cardiac organoid according to claim 1, wherein 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.

3. The cardiac organoid according to claim 1, wherein: The cardiac organoids can reproduce the rhythmic beating of the heart; the cardiac organoids can reproduce the key developmental events in the early development of the embryonic heart during the differentiation process; the cardiac organoids can autonomously and orderly produce heart-specific cells that appear in the early development of the embryonic heart during the differentiation process; the distribution patterns of different cell subtypes produced by the cardiac organoids during the differentiation process.

4. The cardiac organoid according to claim 3, wherein The key developmental events include the specialization process of the first cardiac field progenitor cells and the second cardiac field progenitor cells and the endothelial-mesenchymal transformation 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.

5. The cardiac organoid according to any one of claims 1 to 4, characterized in that: The preparation method of the cardiac organoid 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 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; 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.

6. The cardiac organoid according to claim 5, wherein: 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 second cardiac organoid differentiation medium is the first basal medium additionally supplemented with a WNT pathway inhibitor.

7. The cardiac organoid according to claim 6, wherein: The concentration of the WNT pathway activator in the first cardiac organoid differentiation medium is 3 μM to 12 μM; the concentration of the bone morphogenetic protein 4 in the first cardiac organoid differentiation medium is 1 ng / mL to 3 ng / mL; the concentration of activin A in the first cardiac organoid differentiation medium is 1 ng / mL to 3 ng / mL; The concentration of the WNT pathway inhibitor in the second cardiac organoid differentiation medium is 1 μM to 5 μM.

8. A method for constructing a cardiac organoid according to any one of claims 1 to 7, 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; and performing induction culture on the adherent pluripotent stem cell cluster to obtain a cardiac organoid.

9. 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 described in any one of claims 1 to 7 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.

10. Use of the cardiac organoid according to any one of claims 1 to 7, the method according to claim 8, or the method according to claim 9 in screening drugs for preventing and / or treating heart disease.

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