Maturation medium compositions and methods for human heart organoid maturation

Abstract

Provided herein are maturation media and methods for maturing early embryonic human heart organoids into mature human heart organoids. The maturation media comprises a cell growth medium comprising a media supplement comprising: one or more fatty acids, triiodothyronine (T3) growth hormone, insulin, one or more antioxidants, a sugar, and carnitine; one or more additional fatty acids; additional carnitine or creatine; and additional T3 growth hormone. The maturation media can also comprise one or more additional sugars, additional antioxidants, and growth factors. The methods comprise contacting an early embryonic human heart organoid with one or more maturation media to produce a mature human heart organoid.

Description

[0001] Cross-referencing of related patent applications

[0002] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 391,452, filed July 22, 2022, and U.S. Provisional Patent Application No. 63 / 432,565, filed December 14, 2022. The entire contents of each of the above-cited patent applications are incorporated herein by reference.

[0003] Government rights

[0004] This invention was carried out with government support under licenses HL135464 and HL151505 granted by the National Institutes of Health and the National Heart, Lung and Blood Institute. The government owns certain rights to this invention. Technical Field

[0005] This disclosure relates to maturation culture media, methods, and models for high-throughput production of mature human heart organoids, such as fetal-like human heart organoids, which can be further matured into more adult-like human heart organoids. Background Technology

[0006] Cardiovascular disease (CVD) (including conditions of the heart and blood vessels) is a leading cause of death worldwide, causing an estimated 17.9 million deaths annually. 1 Laboratory models of the heart were used to better and more thoroughly understand the etiology and mechanisms of CVD. Several model systems were used to study CVD, ranging from primary and induced pluripotent stem cell (iPSC)-derived cardiomyocyte cultures to animal models and 3D culture systems such as spheroids and engineered cardiac tissue. 2-7 However, due to various reasons, including the lack of endogenous extracellular matrix (ECM) and non-cardiac cardiomyocyte cell types, as well as the lack of physiological morphology and cellular organization, many aspects of these systems fail to fully reproduce the complex nature of the human heart. 8,9 Furthermore, animal models possess unique non-human physiological, metabolic, electrophysiological, and pharmacokinetic characteristics, which typically prevent them from accurately predicting relevant human responses. 8,9 Therefore, these systems and methods are not suitable for comprehensive research and simulation of human diseases and physiology.

[0007] The introduction of human-related models is crucial for discovering effective, clinically transferable CVD solutions. Over the past decade, human induced pluripotent stem cells (hiPSCs) have been a significant contributor. 10-12 and organoids 13,14 Technological advancements have enabled increasingly sophisticated techniques for simulating and studying human systems with greater precision. Recently, methods for generating human heart organoids from pluripotent stem cells have been reported. Due to their cellular complexity and physiological relevance, these methods allow for the study of human heart development and disease in dishes. 15-19 They are capable of reaching unprecedented levels. However, these systems still fail to reproduce important aspects of late-embryonic human heart and human heart development (such as anterior-posterior patterning and coronary artery formation) and lack important cell populations that contribute to cardiac structures (such as the neural crest).

[0008] Furthermore, the metabolic transition from glycolysis to fatty acid oxidation is a crucial step in the later stages of cardiac development, preparing the heart for increased energy expenditure and inducing transcriptional regulation and stimulating physiological maturation. 28,30,95-97,141 Efforts have been made to simulate these phenomena in vitro using cardiomyocytes and engineered heart tissue, and beneficial effects of altering glucose concentration and adding fatty acids have been discovered. 7,20,21,23,142 However, these systems are simple models and do not possess the high physiological complexity observed in human heart organoids.

[0009] In summary, there is an urgent need to develop more sophisticated and sophisticated in vitro model systems for studying human heart development and disease pathology. Summary of the Invention

[0010] This section provides a general overview of the contents of this disclosure and is not a full disclosure of its entire scope or all its features.

[0011] This document provides a maturation culture medium comprising a cell growth medium containing a culture supplement containing one or more fatty acids, triiodothyronine (T3) growth hormone, insulin, one or more antioxidants, sugars, and carnitine. The maturation culture medium may also contain one or more additional fatty acids, additional carnitine or creatine, and additional T3 growth hormone.

[0012] In another embodiment, a method for maturing early embryonic human heart organoids into mature human heart organoids is provided. The method includes contacting the early embryonic human heart organoids with a maturation culture medium. The maturation culture medium comprises a cell growth medium containing a culture medium supplement comprising one or more fatty acids, triiodothyronine (T3) growth hormone, insulin, one or more antioxidants, sugars, and carnitine. The maturation culture medium also contains one or more additional fatty acids, additional carnitine or creatine, and additional T3 growth hormone.

[0013] In another implementation, a mature human heart organoid produced by the method described herein is provided.

[0014] Other implementation schemes (including specific aspects of the implementation schemes summarized above) will become apparent from the following detailed description. Attached Figure Description

[0015] Figures 1A to 1H A developmental induction method for improving human heart organoid development modeling. Figure 1A This is a schematic diagram illustrating the culture medium conditions for the differentiation protocol used to generate human heart organoids and four maturation strategies (control, MM, EMM1, and EMM2 / 1). Figure 1B These are bright-field images of organoids throughout the 30-day culture period. Two representative organoids for each condition are shown (data represent 23 to 24 organoids per condition). Scale bar = 400 μm. Figure 1C This is a quantification of the long and short diameters of organoids on day 30 of culture for each maturation strategy (n=7 to 8 organoids / each condition). Data are expressed as mean ± sem. Figure 1D This is a quantitative representation of organoid area on day 30 of organoid culture for each mature strategy (n = 7 to 8 organoids / each condition in two independent experiments). Data are represented as a violin plot containing all points. Figure 1E It is a quantification of the percentage of visible pulsating organoids under bright-field microscopy from five different organoid batches under each condition (n=22 to 24 organoids / condition in five independent experiments). Figure 1F These are TEM images showing myosarcomas, myofibrils (M), and I bands (arrows) in organoids from each maturation condition at day 15 and day 30 (n = 4 organoids / each condition). Scale bar = 1 μm. Figure 1G This is a quantitative analysis of sarcomere length in TEM images. Data are expressed as mean ± sem (n = 4 organoids / each condition). One-way ANOVA and Brown-Forsythe and Welch multiple comparison tests were performed. Figure 1H This represents the mRNA expression of key sarcomere genes involved in cardiomyocyte maturation from day 20 to day 30 of culture for each condition (n = 7 to 14 organoids / day / each condition / each gene in three independent experiments). Data are expressed as log2 fold change normalized relative to day 20. Value = mean ± sem.

[0016] Figures 2A to 2D Single-cell RNA sequencing of human heart organoids revealed different populations of heart cells. Figure 2A This is the UMAP projection of k-means clustering of single-cell RNA sequencing data from organoids on day 34 under each condition. Cluster labels are shown in the legend below. Figure 2B It is a quantitative percentage of the total cell count for each cluster. The color of the region corresponds to the area in... Figure 2A The colors present in the legend. Figure 2C This is a heatmap showing the differential expression of the top 10 differentially expressed genes in all clusters. Figure 2D This is a feature map showing the key marker genes for each cluster. The color intensity represents the relative value of gene expression.

[0017] Figures 3A to 3B Cluster identification and intercellular communication networks highlight the importance of self-organization in the development of cardiac organoids. Figure 3A This is a dot plot of differentially expressed genes in each cluster under each condition. The color represents the average expression level across all cells, and the size of the circle represents the percentage of cells in a specific cluster that express the corresponding gene. Figure 3B This is a visualization of the intercellular ligand-receptor communication network under each condition. The clusters (outer) are colored to match the colors of the UMAP projection. Ligands are shown in blue bands and receptors in red bands. Arrows in the figure indicate ligand-receptor pairing.

[0018] Figures 4A to 4K Following developmental induction conditions, human heart organoids have developed an increasingly sophisticated metabolic profile. Figure 4A Mitochondrial markers in human heart organoids on day 30 under each condition (n = 6 organoids / each condition). White = Mitotracker, Blue = NucBlue. Scale bar = 10 μm. Detailed images of mitochondria are shown below each main image. Figure 4B This is a quantification of the mitochondrial area surrounding each individual cell nucleus (n = 6 organoids / condition, n = 50 to 70 measurements / condition). Values ​​= mean ± sem, one-way ANOVA with Brown-Forsythe and Welch multiple comparison tests. Figure 4CThis shows TEM images of mitochondria in organoids from day 15 and day 30 from each maturation condition (n = 4 organoids / condition). Yellow arrows indicate mitochondria, LD = lipid droplets, Gg = glycogen granules. Scale bar = 1 μm. Figure 4D This is a quantification of mitochondrial area from TEM images. Value = mean ± sem, one-way ANOVA with Brown-Forsythe and Welch multiple comparison test (n = 4 organoids / each condition, n = 40 to 144 measured mitochondria / each condition). Figure 4E For each condition, the metabolic genes are metabolized between day 20 and day 30 of culture. PPARGC1A and CPT1B mRNA expression (in three independent experiments, n = 8 organoids / each condition). Data are shown as log2 fold changes relative to day 20. Values ​​= mean ± sem. Figure 4F These are measurements of oxygen consumption rate obtained from the Agilent Seahorse XFe96 metabolic stress test under all conditions (in two independent experiments, n = 8 organoids / condition). Value = mean ± sem. Figure 4G It is a quantification derived from oxygen consumption rate measurements of basal respiration (in two independent experiments, n=8 organoids / each condition). Figure 4H It is a quantification derived from the oxygen consumption rate determination for maximal respiration (in two independent experiments, n=8 organoids / each condition). Figure 4I It is a quantification of oxygen consumption rate measurements for reserve breathing capacity (in two independent experiments, n=8 organoids / each condition). Figure 4J This is a feature map showing the key metabolic genes upregulated in the VCM and ACM clusters. The color intensity represents the relative value of gene expression for each gene. Figure 4K This is a heatmap of expression of key metabolic genes in the VCM and ACM clusters under each condition. Data are presented as log2 fold changes, and normalization is applied to each column (for each gene).

[0019] Figures 5A to 5I Developmental induction conditions promote progressive electrophysiological maturation in human heart organoids. Figure 5A These are representative calcium transient traces from human heart organoids on day 30 under each condition (n=12 organoids / condition in three independent experiments). The traces represent data from individual cardiomyocytes within human heart organoids. Additional traces are... Figure 10A As shown in the image. Figure 5BThis is a quantification of the peak amplitude of the calcium transient trajectory from each condition (n=12 organoids / each condition in three independent experiments). Quantification and averaging were performed on at least two regions and 16 peaks for each organoid. Value = Mean ± sem. Figure 5C This is a quantification of the calcium transient peak frequency for each condition (n=12 organoids / condition in three independent experiments). Quantification and averaging were performed on at least two regions and 16 peaks for each organoid. Value = Mean ± sem. Figure 5D This is a feature map showing the differential expression of key electrophysiological genes in the VCM and ACM clusters under each condition. The color intensity represents the relative value of gene expression for each gene. Figure 5E This represents the mRNA expression of key electrophysiological genes from day 20 to day 30 of culture for each condition (n = 8 organoids / day / each condition / each gene / in three independent experiments). Data are presented as log2 fold changes normalized relative to day 20. Values ​​= mean ± sem. Figure 5F The representative voltage trajectories of organoids under EMM2 / 1 and control conditions depict atrial-like, nodular-like, and ventricular-like action potentials (n=9 single cells (from 3 independent organoids) / each action potential subtype / each condition in 3 independent experiments). Figure 5G This is a representative immunofluorescence image of caveolin-3 spots within the TNNT2+ region of organoids for each condition (n=15 organoids / condition in three independent experiments). Green = caveolin-3, red = TNNT2, blue = DAPI. Scale bar = 20 μm. Figure 5H For each condition, from Figure 5G The images presented show the quantification of the area of ​​caveolin-3 positivity per 400 square μm (in three independent experiments, n=15 organoids / condition). Data are presented as fold changes normalized relative to controls. Values ​​= mean ± sem, one-way ANOVA with Brown-Forsythe and Welch multiple comparison tests. Figure 5I These are representative immunofluorescence images of KCNJ2+ spots within the TNNT2+ region in organoids for each condition (n=14 organoids / condition in 3 independent experiments). KCNJ2 = green, TNNT2 = red, DAPI = blue. Scale bar = 20 μm. Figure 5J It comes from Figure 5IThe total number of KCNJ2+ spots in the images presented was quantified for each condition (n=14 organoids / condition in 3 independent experiments). Data are presented as fold changes normalized relative to controls. Values ​​= mean ± sem, one-way ANOVA and Dunnett's multiple comparison test.

[0020] Figures 6A to 6J Developmental induction promotes the emergence of anterior epicardial organs and the formation of different atrial and ventricular chambers through self-organization. Figure 6A Representative surface and internal immunofluorescence images of individual day 30 organoids under all conditions are shown, illustrating WT1 (green), TNNT2 (red), and DAPI (blue). Three organoids for each condition are shown (n=12 to 15 organoids / condition in two independent experiments). Scale bar = 200 μm. Figure 6B It comes from Figure 6A The images presented quantify the TNNT2+ chamber area under each condition (n = 12 to 15 organoids / condition in two independent experiments). Values ​​are expressed as fold changes relative to controls, normalized. Value = mean ± sem, one-way ANOVA and Dunnett's multiple comparison test. Figure 6C It comes from Figure 6A The images presented quantify the WT1+ chamber area under each condition (n=12 to 15 organoids / condition in two independent experiments). Values ​​are expressed as fold changes relative to controls, normalized. Value = mean ± sem, one-way ANOVA and Dunnett's multiple comparison test. Figure 6D Representative surface and internal immunofluorescence images of a single day 30 organoid under all conditions, showing MYL2 (green), MYL7 (red), and DAPI (blue). Three organoids for each condition are shown (n=13 organoids / condition in three independent experiments). Scale bar = 200 μm. Figure 6E It comes from Figure 6D The images presented quantify the MYL2+ area in each organoid under each condition (n = 9 to 13 organoids / condition in three independent experiments). Values ​​are expressed as fold changes relative to controls, normalized. Value = mean ± sem, one-way ANOVA and Dunnett's multiple comparison test. Figure 6FThis is a representative immunofluorescence image of a single day 30 organoid under all conditions, showing NR2F2 (green), MYL7 (red), and DAPI (blue). Three organoids for each condition are shown (n=12 organoids / condition in three independent experiments). Scale bar = 200 μm. Figure 6G It comes from Figure 6F The images presented in the figure are used to quantify the colocalization (Pearson coefficient) between NR2F2 (green) and MYL3 (red). Value = mean ± sem, unpaired t-test. Figure 6H These are feature maps highlighting the VCM and ACM clusters for further use in 6I and 6J. Figure 6I This is a feature map showing the differentially expressed signature atrial and ventricular identity genes in the ACM cluster. The color intensity represents the relative value of gene expression for each gene. Figure 6J This is a feature map showing differentially expressed ventricular chamber identity genes in the VCM cluster. The color intensity represents the relative value of gene expression for each gene.

[0021] Figures 7A to 7J The endogenous retinoic acid gradient is responsible for the spontaneous formation of the anterior-posterior cardiac tube pattern. Figure 7A It is a schematic diagram depicting the formation of the cardiac tube within the uterus, highlighting the location and intensity of the retinoic acid gradient from the anterior segment (arterial pole) to the posterior segment (venous pole) of the primitive cardiac tube. Figure 7B This is a Raman spectral intensity map of organoids from all four developmental maturation conditions on day 30 of culture. Target peaks, such as DNA, cardiac troponin, and retinoic acid, are labeled. The data presented represent n=3 organoids / each condition. Figure 7C It is on the 30th day under all conditions ALDH1A2 mRNA expression (n=7 organoids / condition in two independent experiments). Data are expressed as log2 fold change relative to control. Values ​​= mean ± sem, one-way ANOVA and Dunnett's multiple comparison test. Figure 7D It is a display ALDH1A2 Characteristic map of gene expression. Color intensity represents the relative value of gene expression. Figure 7E These are representative immunofluorescence images of individual day 30 organoids under all conditions, showing ALDH1A2 (green), TBX18 (red), and DAPI (blue). Three organoids for each condition are shown (representing n=22 to 24 organoids / condition in three independent experiments). Scale bar = 200 μm. Figure 7F yes Figure 7CThe image shows high-magnification images of EMM2 / 1 and control organoids, highlighting ALDH1A2 (green), TBX18 (red), and DAPI (blue). The yellow squares in the top image (scale bar = 200 μm) represent the areas at high magnification. Scale bar = 50 μm. Figure 7G It comes from Figure 7E The images presented show ALDH1A2 within organoids under each condition. + TBX18 + Quantification of area (n=22 to 24 organoids / condition in three independent experiments). Data are presented as fold changes normalized relative to controls. Values ​​= mean ± sem, one-way ANOVA and Dunnett's multiple comparison test. Figure 7H Representative immunofluorescence images of individual EMM2 / 1 organoids on day 30 after exposure to deoxyaminobenzaldehyde (DEAB), retinoic acid (RA), or no treatment. Staining with MYL3 (pink), NR2F2 (green), and DAPI (blue) was performed. Two organoids are shown for each condition (n=9 organoids / condition in two independent experiments). Scale bar = 200 μm. Figure 7I yes Figure 7H Quantification of NR2F2+ area of ​​organoids presented in the study (n=9 organoids / condition in two batches of organoids). Value = mean ± sem, one-way ANOVA and Dunnett's multiple comparison test. Figure 7J yes Figure 7H Quantitative analysis of the MYL3+ area of ​​organoids presented in the study (n=9 organoids / condition in two batches of organoids). Value = mean ± sem, one-way ANOVA and Dunnett's multiple comparison test.

[0022] Figures 8A to 8J Heart organoids treated with ondansetron simulate the morphological and electrophysiological phenotypes of congenital heart disease. Figure 8A Representative immunofluorescence images of individual EMM2 / 1 organoids at day 30 after exposure to different concentrations of ondansetron (1 μM, 10 μM, or 100 μM) or no treatment (untreated) from day 9 to day 30 of culture. MYL2 (green), MYL7 (red), and DAPI (blue) staining were performed. Three organoids for each condition are shown (n=12 organoids / condition in two independent experiments). Scale bar = 200 μm. Figures 8B to 8C They are from Figure 8A Quantification of MYL2+ and MYL7+ areas for each condition in the images presented (n=12 organoids / condition in two independent experiments). Data are presented as fold changes relative to untreated, normalized. Values ​​= mean ± sem, one-way ANOVA and Dunnett's multiple comparison test. Figure 8D It is on the 30th day under all conditions MYL2 mRNA expression (in two independent experiments, n = 6 organoids / condition). Data are presented as log2 fold changes relative to controls, normalized. Values ​​= mean ± sem, one-way ANOVA and Dunnett's multiple comparison test. Figures 8E to 6F These are representative voltage trajectories of organoids, showing three voltage trajectories from independent organoids under each condition (n=6 organoids / each condition in two independent experiments). Figure 8G J is from Figures 8E to 8F The trajectories presented are quantitative representations of voltage trajectories from individual organoids under each condition (n=6 organoids / condition in two independent experiments), showing frequency, amplitude, APD30, and APD90, respectively. Values ​​= mean ± sem, one-way ANOVA and Dunnett's multiple comparison test.

[0023] Figures 9A to 9C Longitudinal assessment of apoptosis under all maturation conditions. Figure 9A These are representative fluorescence images of organoids from day 20 and day 30 of each condition, showing the FlipGFP fluorescence signal (n=12 organoids / each condition / day). Figure 9B This is a representative fluorescence image of EMM2 / 1 organoids on day 30, 48 hours after exposure to doxorubicin, showing the FlipGFP fluorescence signal (n=6 organoids). Figure 9C It comes from Figure 9A Quantitative analysis of fluorescence intensity in the images presented (n = 12 organoids / condition / day). Data are presented as fold changes normalized relative to day 20. Values ​​= mean ± sem, matched with two-way ANOVA and Tukey multiple comparison test.

[0024] Figures 10A to 10C Transcriptome organoid landscape reveals similarities to the developing human heart in vivo. Figure 10A It is a schematic diagram comparing the timeline of embryonic heart development with that of human heart organoids. Figure 10B This is a UMAP projection of scRNAseq datasets of human embryonic hearts and human heart organoids. (Asp et al, Cell The cluster naming in the dataset (2019) is preserved from the original text. Cluster identifiers and colors in the human heart organoid dataset are preserved (…). Figure 2A As shown in the figure. Figure 10C yes Figure 10B The PCA diagram of the dataset presented in the image.

[0025] Figures 11A to 11F Human heart organoids share key gene expression with embryonic human hearts in some heart cell types. Figures 11A to 11F This displays a feature map of each key biomarker gene for each of the following corresponding clusters in the Asp 2019, Cui 2019, and Human Heart Organoid Datasets: atrial cardiomyocytes; ventricular cardiomyocytes; cells derived from anterior epicardium; epicardial cells; valvular cells; and conduction cells. Color intensity represents the relative value of gene expression.

[0026] Figure 12 A. Calcium measurements demonstrate reproducibility in independent organoids. Figure 12 A represents the calcium transient trajectory from human heart organoids on day 30 under each condition (n=12 organoids / condition in three independent experiments). Data from 6 independent organs are shown. The trajectory represents data from a single cardiomyocyte within a human heart organoid.

[0027] Figures 13A to 13D The formation of ventricular and atrial chambers is reproducible in all three hPSC systems. Figure 13A These are representative immunofluorescence images of individual day 30 organoids from cell lines L1, BYS0111, and H9 under both control and EMM2 / 1 conditions, showing NR2F2 (green), MYL3 (red), and DAPI (blue). Three organoids are shown for each cell line under each condition (n=12 organoids / condition in three independent experiments for L1 organoids; n=11 organoids / condition in two independent experiments for BYS0111 organoids; and n=12 organoids / condition in two independent experiments for H9 organoids). Figures 13B to 13D They are from Figure 13A Quantitative analysis of colocalization (Pearson coefficient) of L1, BYS0111, and H9 organoids presented in the images between NR2F2 (green) and MYL3 (red). Value = mean ± sem, unpaired t-test.

[0028] Figures 14A to 14C Real-time longitudinal imaging using optical coherence tomography revealed large, interconnected chambers within human heart organoids. Figure 14AThis is a schematic diagram of a custom optical coherence tomography (OCT) system used for imaging human heart organoids. Figure 14B These are longitudinal OCT cross-sectional scans of human heart organoids taken on days 20 to 30 under each condition. Scale bar = 500 μm. The images shown represent 6 organoids per condition. Figure 14C It comes from Figure 14A The images presented are 3D segmented from OCT scans, revealing temporal dynamic volumetric visualizations of chamber identity under each condition.

[0029] Figures 15A to 15D Interfering with the localization and morphology of endothelial cells through enhanced developmental maturation strategies. Figure 15A These are representative immunofluorescence images of the surface and interior of organoids on day 30 under each condition, using DAPI (blue), TNNT2 (red), and PECAM1 (green) (n=7 to 8 organoids / condition in two independent experiments). Scale bar = 200 μm. Figure 15B It comes from Figure 15A The images presented are representative immunofluorescence images of organoids on day 30 with DAPI (blue), TNNT2 (red), and PECAM1 (green) (n=7 to 8 organoids / condition in two independent experiments). Images are shown as maximum intensity projections. Scale bar = 200 μm. Figure 15C yes Figure 15B Quantification of PECAM1+ area was presented in two independent experiments (n=7 to 8 organoids / condition). Data were presented as logarithmic fold changes relative to controls. Values ​​= mean ± sem, one-way ANOVA and Dunnett's multiple comparison test. Figure 15D The images shown are representative high-magnification immunofluorescence images of organoids using DAPI (blue), TNNT2 (red), and PECAM1 (green) under each condition (n = 7 to 8 organoids / condition in two independent experiments). Scale bar = 50 μm. The top image is a representative low-magnification organoid for each condition (scale bar = 200 μm), where yellow squares represent areas of high magnification. Images are shown as maximum intensity projections.

[0030] Figures 16A to 16D The presence of the ALDH1A2+ anterior epicardial pole was reproducible in all three hPSC lineages by using the EMM2 / 1 developmental maturation strategy. Figure 16AThese are representative immunofluorescence images of individual day 30 organoids from cell lines L1, BYS0111, and H9 under control and EMM2 / 1 conditions, showing ALDH1A2 (green), TBX18 (red), and DAPI (blue). Three organoids for each cell line are shown for each condition (n=22 to 24 organoids / condition in three independent experiments for L1 organoids; n=12 organoids / condition in two independent experiments for BYS0111 organoids; and n=12 organoids / condition in two independent experiments for H9 organoids). Scale bar = 200 μm. Figures 16B to 16D Each comes from Figure 16A Images of L1, BYS0111, and H9 organoids presented in the image show ALDH1A2 within the organoids under each condition. + TBX18 + Quantification of area. Data are presented as fold changes relative to controls, normalized. Values ​​= mean ± sem, unpaired t-test.

[0031] Figures 17A to 17G The transcriptional profiles of key genes in human heart organoids are reproducible across the three hPSC lines. Figures 17A to 17G The mRNA expression of the selection genes from L1, BYS0111, and H9 organoids was measured on day 30 under control and EMM2 / 1 conditions (n=7 to 14 organoids / condition in three independent experiments for L1 organoids; n=8 organoids / condition in two independent experiments for BYS0111 organoids; and n=8 organoids / condition in two independent experiments for H9 organoids). Figure 17A yes MYL2 mRNA expression of genes. Figure 17B yes MYL7 mRNA expression of genes. Figure 17C yes MYH6 mRNA expression of genes. Figure 17D yes MYH7 mRNA expression of genes. Figure 17E yes ALDH1A2 mRNA expression of genes. Figure 17F yes PPARGC1A mRNA expression of genes. Figure 17G yes WT1 mRNA expression of genes. For each cell line, data are shown as a normalized log2 fold change relative to the control. Values ​​= mean ± sem, unpaired t-test.

[0032] Figures 18A to 18C Apoptosis is not a contributing factor to ondansetron-induced cardiac organoid malformations. Figure 18ARepresentative fluorescence images of EMM2 / 1 organoids from each condition on day 30, following ondansetron treatment from day 9 to day 30 and doxorubicin treatment from day 28 to day 30, showing FlipGFP fluorescence signals (in two independent experiments, n = 12 organoids / condition for the ondansetron group; n = 6 organoids for the doxorubicin group). Scale bar = 200 μm. Figure 18B It comes from Figure 18A Quantification of fluorescence intensity in the images presented. Data are shown as fold changes relative to untreated images after normalization. Values ​​= mean ± sem, one-way ANOVA and Games-Howell multiple comparisons. Figure 18C It is a quantification of the percentage of pulsatile organoids during the entire treatment period for each ondansetron condition from day 0 to day 30 (n=23 to 24 organoids / condition in two independent experiments). Detailed Implementation

[0033] The provision of exemplary embodiments makes this disclosure comprehensive and will fully communicate the scope to those skilled in the art. Numerous specific details, such as examples of specific components, parts, apparatuses, and methods, are set forth to provide a comprehensive understanding of embodiments of this disclosure. It will be apparent to those skilled in the art that specific details are not required, that the exemplary embodiments may be embodied in many different forms, and neither should be construed as limiting the scope of this disclosure. In some exemplary embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.

[0034] The terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be restrictive. As used herein, nouns without quantifiers may also be intended to include plural forms unless the context clearly indicates otherwise. The terms “comprising,” “containing,” “including,” and “having” are inclusive and thus indicate the presence of the stated features, elements, components, steps, integers, operations, and / or components, but do not exclude the presence or addition of one or more additional features, integers, steps, operations, elements, components, and / or groups thereof. While the open-ended term “comprising / including” is understood as a non-restrictive term used to describe and claim the multiple embodiments set forth herein, in some respects it may be understood alternatively as a more restrictive and binding term, such as “consisting of” or “substantially consisting of”. Therefore, for any given embodiment that states composition, materials, components, elements, features, integers, operations, and / or process steps, this disclosure also specifically includes embodiments consisting of such stated composition, materials, components, elements, features, integers, operations, and / or process steps, or embodiments substantially consisting of such stated composition, materials, components, elements, features, integers, operations, and / or process steps. In the case of "consisting of," alternative embodiments exclude any additional composition, materials, components, elements, features, integers, operations, and / or process steps, while in the case of "substantially consisting of," any additional composition, materials, components, elements, features, integers, operations, and / or process steps that substantially affect the essential and new characteristics are excluded from such an embodiment, but any composition, materials, components, elements, features, integers, operations, and / or process steps that do not substantially affect the essential and new characteristics may be included in that embodiment.

[0035] Unless explicitly specified as the order of execution, no methodological steps, procedures, or operations described herein should be construed as requiring them to be performed in the particular order discussed or illustrated. It should also be understood that, unless otherwise stated, alternative or alternative steps may be employed.

[0036] While the terms first, second, third, etc., may be used herein to describe multiple steps, elements, components, regions, layers, and / or portions, these steps, elements, components, regions, layers, and / or portions should not be limited by these terms unless otherwise indicated. These terms may be used only to distinguish one step, element, component, region, layer, or portion from another. Unless the context clearly indicates otherwise, terms such as “first,” “second,” and other numerical terms used herein do not imply order or sequence. Therefore, without departing from the teachings of the exemplary embodiments, the first step, element, component, region, layer, or portion discussed below may be referred to as the second step, element, component, region, layer, or portion.

[0037] For ease of description, spatially or temporally related terms such as "before," "after," "inside," "outside," "below," "below," "lower," "above," and "upper" may be used herein to describe the relationship between one element or feature as illustrated in the accompanying drawings and another element or feature. Spatially or temporally related terms may be intended to cover different orientations of the apparatus or system in use or operation other than those depicted in the accompanying drawings.

[0038] Throughout this disclosure, numerical values ​​represent approximate measurements or limitations on ranges to cover small deviations from a given value, as well as embodiments having approximately the mentioned value and those having the exact mentioned value. Except for the working example provided at the end of the detailed description, all numerical values ​​of parameters (e.g., quantities or conditions) in this specification (including the appended claims) should be understood to be modified in all cases by the term “about,” regardless of whether “about” actually appears before the numerical value. “About” indicates that the numerical value allows for some slight inaccuracy (some of which are close to the accuracy of the value; approximately or reasonably close to the value; almost). If the inaccuracy provided by “about” is not otherwise understood in this ordinary sense in the art, then “about” as used herein at least indicates a variation that can be caused by common methods of measuring and using such a parameter. For example, “about” can include variations such as: less than or equal to 5%, optionally less than or equal to 4%, optionally less than or equal to 3%, optionally less than or equal to 2%, optionally less than or equal to 1%, optionally less than or equal to 0.5%, and in some respects, optionally less than or equal to 0.1%.

[0039] In addition, the disclosure of the range includes all values ​​within the entire range and the disclosure of further subdivided ranges, including the endpoints and subranges given by the range.

[0040] Exemplary embodiments will now be described more fully with reference to the accompanying drawings.

[0041] As mentioned above, laboratory models of the human heart have made considerable progress over the past few decades, starting with animal models and primary cardiomyocyte cultures, and continuing to advance to induced pluripotent stem cell-derived heart tissues (e.g., cardiomyocytes) and tissue engineering methods (3D printing, biomaterials). The latest advancement in human heart models is the development of cardiac organoids derived from pluripotent stem cells. 15-17,115 However, due to a lack of maturity and faithfulness regarding human physiology, morphology, cellular organization, and function, these systems do not possess the true complexity of the human heart in utero. These limitations severely restrict the relevance of traditional model systems.

[0042] Therefore, there is a need for developmental and physiologically relevant models of the adult heart, such as those derived from hiPSCs, which can be achieved efficiently and in a high-throughput manner. This paper presents maturation media and methods for generating mature human heart organoids in a reproducible and high-throughput manner using developmental induction strategies inspired by intrauterine biological steps, to produce human heart organoids with greater anatomical complexity and physiological relevance (as associated with early fetal development in pregnancy). The maturation media and methods described herein advantageously reproduce in vitro heart development and enable organoids to achieve high levels of complexity and anatomical relevance by inducing progressive mitochondrial and metabolic maturation, electrophysiological maturation, enhanced morphological and cellular complexity, and reproducing anterior-posterior tubular pattern building through endogenous retinoic acid signaling and self-organization.

[0043] A. Maturation culture medium

[0044] This article provides maturation culture media, which can be used, for example, to induce early embryonic human heart organoids to develop into mature human heart organoids or to mature early embryonic human heart organoids into mature human heart organoids. The maturation culture media comprises cell growth medium and culture medium supplements.

[0045] Examples of suitable cell growth media include, but are not limited to: Roswell Park Memorial Institute (RPMI) media, such as RPMI 1640, which contains various formulations such as those containing D-glucose, D-glucose-free, L-glutamine-containing, L-glutamine-free, sodium bicarbonate-containing, sodium bicarbonate-free, and HEPES-modified; Dulbecco's Modified Eagle's Medium (DMEM), which contains various formulations such as high glucose, low glucose, and HEPES-containing; DMEM derivatives, such as Iscove's Modified Dulbecco's Medium (IMDM) or Advanced Dulbecco's Modified Eagle's Medium (ADMEM); or combinations thereof. These culture media are available from ThermoFisher Scientific, Sigma-Aldrich, Millipore Sigma, etc., and the same or identical trade names of the culture media indicate the same culture media composition, regardless of the manufacturer.

[0046] In any embodiment, based on the total volume of the maturation medium, the cell growth medium may be present in the maturation medium in an amount greater than or equal to about 90 v / v%, greater than or equal to about 95 v / v%, greater than or equal to about 96 v / v%, greater than or equal to about 97 v / v%, greater than or equal to about 98 v / v%, or about 99 v / v; or about 90 v / v% to about 99 v / v%, about 95 v / v% to about 99 v / v%, about 96 v / v% to about 99 v / v%, or about 97 v / v% to about 98 v / v%.

[0047] Culture medium supplements may contain one or more fatty acids, triiodothyronine (T3) growth hormone, insulin, one or more antioxidants, sugars, and carnitine. For example, culture medium supplements may contain one or more of the following: biotin, L-carnitine, corticosterone, ethanolamine, D(+)-galactose, glutathione (reduced), linoleic acid, linolenic acid, oleic acid, piperidinic acid, progesterone, putrescine, retinyl acetate, sodium selenite, T3 growth hormone, DL-α-tocopherol (vitamin E), DL-α-tocopherol acetate, protein, bovine albumin, catalase, insulin, superoxide dismutase, and transferrin. Exemplary suitable commercially available culture medium supplements include a variety of B-27™ supplement formulations (available from ThermoFisher Scientific), such as B-27™ Supplement (50×), serum-free; B-27™ Supplement, insulin-free; B-27™ Plus Supplement (50×); B-27™ Supplement (50×), vitamin A-free; B-27™ Supplement (50×), antioxidant-free; etc.

[0048] In any implementation, based on the total volume of the mature culture medium, the culture medium supplement may be present in the mature culture medium in an amount less than or equal to about 5 v / v%, less than or equal to about 4 v / v%, less than or equal to about 3 v / v%, greater than or equal to about 1 v / v%, or greater than or equal to about 2 v / v; or about 1 v / v% to about 5 v / v%, about 1 v / v% to about 4 v / v%, about 1 v / v% to about 3 v / v%, or about 1 v / v% to about 2 v / v%.

[0049] Alternatively or concurrently, the maturation medium may also contain antibiotics. Any suitable antibiotic used for cell culture may be included. For example, antibiotics may include amphotericin B, ampicillin, cephalosporins, dihydrostreptomycin, gentamicin sulfate, penicillin streptomycin, kanamycin sulfate, lincomycin hydrochloride, neomycin sulfate, nystatin, paromomycin sulfate, penicillin-G, phenoxymethylpenicillic acid, polymyxin B sulfate, spectinomycin, streptomycin, tetracycline hydrochloride, tylosin tartrate, or combinations thereof. It is contemplated herein that antibiotics may be optional and are not required to be present in the maturation medium. When present in a mature culture medium, the antibiotic may be present in amounts less than or equal to about 5 v / v%, less than or equal to about 4 v / v%, less than or equal to about 3 v / v%, greater than or equal to about 1 v / v%, or greater than or equal to about 2 v / v, based on the total volume of the mature culture medium. Alternatively, it may be present in amounts of about 1 v / v% to about 5 v / v%, about 1 v / v% to about 4 v / v%, about 1 v / v% to about 3 v / v%, or about 1 v / v% to about 2 v / v%.

[0050] In any embodiment, the maturation medium also contains one or more of the following additional components: one or more additional fatty acids, additional carnitine or creatine, additional T3 growth hormone, additional sugars, and additional antioxidants. As used herein, "additional component" means a component present in a certain amount in the culture medium supplement in addition to the components or types of components already present in the culture medium supplement. For example, "additional T3 growth hormone" means T3 growth hormone present in the maturation medium in addition to the T3 growth hormone present in the culture medium supplement.

[0051] Suitable fatty acids include, but are not limited to, palmitic acid, oleic acid, linoleic acid, stearic acid, or combinations thereof. For example, a maturation medium may include palmitic acid, oleic acid, and linoleic acid. In any embodiment, oleic acid and linoleic acid may be present in the medium supplement, as well as in additional amounts in the maturation medium. It is also contemplated herein that one or more fatty acids may be mixed with bovine serum albumin (BSA), wherein the BSA is present in a negligible amount.

[0052] In any embodiment, one or more additional fatty acids, alone or in combination, may be present in the maturation medium in amounts greater than or equal to about 10 µM, greater than or equal to about 20 µM, greater than or equal to about 40 µM, greater than or equal to about 50 µM, less than or equal to about 100 µM, less than or equal to about 90 µM, less than or equal to about 80 µM, less than or equal to about 70 µM, or less than or equal to about 60 µM; or about 10 µM to about 100 µM, about 10 µM to about 80 µM, about 10 µM to about 60 µM, about 10 µM to about 40 µM, about 20 µM to about 100 µM, about 20 µM to about 80 µM, or about 20 µM to about 60 µM. For example, in addition to oleic acid present in the culture medium supplement, the maturation medium may also contain about 20 to 60 µM of oleic acid, and in addition to linoleic acid present in the culture medium supplement, the maturation medium may also contain about 10 to 40 µM of linoleic acid.

[0053] Alternatively or concurrently, one or more fatty acids, alone or in combination, may be present in the maturation medium in a total amount greater than or equal to about 10 µM, greater than or equal to about 20 µM, greater than or equal to about 40 µM, greater than or equal to about 50 µM, less than or equal to about 100 µM, less than or equal to about 90 µM, less than or equal to about 80 µM, less than or equal to about 70 µM, or less than or equal to about 60 µM; or about 10 µM to about 100 µM, about 10 µM to about 80 µM, about 10 µM to about 60 µM, about 20 µM to about 100 µM, about 20 µM to about 80 µM, or about 20 µM to about 60 µM. For example, the maturation medium may include the following total amounts: about 20 to 80 µM palmitic acid, about 20 to 80 µM oleic acid, and about 10 to 60 µM linoleic acid.

[0054] Suitable carnitines include, but are not limited to, L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, or combinations thereof. For example, a maturation culture medium may contain additional L-carnitine.

[0055] In any embodiment, additional carnitine and / or creatine may each be present in the maturation medium in amounts greater than or equal to about 60 µM, greater than or equal to about 80 µM, greater than or equal to about 100 µM, greater than or equal to about 120 µM, less than or equal to about 200 µM, less than or equal to about 180 µM, less than or equal to about 160 µM, or less than or equal to about 140 µM; or about 60 µM to about 200 µM, about 60 µM to about 180 µM, about 60 µM to about 160 µM, about 80 µM to about 160 µM, about 100 µM to about 140 µM, or about 100 µM to about 130 µM. For example, in addition to the carnitine present in the culture medium supplement, the maturation medium may also contain about 60 µM to about 160 µM of carnitine.

[0056] Alternatively or additionally, carnitine and / or creatine may each be present in the maturation medium in a total amount greater than or equal to about 60 µM, greater than or equal to about 80 µM, greater than or equal to about 100 µM, greater than or equal to about 120 µM, less than or equal to about 200 µM, less than or equal to about 180 µM, less than or equal to about 160 µM, or less than or equal to about 140 µM; or about 60 µM to about 200 µM, about 60 µM to about 180 µM, about 60 µM to about 160 µM, about 80 µM to about 160 µM, or about 100 µM to about 140 µM. For example, the maturation medium may contain a total of about 60 µM to about 200 µM of carnitine and / or creatine.

[0057] In any embodiment, additional T3 growth hormone may be present in the maturation medium in amounts greater than or equal to about 10 nM, greater than or equal to about 15 nM, greater than or equal to about 20 nM, greater than or equal to about 25 nM, less than or equal to about 50 nM, less than or equal to about 45 nM, less than or equal to about 40 nM, less than or equal to about 35 nM, or less than or equal to about 30 nM; or about 10 nM to about 60 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 20 nM to about 60 nM, about 20 nM to about 50 nM, or about 20 nM to about 40 nM. For example, in addition to the T3 growth hormone present in the medium supplement, the maturation medium may also contain about 10 nM to about 50 nM of T3 growth hormone.

[0058] Alternatively or concurrently, T3 growth hormone may be present in the maturation medium in a total amount greater than or equal to about 10 nM, greater than or equal to about 15 nM, greater than or equal to about 20 nM, greater than or equal to about 25 nM, less than or equal to about 50 nM, less than or equal to about 45 nM, less than or equal to about 40 nM, less than or equal to about 35 nM, or less than or equal to about 30 nM; or about 10 nM to about 60 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 20 nM to about 60 nM, about 20 nM to about 50 nM, or about 20 nM to about 40 nM. For example, the maturation medium may contain a total of about 10 nM to about 60 nM of T3 growth hormone.

[0059] Suitable sugars include, but are not limited to, glucose, fructose, galactose, and combinations thereof. For example, a mature culture medium may contain glucose, which may supplement other sugars present in the culture medium supplement.

[0060] In any embodiment, additional sugars (e.g., glucose) may be present in the maturation medium in amounts greater than or equal to about 1 mM, greater than or equal to about 2 mM, greater than or equal to about 3 mM, greater than or equal to about 4 mM, greater than or equal to about 5 mM, less than or equal to about 10 mM, less than or equal to about 9 mM, less than or equal to about 8 mM, less than or equal to about 7 mM, or less than or equal to about 6 nM; or about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 5 mM, about 2 mM to about 8 mM, or about 2 mM to about 6 mM. It is contemplated herein that the aforementioned amounts of additional sugars correspond to the total amount of said sugars (e.g., glucose) present in the maturation medium.

[0061] Suitable antioxidants include, but are not limited to, ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid, carotene, tocopherol (vitamin E), and panthenol, and combinations thereof. For example, a mature culture medium may contain ascorbic acid (vitamin C), which may supplement other antioxidants present in the culture medium supplement.

[0062] In any embodiment, the antioxidant (e.g., ascorbic acid (vitamin C)) may be present in the maturation medium in amounts greater than or equal to about 0.1 mM, greater than or equal to about 0.2 mM, greater than or equal to about 0.3 mM, greater than or equal to about 0.4 mM, greater than or equal to about 0.5 mM, less than or equal to about 1 mM, less than or equal to about 0.9 mM, less than or equal to about 0.8 mM, less than or equal to about 0.7 mM, or less than or equal to about 0.6 mM; or in amounts from about 0.1 mM to about 1 mM, from about 0.1 mM to about 0.8 mM, from about 0.1 mM to about 0.6 mM, from about 0.1 mM to about 0.5 mM, from about 0.2 mM to about 0.8 mM, or from about 0.2 mM to about 0.6 mM. It is contemplated herein that the amounts of the aforementioned additional antioxidants may correspond to the total amount of the antioxidants (e.g., ascorbic acid (vitamin C)) present in the maturation medium.

[0063] Alternatively or concurrently, the mature culture medium may also contain growth factors such as IFG-1, IFG-2, or combinations thereof. In any embodiment, the growth factor may be present in the maturation medium in amounts greater than or equal to about 5 ng / mL, greater than or equal to about 10 ng / mL, greater than or equal to about 20 ng / mL, greater than or equal to about 30 ng / mL, greater than or equal to about 40 ng / mL, greater than or equal to about 50 ng / mL, less than or equal to about 110 ng / mL, less than or equal to about 100 ng / mL, less than or equal to about 90 ng / mL, less than or equal to about 80 ng / mL, less than or equal to about 70 ng / mL, or less than or equal to about 60 ng / mL; or about 5 ng / mL to about 110 ng / mL, about 10 ng / mL to about 100 ng / mL, about 20 ng / mL to about 90 ng / mL, about 30 ng / mL to about 70 ng / mL, or about 40 ng / mL to about 60 ng / mL.

[0064] In several respects, the maturation medium may comprise cell growth media as described herein (e.g., RPMI 1640), and culture medium supplements as described herein (e.g., B-27). TM Supplements), one or more additional fatty acids as described herein (e.g., palmitic acid, oleic acid, linoleic acid), additional carnitine (e.g., L-carnitine) or creatine as described herein, additional T3 growth hormone, and optionally antibiotics as described herein (e.g., penicillin-streptomycin). For example, the maturation medium may contain approximately 97% RPMI 1640 medium; approximately 2% medium supplement (e.g., B-27). TM(Supplements); approximately 1% penicillin and streptomycin; approximately 52.5 μM palmitic acid in total; approximately 43.95 μM oleic acid in total; approximately 26 μM linoleic acid in total; approximately 132.2 μM L-carnitine in total; and approximately 33.01 nM T3 hormone in total.

[0065] In another aspect, the maturation medium may comprise cell growth media as described herein (e.g., RPMI 1640) and medium supplements as described herein (e.g., B-27). TM Supplements), one or more additional fatty acids as described herein (e.g., palmitic acid, oleic acid, linoleic acid), additional carnitine (e.g., L-carnitine) or creatine as described herein, additional T3 growth hormone, additional sugars as described herein (e.g., glucose), additional antioxidants as described herein (e.g., ascorbic acid (vitamin C)), and optionally, antibiotics as described herein (e.g., penicillin and streptomycin). For example, the maturation medium may contain approximately 97% RPMI 1640 medium (without D-glucose); approximately 2% medium supplements (e.g., B-27). TM (Supplements); approximately 1% penicillin and streptomycin; approximately 52.5 μM palmitic acid in total; approximately 43.95 μM oleic acid in total; approximately 26 μM linoleic acid in total; approximately 132.2 μM L-carnitine in total; approximately 33.01 nM T3 hormone in total; approximately 0.4 mM ascorbic acid in total; and approximately 4 mM glucose in total.

[0066] In another aspect, the maturation medium may comprise cell growth media as described herein (e.g., RPMI 1640) and medium supplements as described herein (e.g., B-27). TM Supplements), one or more additional fatty acids as described herein (e.g., palmitic acid, oleic acid, linoleic acid), additional carnitine (e.g., L-carnitine) or creatine as described herein, additional T3 growth hormone, additional sugars as described herein (e.g., glucose), additional antioxidants as described herein (e.g., ascorbic acid (vitamin C)), growth factors as described herein (e.g., IGF-1), and optionally antibiotics as described herein (e.g., penicillin-streptomycin). For example, the maturation medium may contain approximately 97% RPMI 1640 medium (without D-glucose); approximately 2% medium supplements (e.g., B-27). TM(Supplements); approximately 1% penicillin and streptomycin; approximately 52.5 μM palmitic acid; approximately 43.95 μM oleic acid; approximately 26 μM linoleic acid; approximately 132.2 μM L-carnitine; approximately 33.01 nM T3 hormone; approximately 0.4 mM ascorbic acid; approximately 4 mM glucose; and approximately 50 ng / mL IGF-1.

[0067] In any implementation, the maturation medium may not contain exogenous retinoic acid and / or extracellular matrix materials, such as hydrogels (e.g., Matrigel® matrix). As used herein, “exogenous retinoic acid” means retinoic acid that is not naturally present in or produced from human heart organoids.

[0068] B. Maturation Methods

[0069] This document also provides methods for maturing early embryonic human heart organoids into mature human heart organoids. These methods may also be referred to as developmental induction strategies. The methods involve contacting the early embryonic human heart organoid with the maturation culture medium described herein. As used herein, "early embryonic human heart organoid" refers to a three-dimensional body having an interior comprising cardiac muscle tissue and an outer surface comprising epicardial tissue, exhibiting both first and second cardiac regions and cardiac chambers. The early embryonic heart organoid may also comprise at least one chamber or microchamber defined by cardiac muscle tissue, said at least one chamber or microchamber lined with endocardial cells. Epicardial tissue (containing epicardial cells) may be disposed on at least a portion of this surface. The early embryonic heart organoid may also comprise cardiac fibroblasts and an endothelial vascular system and be pulsating. As used herein, "mature human heart organoid" encompasses fetal-like human heart organoids and adult human heart organoids, which can be obtained through a relatively long culture period. "Fetal-like human heart organoid" refers to an organoid having clearly defined atrial and ventricular chambers. For example, fetal-like human heart organoids can be considered equivalent to fetal human hearts from approximately day 45 to approximately day 90 of gestation. “Adult human heart organoids” refer to heart organoids with well-defined atrial and ventricular chambers and metabolic and electrophysiological profiles characteristic of the adult heart (e.g., fatty acid metabolism, presence of atria, ventricles, and conduction action potentials).

[0070] In any embodiment, early embryonic human heart organoids can be formed by methods known in the art. For example, early embryonic human heart organoids can be formed from the differentiation of human induced pluripotent stem cells (hiPSCs), as described in International Patent Publication No. WO 2021 / 257812, which is incorporated herein by reference in its entirety.

[0071] After the onset of hiPSC differentiation, early embryonic human heart organoids may be contacted with the maturation medium described herein. Differentiation of hiPCS may begin on day zero (0). Early embryonic human heart organoids may be contacted with the maturation medium described herein after day zero of hiPSC differentiation. For example, early embryonic human heart organoids may be contacted with the maturation medium described herein on any day between day 15 and day 25 after day zero of hiPSC differentiation. In several respects, embryonic human heart organoids may be contacted with the maturation medium described herein on day 20 after day zero of hiPSC differentiation.

[0072] In any embodiment, early embryonic human heart organoids may be exposed to the maturation medium described herein for an appropriate period of time to mature into mature human heart organoids. For example, early embryonic human heart organoids may be exposed to the maturation medium described herein for greater than or equal to about 4 days, greater than or equal to about 6 days, greater than or equal to about 8 days, greater than or equal to about 9 days, less than or equal to about 16 days, less than or equal to about 14 days, less than or equal to about 12 days, less than or equal to about 11 days, or less than or equal to about 10 days; about 4 days to about 16 days, about 4 days to about 14 days, about 6 days to about 12 days, or about 8 days to about 10 days. In any embodiment, early embryonic human heart organoids may be exposed to the maturation medium described herein for about 10 days, for example, from about day 20 to about day 30 after day 0 of hiPSC differentiation. As needed, for example, every 24 to 72 hours (e.g., every 24 hours, every 48 hours, every 72 hours), at least a portion of the maturation medium exposed to the early embryonic human heart organoids may be replaced with fresh maturation medium. Fresh maturation media may have the same or different composition as the maturation media being replaced. It is also anticipated in this paper that a portion of the replaced maturation media remains in contact with the early embryonic human heart organoids. Alternatively, essentially all maturation media in contact with the early embryonic human heart organoids may be replaced with fresh maturation media.

[0073] In several respects, early embryonic human heart organoids may be contacted with a maturation culture medium at any time, for example, from approximately day 20 to approximately day 30, wherein the maturation culture medium comprises a cell growth medium (e.g., RPMI 1640) as described herein, and a culture medium supplement (e.g., B-27) as described herein. TM Supplements), one or more additional fatty acids as described herein (e.g., palmitic acid, oleic acid, linoleic acid), additional carnitine (e.g., L-carnitine) or creatine as described herein, additional T3 growth hormone, and optionally an antibiotic as described herein (e.g., penicillin-streptomycin). For example, the maturation medium may contain approximately 97% RPMI 1640 medium; approximately 2% medium supplements (e.g., B-27).TM (Supplements); approximately 1% penicillin and streptomycin; approximately 52.5 μM palmitic acid in total; approximately 43.95 μM oleic acid in total; approximately 26 μM linoleic acid in total; approximately 132.2 μM L-carnitine in total; and approximately 33.01 nM T3 hormone in total.

[0074] In another aspect, early embryonic human heart organoids may be contacted with a maturation medium at any time, for example, from about day 20 to about day 30, said maturation medium comprising cell growth medium (e.g., RPMI 1640) as described herein and medium supplements (e.g., B-27) as described herein. TM Supplements), one or more additional fatty acids as described herein (e.g., palmitic acid, oleic acid, linoleic acid), additional carnitine (e.g., L-carnitine) or creatine as described herein, additional T3 growth hormone, additional sugars as described herein (e.g., glucose), additional antioxidants as described herein (e.g., ascorbic acid (vitamin C)), and optionally, antibiotics as described herein (e.g., penicillin-streptomycin). For example, the maturation medium may contain approximately 97% RPMI 1640 medium (without D-glucose); approximately 2% medium supplement (e.g., B-27). TM (Supplements); approximately 1% penicillin and streptomycin; approximately 52.5 μM palmitic acid in total; approximately 43.95 μM oleic acid in total; approximately 26 μM linoleic acid in total; approximately 132.2 μM L-carnitine in total; approximately 33.01 nM T3 hormone in total; approximately 0.4 mM ascorbic acid in total; and approximately 4 mM glucose in total.

[0075] In another aspect, early embryonic human heart organoids may be contacted with a maturation medium at any time, for example, from day 20 to day 30, said maturation medium comprising cell growth media as described herein (e.g., RPMI 1640) and culture medium supplements as described herein (e.g., B-27). TM Supplements), one or more additional fatty acids as described herein (e.g., palmitic acid, oleic acid, linoleic acid), additional carnitine (e.g., L-carnitine) or creatine as described herein, additional T3 growth hormone, additional sugars as described herein (e.g., glucose), additional antioxidants as described herein (e.g., ascorbic acid (vitamin C)), growth factors as described herein (e.g., IGF-1), and optionally antibiotics as described herein (e.g., penicillin-streptomycin). For example, the maturation medium may contain approximately 97% RPMI 1640 medium (without D-glucose); approximately 2% medium supplements (e.g., B-27). TM(Supplements); approximately 1% penicillin and streptomycin; approximately 52.5 μM palmitic acid; approximately 43.95 μM oleic acid; approximately 26 μM linoleic acid; approximately 132.2 μM L-carnitine; approximately 33.01 nM T3 hormone; approximately 0.4 mM ascorbic acid; approximately 4 mM glucose; and approximately 50 ng / mL IGF-1.

[0076] This document also considers the possibility that early embryonic human heart organoids may be contacted with more than one maturation culture medium as described herein, for example, during day 20 to day 30. In any embodiment, early embryonic human heart organoids may be contacted with a maturation culture medium (first maturation culture medium) for example, during day 20 to day 26, said maturation culture medium (first maturation culture medium) comprising a cell growth medium (e.g., RPMI 1640) as described herein, and a culture medium supplement (e.g., B-27) as described herein. TM The supplement contains one or more additional fatty acids as described herein (e.g., palmitic acid, oleic acid, linoleic acid), additional carnitine (e.g., L-carnitine) or creatine as described herein, additional T3 growth hormone, and also contains additional antioxidants as described herein (e.g., ascorbic acid), additional sugars as described herein (e.g., glucose), growth factors as described herein (e.g., IGF-1), and optionally antibiotics as described herein (e.g., penicillin-streptomycin). Additionally, the embryonic human heart organoid may, for example, be contacted with a maturation medium (second maturation medium) from day 26 to day 30, said maturation medium (second maturation medium) containing cell growth media as described herein (e.g., RPMI 1640), and culture medium supplements as described herein (e.g., B-27). TM The culture medium contains supplements, one or more additional fatty acids as described herein (e.g., palmitic acid, oleic acid, linoleic acid), additional carnitine (e.g., L-carnitine) or creatine as described herein, additional T3 growth hormone, and also contains additional antioxidants as described herein (e.g., ascorbic acid), additional sugars as described herein (e.g., glucose), wherein the maturation medium does not contain growth factors as described herein, such as IGF-1. In any embodiment, a portion of the maturation medium (first maturation medium) from day 20 to day 26 may be exposed to early embryonic human heart organoids starting from day 26 (e.g., from day 26 to day 30). In other words, when the first maturation medium is replaced on day 26, not all of the first maturation medium used from day 20 to day 26 is removed, but only a portion of the first maturation medium is removed and replaced with a second maturation medium.

[0077] In any implementation, exogenous retinoic acid and / or extracellular matrix material may not be added during the methods described herein.

[0078] Mature human heart organoids produced by the methods described herein are also provided.

[0079] The method described herein, utilizing the maturation culture medium, has been found to advantageously produce human heart organoids possessing several unique and key features representative of the early fetal human heart, including anterior-posterior pattern formation with a retinoic acid gradient at the posterior pole and polarity separation of the atrioventricular chambers with an epicardial layer at the anterior end. Furthermore, the disclosed method can also produce human heart organoids containing valvular cells, conduction cells, anterior epicardial cells, etc., as well as large hollow chambers, functional electrophysiology, increased mitochondrial density, and a metabolic transcriptional profile similar to that of the pregnant human heart. For example, single-cell gene expression of key genes associated with multiple cell clusters revealed that human heart organoids produced by exposing early embryonic human heart organoids to two different maturation culture media from day 20 to day 30 as described above exhibit the highest similarity to the human heart developing in vivo at 6.5 weeks post-conception (GD45). 41 Furthermore, the strategies described in this paper lead to the expansion and reduction of certain cardiac cell type populations, such as cardiomyocytes in the atria and ventricles, and mesenchymal cell types (stromal cells), which are considered to be processes of fine-tuning and remodeling. Interestingly, the emergence of valve and conduction cell types from the human cardiac organoids generated in this paper was observed.

[0080] The mature human heart organoids generated in this study showed a significant response to developmental maturation stimuli and were metabolically mature, exhibiting enhanced mitochondrial growth, dense respiration rate, and gene expression. Compared to conventional methods, these significant responses may be the result of synergistic effects among multiple cardiac cell subtypes, such as epicardial cells and cardiac fibroblasts, which have been shown to stimulate cardiomyocyte growth and function. 121,122 .

[0081] Furthermore, proper and gradual electrophysiological maturation throughout the cardiac syncytium (including the complex interactions between various ion channels and their subtypes, as well as depolarization via the t-tubule) encompasses key aspects of cardiac development and function. 103-106,126,127,145 Human heart organoids produced using the methods described herein (e.g., by exposing early embryonic human heart organoids to two different maturation media on days 20 to 30 as described above) exhibited significant calcium transients with enhanced physiological mimicry (due to increased amplitude and decreased frequency). Furthermore, these organoids produced higher levels of transverse tubes, inward rectifier potassium channels, and hERG channels compared to other maturation strategies. In fact, many efforts to induce transverse tubes in vitro have struggled or failed to do so. 146,147And inward rectifying potassium ion channels, which remain crucial for establishing low resting membrane potential. 148,149 The existence of [the specific pathway / channel] is also important. Furthermore, the cardiac hERG channel represents a crucial pathway for pharmacological screening because its disruption carries a high potential for arrhythmogenicity. 116,117 Nevertheless, the sum of multiple ion transients generates cardiac action potentials, the ultimate driving force for human cardiac contraction and function. Furthermore, these generated organoids exhibit ventricular, atrial, and nodular action potentials, opening the door to electrophysiological applications in drug screening.

[0082] The embryonic heart begins as an unpatterned cardiac tube and undergoes cellular and structural changes through morphogenetic signal transduction events, eventually forming a four-chambered heart along an anterior-posterior axis. 150,151 Unexpectedly, human heart organoids produced using the maturation method described herein, for example, when early embryonic human heart organoids, as described above, are exposed to two different maturation media on days 20 to 30, form a two-chambered structure with cardiomyocytes, creating one chamber with atrial identity and another with ventricular fate. The dense epicardial layer at the atrial chamber defines the anterior epicardial organoid as the posterior pole of the cardiac tube. 152 Furthermore, it was revealed that these organoids spontaneously form along the aforementioned anterior-posterior axis. It was also found that this self-organization and pattern formation in these organoids is driven by an endogenous retinoic acid signaling gradient. The enzyme ALDH1A2, required for retinoic acid synthesis, was observed to be spatially confined to the posterior end of the organoid and co-localized with TBX18, a transcription factor that identifies anterior epicardial organoids as functional epicardial organs. Therefore, in several aspects, the methods described herein can produce mature human cardiac organoids containing one or more of the following features: (i) endogenous retinoic acid; (ii) at least two chambers of the heart (e.g., atrial and ventricular chambers); (iii) a posterior anterior epicardial pole (see [link to previous section]). Figure 7A ); and (iv) anterior-posterior cardiac tube patterning (e.g., anterior-posterior patterning of the ventricle (anterior pole) and atrium (posterior) chambers) (see Figure 7AFurthermore, the methods described herein can produce human heart organoids similar to those of a pregnant human heart, possessing valve cells, conduction cells, anterior epicardial cells, large hollow chambers, functional electrophysiology, and increased mitochondrial density and metabolic transcription profile. As used herein, “endogenous retinoic acid” refers to retinoic acid naturally present in mature human heart organoids or, for example, retinoic acid produced by mature human heart organoids by day 30 of the method. Endogenous retinoic acid can be present in a gradient within mature human heart organoids. As discussed above, retinoic acid is a potent morphogenetic factor involved in heart development and provides instructions for cellular development and pattern building in the heart. The retinoic acid gradient can originate from and / or be located at the posterior pole (anterior epicardial pole / atrial pole) of the obtained human heart organoid. Additionally, the mature human heart organoid can be pulsating, for example, at 60 to 80 beats per minute. In this case, the mature human heart organoids described herein can be considered equivalent to the fetal human heart from approximately day 45 to approximately day 90 of gestation.

[0083] In summary, the human heart organoids generated by the method described in this paper are believed to reproduce events occurring during intrauterine pregnancy, wherein anterior epicardial organs surround the posterior pole of a pattern-built cardiac tube, in which posterior atrial cardiomyocytes and anterior epicardial cells produce retinoic acid to form a signal transduction gradient, which further guides the remainder of the cardiac tube with pattern-building and specialization information. 41,56,131,134,152 .

[0084] Example

[0085] Example 1

[0086] Methods and Operations

[0087] stem cell cultureThe following human induced pluripotent stem cell (hiPSC) lines and human embryonic stem cell (ESC) lines were used in this study: iPSC-L1 (iPSC) (sex: male), ATCC-BYS0111 (iPSC) (sex: male) (alias: ATCC), and H9 (ESC) (sex: female) (WiCell, WA09). Pluripotency and genomic stability of all hiPSC lines used were tested. hiPSCs were cultured in Essential 8 Flex medium containing 1% penicillin-streptomycin (Gibco) in 6-well plates on Corning gel with reduced growth factors, inside an incubator at 37°C and 5% CO2. hiPSCs were passaged using ReLeSR passage reagent (STEMCELL Technologies) when they reached 60% to 80% confluence. Unless otherwise stated, all data in the following results are from the iPSC-L1 line.

[0088] Differentiation of self-assembled human heart organoids. It provides a step-by-step, detailed scheme describing the origin and differentiation of human heart organoids. 14 In short, hiPSCs were grown to 60% confluence in 6-well plates and dissociated using Accutase (Innovative Cell Technologies) to obtain a single-cell solution. The hiPSCs were collected and diluted at 300 mL / min. g Centrifuge for 5 minutes and resuspend in Essential 8 Flex medium containing 2 μM ROCK inhibitor (Thiazovivin) (Millipore Sigma). hiPSCs were counted using a Moxi cell counter (Orflo Technologies) and seeded at a concentration of 10,000 cells / well in 100 μL volumes on day -2 in round-bottom 96-well ultra-low adhesion plates (Costar). The plates were then centrifuged at 100... gCentrifuge for 3 minutes and then incubate at 37°C with 5% CO2. After 24 hours (Day -1), remove 50 μL from each well and add 200 μL of fresh Essential 8 Flex medium to each well to obtain a final volume of 250 μL per well. Then incubate the plate at 37°C with 5% CO2. After 24 hours (Day 0), remove 166 μL of medium from each well. Then, add 166 μL of RPMI (Gibco) containing B27 supplemented with 1% penicillin-streptomycin (Gibco) and containing CHIR99021, BMP4, and activin A to each well to obtain a final concentration of 4 μM CHIR99021, 36 pM (1.25 ng / mL) BMP4, and 8 pM (1.00 ng / mL) activin A. The plates were then placed in an incubator at 37°C and 5% CO2. After 24 hours (Day 1), 166 μL of medium was removed from each well and replaced with 166 μL of fresh insulin-removed RPMI / B27. On Day 2, 166 μL of medium was removed from each well and 166 μL of insulin-removed RPMI / B27 containing Wnt-C59 (Selleck) was added to obtain a final concentration of 2 μM Wnt-C59 in each well. The plates were then incubated for 48 hours. On Day 4, 166 μL was removed and replaced with fresh insulin-removed RPMI / B27, and the plates were incubated for 48 hours. On Day 6, 166 μL was removed and replaced with 166 μL of RPMI containing B27 supplement (insulin) and 1% penicillin-streptomycin (hereinafter referred to as "RPMI / B27"). The plates were incubated for 24 hours. On day 7, remove 166 μL of medium from each well and add 166 μL of RPMI / B27 containing CHIR99021 to obtain a final concentration of 2 μM CHIR99021 per well. Incubate the plate for 1 hour. After 1 hour, remove 166 μL of medium from each well and add 166 μL of fresh RPMI / B27 to each well. Incubate the plate for 48 hours. From day 9 to day 19, change the medium every 48 hours by removing 166 μL of medium from each well and adding 166 μL of fresh RPMI / B27.

[0089] Developmental induction conditions. Organoids were generated and differentiated according to the previously outlined protocol. Starting on day 20, the organoids were subjected to various maturation culture medium conditions. The control strategy involved continued culture in RPMI / B27 from day 20 to day 30, with standard medium replaced every 48 hours.

[0090] From day 20 to day 30, a maturation medium (MM) strategy was employed, with the medium being replaced every 48 hours using MM medium. The MM medium consisted of a stock solution of RPMI / B27 (containing insulin) with 52.5 μM palmitate-BSA, 40.5 μM oleate-BSA (Sigma), 22.5 μM linoleate-BSA (Sigma), 120 μM L-carnitine (Sigma), and 30 nM T3 hormone (Sigma). Details of the MM medium formulation are provided in Table 1 below.

[0091] Table 1. MM culture medium

[0092]

[0093] From day 20 to day 30, an enhanced maturation medium 1 (EMM1) strategy was employed, with the medium being replaced every 48 hours using EMM1. EMM1 consisted of a stock solution of RPMI 1640 (Gibco) supplemented with the following glucose-free stock solution: B27 (containing insulin), 1% penicillin-streptomycin (Gibco), 52.5 μM palmitate-BSA, 40.5 μM oleate-BSA (Sigma), 22.5 μM linoleate-BSA (Sigma), 120 μM L-carnitine (Sigma), 30 nM T3 hormone (Sigma), 0.4 mM ascorbic acid (Thermo Fisher Scientific), and 4 mM glucose (Gibco). Details of the EMM1 medium formulation are provided in Table 2 below.

[0094] Table 2. EMM1 culture medium

[0095]

[0096] From day 20 to day 30, an enhanced maturation medium 2 / 1 (EMM2 / 1) strategy was employed, with the medium being changed every 48 hours using a combination of the two media. From day 20 to day 26, EMM2 medium was used, consisting of a glucose-free stock solution of RPMI 1640 (Gibco) supplemented with the following: B27 (containing insulin), 1% penicillin-streptomycin (Gibco), 52.5 μM palmitate-BSA, 40.5 μM oleate-BSA (Sigma), 22.5 μM linoleate-BSA (Sigma), 120 μM L-carnitine (Sigma), 30 nM T3 hormone (Sigma), 0.4 mM ascorbic acid (Thermo Fisher Scientific), 4 mM glucose (Gibco), and 50 ng / mL IGF-1. The EMM2 / 1 strategy was continued, and from day 26 to day 30, EMM1 medium was used. The formulation details of EMM2 medium are provided in Table 3 below.

[0097] Table 3. EMM2 culture medium

[0098]

[0099] Organoids were collected on day 30 for analysis.

[0100] Immunofluorescence. Human heart organoids were transferred from round-bottom ultra-low adhesion 96-well plates to 1.5 mL microcentrifuge tubes (Eppendorf) using chopped 200 μL pipette tips (to increase the tip diameter and avoid damaging the organoids). The organoids were fixed in 4% paraformaldehyde (VWR) in PBS for 30 min. Subsequently, the organoids were washed three times with PBS-glycine (1.5 g / L), 5 min each time. Then, the organoids were blocked and permeabilized overnight on a heat mixer at 300 rpm and 4 °C using a solution containing 0.5% BSA (Thermo Fisher Scientific), 0.5% Triton X-100 (Sigma), and 10% donkey normal serum (Sigma) in PBS. The organoids were then washed three times with PBS and incubated together with the primary antibody (Table 4) in a solution containing 0.5% BSA, 0.5% Triton X-100, and 1% normal donkey serum in PBS (hereinafter referred to as the “antibody solution”) at 4°C and 300 rpm for 24 hours on a heat mixer.

[0101] Table 4. Antibodies used for immunofluorescence.

[0102]

[0103]

[0104] The organoids were then washed three times with PBS for 5 minutes each time. The organoids were then incubated with the secondary antibody in the antibody solution (Table 4) on a hot mixer at 300 rpm in the dark at 4°C for 24 hours. The organoids were then washed three times with PBS for 5 minutes each time and mounted on a glass microscope slide (Fisher Scientific). 90 μm Polybead microspheres (Polyscience, Inc.) were placed between the slide and a No. 1.5 coverslip (VWR) to provide a support pillar, allowing the organoids to retain their three-dimensionality. The organoids were transferred to the glass microscope slide using a diced 200 μL pipette tip and mounted with the previously described clear solution. 153 Transverse tube staining was performed using FITC-conjugated wheat germ agglutinin (WGA) lectin (Sigma).

[0105] Confocal microscopy and image analysis.Immunofluorescence images were acquired using a confocal laser scanning microscope (Nikon Instruments A1 confocal laser microscope). Images were analyzed using Fiji. When comparing images within or between conditions, for each channel of the measured image, the pixel intensity values ​​were equalized to those of the control or EMM2 / 1 condition, where appropriate. Linear and freehand tools were used to measure organoid diameter and area, respectively. Automatic thresholding was used to measure mitochondrial (MitoTracker) area, Cav-3+ area, KCNJ2+ spots, MYL2+ area, MYL7+ area, PECAM1+ area, and ALDH1A2+ TBX18+ area. The elliptical selection tool was used to measure TNNT2+ and WT1+ chamber dimensions, with the organoid walls used as the boundary regions for the corresponding areas to be plotted. For area measurements using the entire organoid (low magnification images), data points were normalized to organoid area. The average gray value was calculated to measure FlipGFP fluorescence intensity. To measure the Pearson coefficient, the JaCOP colocalization plugin was used (Bolte, S., & Cordelières, FP (2006). A guided tour into subcellular colocalization analysis in light microscopy. Journal of Microscopy, 224(3), 213–232. doi:10.1111 / j.1365-2818.2006.01706.x). A threshold for equalized image intensity values ​​was generated. A spatial resolution of 1.243 μm per pixel was used.

[0106] Organoid dissociation of the heart.On day 30, organoids were collected from each mature strategy (control, MM, EMM1, EMM2 / 1). Organoids were dissociated and pooled in separate 1.5 mL microcentrifuge tubes (Eppendorf). Organoids were dissociated into single-cell suspensions using a modified STEMdiff cardiomyocyte dissociation kit (STEMCELL Technologies). After transfer to microcentrifuge tubes, organoids were washed with PBS, immersed in 200 μL of warm dissociation medium (37°C), and placed on a heat mixer at 37°C and 300 rpm for 5 minutes. The supernatant was then collected and transferred to 15 mL Falcon tubes (Corning) containing 5 mL of the appropriate medium (control, MM, EMM1, etc.) containing 2% BSA (Thermo Fisher Scientific). An additional 200 μL of warm dissociation medium (37°C) was then added back to the organoids on the heat mixer (37°C). Then, gently aspirate the organoid dissociation medium solution up and down 3 to 5 times. Place the organoids on a heat mixer for another 5 minutes. If the organoids are still visible, repeat the process. Once the organoids are no longer visible, gently aspirate the microcentrifuge tube solution up and down 3 to 5 times and transfer all its contents to 15 mL Falcon tubes containing the appropriate medium + 2% BSA and cells. Then, centrifuge these tubes at 300... g Centrifuge for 5 minutes. Aspirate the supernatant and resuspend the cell pellet in the appropriate culture medium + 2% BSA. Use a hemocytometer to obtain viability, cell count, and aggregation percentage.

[0107] Single-cell RNA sequencing.Library preparation was performed using the 10× Chromium Next GEM Single Cell 3' Kit, v3.1, and related components. Complete libraries were QC'd and quantified using a combination of Qubit dsDNA HS, Agilent 4200 TapeStation HS DNA1000, and Invitrogen Collibri library quantification qPCR assay. Libraries were pooled in equimolar proportions and the pooled copies were quantified again using the Invitrogen Collibri qPCR assay. The pooled copies were loaded into two lanes of an Illumina NovaSeq 6000 SP flow cell (v1.5) and sequenced with custom paired ends for 28 cycles for read 1, 210 cycles for indexed reads, and 90 cycles for read 2. Sequencing was performed using the v1.5 100-cycle NovaSeq kit. Read 1, a 28 bp read, contained 10× cell barcodes and UMI, and read 2 was a cDNA read. The output of the Real-Time Analysis (RTA) was demultiplexed and converted to FastQ format using Illumina Bcl2fastq v2.20.0. After demultiplexing, readings from each sample library were further processed using 10× Genomics cellranger count (v 6.1.2). The files were analyzed using 10× Genomics LoupeBrowser v6.3.0, employing k-means clustering of 8 clusters and UMAP visualization. Enrichr... 154-156 Used for evaluating gene ontology. Pathview Web is used to generate biological pathway maps. 101,102 Using Liana 157 and Celltalker 158 Perform intercellular communication analysis (https: / / github.com / arc85 / celltalker). To accomplish this task, count and clustering data from Loupe Browser were imported into Seurat. 159 Both programs used the standard Seurat findmarkers differential expression function for inter-cluster differential expression and then ranked ligand and receptor pairs by significant p-values ​​and log2 fold changes. To identify ligand and receptor pairs, Liana used the OmniPath database. 160 Furthermore, Celltalker uses the Ramilowski-pairs database of ligand-receptor interactions. 161 .

[0108] Transmission Electron Microscopy (TEM) On days 15 and 30, human heart organoids were fixed in 2.5% glutaraldehyde (electron microscopy solution) in PBS for 45 minutes, washed three times in PBS for 5 minutes each time, and then stored at 4°C. The samples were then washed with 100 mM phosphate buffer and post-fixed with 1% osmium tetroxide in 100 mM phosphate buffer, dehydrated in a gradient series of acetone, and infiltrated and embedded in a Spurr (Electron Microscopy Sciences). Thin sections of 70 nm were obtained using a Power Tome microtome (RMC, Boeckeler Instruments, Tucson, AZ) and post-stained with uranyl acetate and lead citrate. Images were acquired using a JEOL 1400 Flash transmission electron microscope (Japan Electron Optics Laboratory, Japan) at an accelerating voltage of 100 kJ.

[0109] Calcium imaging. Calcium transient activity in human cardiac organoids was assessed using Fluo-4 AM (Thermo Fisher Scientific). Fluo-4 AM was dissolved in DMSO according to the manufacturer's instructions. 1.5 μM Fluo-4 solutions were prepared in the appropriate media (control, MM, EMM1, etc.). Organoids were washed twice with RPMI 1640 basal medium, then Fluo-4 AM was added to a final concentration of 1 μM, and incubated at 37 °C and 5% CO2 for 30 min. Organoids were then washed twice with their respective media (control, MM, EMM1, etc.) and transferred to chamber coverslips (Cellvis) using a chopped 200 μL pipette tip. Images were acquired as an image stack at 100 frames per second for a total of 10 seconds using a Cellvivo microscope (Olympus). Samples were excited at 494 nm, and emission at 506 nm was collected. Data were processed using Fiji and Microsoft Excel. The baseline F0 of fluorescence intensity F was calculated using the average of the lowest 50 intensity values ​​obtained from the dataset. Calculate the fluorescence change ΔF / F0 using the following equation:

[0110] .

[0111] Voltage imagingVoltage activity in human cardiac organoids was assessed using di-8-ANEPPS (Thermo Fisher Scientific). Di-8-ANEPPS was dissolved in DMSO according to the manufacturer's instructions. A 15 μM di-8-ANEPPS solution was prepared in the appropriate medium (control, MM, EMM1, etc.). The organoids were washed twice with RPMI 1640 basal medium, then di-8-ANEPPS was added to a final concentration of 10 μM, and incubated at 37 °C and 5% CO2 for 30 min. The organoids were then washed twice with their respective medium (control, MM, EMM1, etc.) and transferred to chamber coverslips (Cellvis) using a chopped 200 μL pipette tip. Images were acquired as an image stack at 100 frames per second for a total of 10 seconds at 20× magnification (Ondansetron) or 100× magnification (Mature Electrophysiology) using a Cellvivo microscope (Olympus). Samples were excited at 465 nm excitation, and 630 nm emission was collected. Data were processed using Fiji and Microsoft Excel. The baseline F0 of fluorescence intensity F was calculated using the average of the lowest 50 intensity values ​​in the obtained dataset. Fluorescence change ΔF / F0 was calculated using the same method as described for calcium imaging. APD30 and APD90 were measured from the midpoint of the upstroke up to 30% or 90% of the repolarization, respectively. 162 .

[0112] Real-time RT-PCROrganoids were collected on days 20, 21, 23, 25, and 30 and stored at -20°C in RNAprotect (Qiagen). RNA was extracted primarily according to the manufacturer's instructions using the Qiagen RNEasy Mini kit. Organoids were lysed for 30 seconds at speed 2 using a Bead Mill 4 homogenizer (Fisher Scientific). RNA concentration was measured using NanoDrop One (Thermo Fisher Scientific). A minimum threshold of 10 ng / μL was required for reverse transcription. cDNA was generated using the Quantitect Reverse Transcription Kit (Qiagen) and stored at -20°C. Primers for real-time qPCR were designed using the Primer Quest tool (Integrated DNA Technologies). SYBR Green (Thermo Fisher Scientific) was used as the amplifier for the DNA intercalation dye and reaction vessel. Real-time qPCR was performed using the QuantStudio 5 Real-Time PCR system (Applied Biosystems) with a total reaction volume of 20 μL. Gene expression levels were normalized relative to HPRT1 expression in each individual sample. Logarithmic fold change to base 2 was obtained using the double delta-CT method. At least four independent samples were run for each gene expression assay at each time point under each condition. mRNA expression data were displayed as a logarithmic fold change to base 2 relative to the control.

[0113] Mitochondrial imaging.The presence of intracellular mitochondria in human heart organoids was visualized using a Mitotracker Deep Red FM (Thermo Fisher Scientific). Mitotracker was prepared according to the manufacturer's instructions. 150 nM Mitotracker was prepared in the appropriate medium (control, MM, EMM1, etc.). NucBlue (Thermo Fisher Scientific) was used for visualization of the cell nucleus. NucBlue was prepared by adding 2 drops per mL of 150 nM Mitotracker solution (as described above). The organoids were washed twice with 166 μL of RPMI 1640 basal medium, then 166 μL of Mitotracker was added to achieve a final concentration of 100 nM, and incubated at 37°C and 5% CO2 for 30 min. The organoids were then washed twice with their respective medium (control, MM, EMM1, etc.) and transferred to chamber coverslips (Cellvis) using a chopped 200 μL pipette tip. Images were acquired using a Cellvivo microscope (Olympus). Data were processed using Fiji.

[0114] Raman microscopy. Raman spectra of organoids were acquired using a Renishaw inVia confocal Raman spectrometer connected to a Leica microscope (Leica DMLM, Leica Microsystems, Buffalo Grove, IL, USA). A 785 nm near-IR laser, a Nikon Flour 60×NA=1.00 water immersion objective, an exposure time of 1000 ms, and an average of 100 cumulative exposures were used to acquire data for each scan location of the organoids. To avoid strong background signals, a quartz slide (Chemglass Life Sciences, NJ, USA) was used as the substrate for Raman spectroscopy acquisition. Organoids were collected on day 30 for analysis. Optical coherence tomography This will utilize a system similar to the Spectral-Domain Optical Coherence Tomography (SD-OCT) system used in previous work. 128,129Label-free longitudinal imaging of cardiac organoids was performed. A superluminescent diode (EXALOS, EXC250023-00) was used as the light source, with a center wavelength of approximately 1300 nm and a 3 dB spectral range of approximately 180 nm. A spectrometer (Wasatch Photonics, Cobra 1300) based on a 2048-pixel InGaAs line scan camera (SensorsUnlimited, GL2048) was used to provide a maximum A-scan rate of 147 kHz. A 5× objective lens was used, and the lateral and axial resolutions measured in tissue were approximately 2.83 μm and 3.04 μm, respectively. Longitudinal 3D OCT imaging was performed every other day from day 20 to day 30. Each 3D OCT scan consisted of 600 A scans / one B scan and 600 B scans. Using an exposure time of approximately 40 microseconds per A scan, approximately 22 seconds were required for image acquisition per organoid. Eight organoids from each group were imaged and used for analysis. During imaging, the culture medium level in each well was adjusted to reduce image artifacts and minimize light absorption. Acquired OCT images were rescaled using ImageJ to obtain isotropic pixel dimensions in the xyz dimensions (Schneider et al., 2012). Registration, cavity segmentation, and 3D rendering of the same organoids from different days were performed using Amira software (Thermo Fisher Scientific). The cavities and total volume within the organoids were quantified based on the segmentation data.

[0115] Ondanstron processing Ondansetron hydrochloride (Sigma) was prepared in DMSO at 200 μM and further diluted in DMEM / F12 (Dulbecco modified Eagle medium / nutrient mixture F-12), then aseptically filtered through a 0.22 μM PVDF filter (Sigma). Ondansetron was applied to cardiac organoids at final concentrations of 1 μM, 10 μM, and 100 μM in EMM2 / 1 medium on days 9 through 20 of culture. Organoids were collected on day 30 for analysis.

[0116] Lentiviral transduction. HEK293T (Horizon Inspired Cell Solutions) cells were transfected with the Flip-GFP plasmid (VectorBuilder) and packaging plasmids pMD2 and psPAX2 using lipofectamine and Plus reagent (Thermo) to generate lentivirus. The lentivirus was added to iPSC-L1 cells containing 8 μg / ml polybrene (Fisher Scientific) and incubated overnight. Puromycin selection was performed for 3 to 5 days until all lentivirus-deficient cells disappeared from the wells. Surviving clones were selected, collected, replated, and further amplified to generate the FlipGFP line.

[0117] Dorothy Star Processing Doxorubicin hydrochloride (Sigma) was diluted to 1 mM in DMEM / F12 and applied to the cardiac organoids at a final concentration of 10 μM for 48 hours from day 28 to day 30 of culture.

[0118] DEAB and retinoic acid treatment 4-Diethylaminobenzaldehyde (DEAB) (Sigma) was prepared in DMSO at 1 M, further diluted to 10 mM using DMSO, and then finally diluted to 1 mM in DMEM / F12. Retinic acid (RA) (Sigma) was prepared in DMSO at 1 M and diluted to 100 μM in DMEM / F12. The diluted solutions of DEAB and RA were aseptically filtered through a 0.22 μm PVDF filter (Sigma). DEAB was applied to the cardiac organoids at a final concentration of 10 μM. RA was applied to the cardiac organoids at a final concentration of 1 μM. DEAB, RA, and DEAB+RA were applied to the cardiac organoids cultured from day 20 to day 30 using an EMM2 / 1 strategy. Organoids were collected on day 30 for analysis.

[0119] Agilent SeahorseMetabolic assays. Real-time extracellular throughput was measured using the Agilent Seahorse XFe96 (Agilent). One day prior to the assay, 200 μL of XF calibrator was loaded into each well of a 96-well practical plate contained in the sensor cartridge, and the sensor was immersed overnight in a CO2-free incubator at 37°C. Also one day prior to the assay, XFe96 spherical microplates were coated with polylysine (Sigma). Specifically, 100 μg / mL of polylysine was prepared in water, and 30 μL of this solution was added to each well of the microplate. After standing for 20 minutes, the polylysine solution was aspirated from the wells and washed twice with sterile water. The plate was then air-dried for at least 30 minutes. The plate was then incubated at 37°C in a CO2-free incubator for 30 minutes. Finally, 100 μL of DMEM / F12 at 37°C was added to each well of the microplate, and the microplate was returned to a CO2-free incubator overnight. The following steps describe the procedures performed on the day of the assay in sequence. XF RPMI (phenol red-free) (Agilent) was prepared as the basal medium for the assay, supplemented with 1 mM pyruvate (Agilent), 2 mM glutamine (Agilent), 11.1 mM glucose (Gibco), and 12.2 μM L-carnitine (Sigma). Using this prepared XF RPMI, the drug solution from the Cell Mito Stress Test kit (Agilent) was resuspended, vortexed for 1 minute, and allowed to stand at room temperature for 1 hour. At this time, the polylysine-coated XFe96 spherical microplate was removed from the incubator, DMEM / F12 was removed from the plate, the plate was washed once with 166 μL of the prepared XF RPMI, and finally, 175 μL of the prepared XF RPMI was added to each well. Then, organoids from day 30 under each condition were washed twice with 166 μL of prepared XF RPMI and transferred to polylysine-coated XFe96 spherical microplates. Organoids were transferred to wells using a diced p200 pipette tip, ensuring the organoid was centered in the well. The plates were then incubated at 37°C in a CO2-free environment. 2 The incubator was incubated for 1 hour. Drug solutions (oligomycin, FCCP, and Rot / AA) were loaded into ports A, B, and C, respectively. The channel concentrations of oligomycin, FCCP, and Rot / AA were 25 μM, 20 μM, and 20 μM, respectively, resulting in final concentrations of 2.5 μM, 2 μM, and 2 μM in solution. The assay was configured to run for 6 cycles at baseline and 10 cycles each for the oligomycin, FCCP, and Rot / AA phases. Each cycle consisted of a 3-minute mixing phase, a 0-minute wait phase, and a 3-minute measurement phase. Data were normalized to organoid area.

[0120] Statistics and reproducibility Raw data were collected using Microsoft Excel. Graphpad Prism 9 software was used for all analyses. Data were presented as normally distributed. Statistical significance (p < 0.05) was assessed using one-way ANOVA with post-correction using Dunnett or Brown-Forsyth and Welch tests, or, where appropriate, unpaired t-tests. All data were presented as mean ± sem. Statistical methods are illustrated in the legend. The number of independent organoids used for each quantitative and statistical test is shown in the legend. When more than one independent experiment (plates / batches of organoids) was performed (as is the case for most of the data in this manuscript), this is appropriately shown in the legend.

[0121] result

[0122] Extended modeling of heart development through improved developmental induction strategies

[0123] A detailed protocol for generating self-organized early embryonic human heart organoids has been previously described. 15 This scheme constitutes the initial step of the following method. In short, through a time-wise, three-step Wnt pathway regulation strategy, cardiac organoids differentiate from hiPSC embryoids into cardiac lineages from day 0 to day 7, and are then cultured in RPMI until day 20. 15 To examine the effects of more advanced organoid culture strategies simulating intrauterine conditions on cardiac organoid development, early embryonic cardiac organoids at day 20 were subjected to four different developmental induction strategies applied from day 20 to day 30. Figure 1A ). Compared with previous human and animal developmental studies 20-24 In comparison, these strategies represent steps with progressively increasing complexity related to intrauterine conditions (in order of less complex to more complex: control, maturation medium, enhanced maturation medium 1, enhanced maturation medium 2 / 1). The "control strategy" represents the continuation of organoid culture in the basal medium RPMI / B27 used for organoid formation. The "maturation medium (MM) strategy" uses fatty acids supplemented (embryo-relevant concentrations of oleic acid, linoleic acid, and palmitic acid). 23,24 and L-carnitine 25 RPMI / B27 promotes the developmental transition of the fetal human heart, a characteristic shift from glucose utilization to fatty acid metabolism. 26-30 The MM strategy also utilizes T3 hormones, which are potent activators of organ growth during embryonic development and metabolic maturation, and have been shown to stimulate cardiovascular growth. 31,32The "Enhanced Maturation Medium 1 (EMM1) Strategy" uses the same basic composition as MM, but reduces the glucose concentration to cardiac physiological levels. 33-35 (From 11.1 mM to 4 mM to further promote the transition to fatty acid oxidation), and ascorbic acid was added as a reactive oxygen species scavenger to counteract the increased oxidative stress. 36,37 The "Enhanced Maturation Culture Medium Strategy 2 / 1 (EMM2 / 1)" utilizes a combination of two different culture medium formulations. From days 20 to 26, EMM2 medium was used, and the basic composition of EMM2 medium was identical to that of EMM1 supplemented with IGF-1. IGF-1 plays an important role in tissue growth and maturation during embryonic and fetal development, particularly in the heart, as demonstrated in studies in mice and humans. 38-40 From day 26, EMM1 medium was used in the EMM2 / 1 strategy. The EMM2 / 1 strategy represents state-of-the-art conditions and best mimics intrauterine heart development. More detailed descriptions of all developmental induction strategies and the concentrations of the corresponding culture medium formulations can be found in the Materials and Methods section above.

[0124] Heart organoids treated with different developmental induction strategies continued to grow and develop, with morphological changes occurring drastically depending on the conditions. Figures 1B to 1D From day 0 to day 10, the organoids underwent a period of rapid growth, increasing in diameter while maintaining their spherical structure. Figure 1B ), and continued to grow until day 30. After day 20, the organoids developed a distinctly elliptical morphology, as observed by bright-field microscopy, exhibiting elongation and deformation, and by day 30 grew to a long diameter of 1000 to 1600 μm, while the short diameter was 600 μm to approximately 1000 μm. Figures 1B to 1C Organoid area measured by bright-field microscopy revealed a similar trend under each condition, at 0.6 mm. 2 up to 0.9 mm 2 ( Figure 1D By day 30 of culture, nearly 100% organoid pulsation was observed in five independent experiments under each condition (n=22 to 24 organoids / each condition / each experiment). Figure 1E Transmission electron microscopy (TEM) images showed the presence of well-developed myofibrils and sarcomere formation within the organoids under all conditions. Figure 1F ), among which the sarcomeres under EMM1 conditions showed a significantly increased sarcomere length compared to the control (1.58 ± 0.323 μm) ( Figure 1GqRT-PCR revealed the expression of signature cardiomyocyte sarcomere genes from day 20 to day 30, as expected. Interestingly, different conditions showed expression at different developmental time points. MYL2 , MYL7 , MYH7 and MYH6 The differential expression indicates that the developmental maturation strategy of this invention triggers different transcriptomic effects on the growth of cardiac organoids. Figure 1H ).

[0125] Development-induced single-cell RNA sequencing (scRNA-seq) of human heart organoids revealed cell types Differences in complexity and cellular composition

[0126] To characterize the cellular and transcriptomic composition of cardiac organoids under each developmental induction condition, scRNA-seq was performed on day 34 of organoid culture. UMAP projections were used to visualize unsupervised K-means clustering analysis under each condition. Figure 2A The cardiac organoids revealed ventricular and atrial cardiomyocytes (VCM and ACM, respectively), valve cells (VC), proepicardial derived cells (PEDC), epicardial cells (EC), stromal cells (SC), cardiac progenitor cells (CPC), conduction cells (CC), and endothelial cells (EC) under all conditions. The abundance of several important cell groups varied depending on the developmental culture conditions. The control organoids consisted of 17% VCM, 17% ACM, 3% VC, 17% PEDC, 1% EPC, 18% SC, 10% CPC, 5% CC, and 1% EC. Figure 2BCompared to the control, MM organoids showed increased percentages of both VCM and ACM (27% and 34%, respectively), increased VC (10%), decreased PEDC (12%), 1% EPC, decreased SC (9%), decreased CPC (6%), and decreased CC (1%). Compared to the control, EMM1 organoids showed increased percentages of VCM (22%), increased ACM (31%), increased VC (10%), decreased PEDC (16%), increased EPC (4%), decreased SC (9%), decreased CPC (7%), and decreased CC (1%). Compared to the control, EMM2 / 1 organoids showed decreased percentages of VCM (13%), increased ACM (20%), increased VC (18%), decreased PEDC (15%), increased EPC (3%), 18% SC, 10% CPC, and decreased CC (2%). Differential gene expression analysis identified characteristic genes used to identify clusters. Figures 2C to 2D ACM has MYH6 , MYL7 , NPPA and GJA5 high expression 4,41-43 。 VCM displays MYL3, MYH7, TNNC1 and HSPB7 high expression 41,42,44-47 PEDC shows PDGFRB , SEMA3D , POSTN and TCF21 high expression 48-52 EPC and PEDC share some similarities, but also exhibit differentially expressed genes, including... WT1 , TBX18 , ITLN1 and TNNT1 41,53-56 CC shows STMN2 , CHGA , SCG2 and INSM1 High expression of these genes; these genes are involved in neuronal growth, development, and neuroendocrine signal transduction. 57–62 Furthermore, it shares similarities with the human embryonic heart dataset in neural crest cells and Schwann cell clusters. 41 EC has PECAM1 , ESAM , SOX18 and FLT high expression 41,63-66 。 SC passed SOX2, ANXA4, SOX9, CD24 The expression determines 41,67-71 VC through SOX9 , UGDH , ID2 and FLRT2 The expression determines 42,69,72-78 In summary, these results indicate that cardiac organoids possess cell types similar to those present in the developing human heart, and demonstrate consistency with previous studies on heart development. 41,42 By day 20, the major cardiac cell lineages had been identified and developmental induction conditions could exert a significant effect on the expansion and maturation of these cell types to better reflect in vivo cardiac development.

[0127] To investigate whether differences in cell type ratios in organoids under different conditions are caused by apoptosis, a genetic reporter iPSC line named FlipGFP was created. This iPSC line fluoresces when the active form of caspase 3, a major regulator of apoptosis, is present. 79 It was found that from day 20 to day 30, the level of apoptosis in cardiac organoids was very low. Figure 9A ), and there was no difference in apoptosis levels between conditions ( Figure 9B ). 48-hour doxorubicin treatment was used as a positive control and showed high levels of fluorescence (). Figure 9C This data suggests that the proportion of cell types in organoids from different conditions is not driven by apoptosis.

[0128] Several additional specific biomarkers were identified in the cardiac organoid dataset. Cardiac fibroblasts were identified in the PEDC cluster, indicating that... DCN , LUM , OGN ,and POSTN as well as COL1A1 expression 41,80 (Data not shown). The organoids also reproduced key genes involved in left-right asymmetry under all conditions, such as... PITX2 , PRRX2 , LEFTY1 and PRRX1 81-84 (Data not shown). Additionally, organoids exhibited proliferation markers under all conditions, such as... MKI67 , PCNA , AURKB and CDK1 The high upregulation indicates that significant growth and remodeling are still underway on day 34 of differentiation. 85-87 (Data not shown). Cells in the first heart field (FHF) and second heart field contribute to the expansion of the linear cardiac tubes and subsequent chamber formation, and are important for proper cardiac morphogenesis. 88 Multiple FHF and SHF biomarkers were observed in organoids under all conditions. 89,90 (Data not displayed). For all conditions, HAND1 , HAND2 , TBX5 and HCN4 Upregulated in both the VCM and ACM clusters. For each condition, ISL1 Upregulated in the VCM and ACM clusters, as well as the CC cluster. Additionally, for all conditions, outflow markers such as... RSPO3 91 and WNT5A 92,93 Upregulated in the PEDC, ACM, VCM, and SC clusters (data not shown).

[0129] These analyses were expanded to begin at day 45 of gestation (GD45) and span from week 5 to week 13 of gestation. 42 Starting with the human embryonic heart, using publicly available data from the Human Cell Atlas project. 41 To compare human heart organoids with developing human hearts ( Figures 10A to 10C Based on their time in culture, human heart organoids should most closely resemble the human fetal heart at GD45 or 6 to 7 weeks of gestation. Figure 10A These scRNAseq datasets were integrated, and a high degree of overlap was found between the cell type annotations and those presented by the Human Cell Atlas project. Figure 10B In this dataset, atrial and ventricular cardiomyocytes, anterior epicardial-derived cells (named fibroblast-like, smooth muscle, and epicardial-derived cells in the Human Cell Atlas Project dataset), endothelial cells, and epicardial cells showed high clustering across datasets. Interestingly, valvular cells mapped closely to capillary endothelial cells, the stromal cells of this invention mapped closely to immune cells, and conduction cells showed no clear mapping correlation, even though conduction cell clusters exhibited gene expression profiles similar to cardiac neural crest clusters in the Human Cell Atlas Project dataset. 41 Then, these datasets were used to compare gene expression profiles at the single-cell level. Figure 10C and Figures 11A to 11F Using the top 1000 differentially expressed genes from each dataset of the VCM, ACM, PEDC, and EPC mapping regions, a high degree of similarity between organoids from each condition and those from the embryonic heart was shown. Figure 10CThe control and EMM2 / 1 organoids clustered closely around the embryonic heart at week 6, while the MM and EMM1 organoids clustered more closely around the embryonic heart at weeks 7–9. This suggests that the MM and EMM1 maturation strategies accelerate the developmental transcriptome in organoids at a rate exceeding that of the conventional developmental paradigm, compared to the control and EMM2 / 1 organoids (which are consistent with the expected developmental stage). Individual gene expression levels within the clusters of embryonic and human heart organoids were also assessed, and high similarity was shown across all major clusters. Figures 11A to 11F ).

[0130] To complement the above scRNA-seq analysis, dot plots describing the mean and percentage expression of differentially expressed genes defined by key lineages for each developmental induction condition were created, illustrating the cellular complexity of heart organoids obtained on day 34. Figure 3A As has been shown before. 13–15,17,94 The high cellular complexity of organoids drives self-organization and intercellular communication. Computational analysis was performed on intercellular communication networks of key genes identified in organoids. Multiple complex receptor-ligand communication pathways were identified in the obtained human heart organoids under each condition. Figure 3B The receptor-ligand network included JAG1-NOTCH1, PDGFR, IGF2-IGF2R, INSR, and VEGF. Gene ontology (GO) analysis was also performed targeting biological process terms corresponding to the most differentially expressed genes contributing to the ontology of each cluster, as well as the most shared genes for each cluster across all four conditions (data not shown). To further investigate the intercellular communication network, scRNA-seq data were used to highlight key receptor-ligand pairs from each maturation condition (data not shown). This data highlights the ability and sensitivity of the obtained organoids to respond to a variety of developmental maturation stimuli surrounding intercellular communication paradigms.

[0131] Mitochondrial maturation and oxidative metabolism of human heart organoids under developmental induction conditions

[0132] In early-developing humans, the heart relies heavily on glycolysis for energy. As it continues to grow, its dependence on glycolysis decreases, and it shifts to fatty acid oxidation for the majority of its energy expenditure. 28,30,95-97 Therefore, we sought to determine the effects of the disclosed developmental induction conditions, and in particular EMM2 / 1, on mitochondrial growth and metabolic transcriptional activity in cardiac organoids. Real-time mitochondrial content in cardiac organoids at day 30 of culture was visualized by adding a mitochondrial-permeable fluorescent MitoTracker. Figure 4AControl organoids showed few and scattered mitochondria, while EMM2 / 1 organoids had the best mitochondrial content (abundance, morphology) under all conditions. Figures 4A to 4B Compared to the control, the increasing trends in mitochondrial content in MM, EMM1, and EMM2 / 1 organoids (fold changes of 1.73 ± 0.10, 2.60 ± 0.11, and 3.10 ± 0.18, respectively) were quantified, indicating that mature organoids have increasingly higher aerobic respiration capacity and actively respond to maturation stimuli. Figure 4B TEM revealed high-magnification details of the presence of mitochondria in organoids cultured for 30 days. Figure 4C The mitochondrial size of the control organoids was similar to that of the mitochondria on day 15. Figure 4D However, compared to the control organoids, the mitochondrial size in MM, EMM1, and EMM2 / 1 organoids was significantly increased. At different time points from day 20 to day 30 of organoid culture, qRT-PCR was used to investigate the differential gene expression of the following two key OXPHOS genes in cardiac metabolic maturation: PPARGC1A (The main regulator of mitochondrial biogenesis) 98 )and CPT1B (Key rate-limiting fatty acid transporter) 99,100 () Figure 4E Compared to the control group, CPT1B Expression increased 1.5-fold on day 30 under EMM2 / 1 conditions, while it decreased approximately 1-2-fold in MM and EMM1 organoids. From day 21 to day 25, expression in EMM2 / 1 organoids... PPARGC1A The levels were up to 2.5 times higher than the control, and by day 30, the increase ended with a 1.5-fold higher fold. From day 21 to day 25, expression in MM and EMM1 organoids also showed an increase relative to the control, but not as high as in EMM2 / 1. By day 30, expression in EMM1 organoids remained similar to the control, while MM and EMM2 / 1 organoids showed 1.2-fold and 1.7-fold higher levels, respectively. On day 30, under EMM2 / 1 conditions, compared to the control, CPT1B The expression of MM increased by 1.5 times; however, for EMM1 organoids, the expression in MM remained similar or decreased.

[0133] To investigate real-time metabolic parameters, the organoids under each condition were measured using the Agilent Seahorse MitoStress Test. Figure 4F Compared to the control, organoids under the EMM2 / 1 condition exhibited basal respiration (…). Figure 4G ), maximum breathing ( Figure 4H) and percentage of spare respiratory capacity () Figure 4I The significant increase in ) is closely related to the enhanced metabolism present in EMM2 / 1 organoids as previously shown in mitochondrial and metabolic data.

[0134] These findings were supported by scRNAseq data, which revealed that key genes involved in cardiac metabolism were upregulated in organoids from MM, EMM1, and EMM2 / 1 conditions. Figure 4J The key genes included: CKMT2, encoding mitochondrial creatine kinase, which is important for metabolic efficiency and involved in cardiac maturation; NMRK2, involved in cardiac maturation and lipid metabolism, and activated under high-energy conditions; and KLF9, a gene associated with adipogenesis and cardiac metabolic maturation. These genes were largely upregulated in the ACM and VCM clusters. Gene expression data from the ACM and VCM clusters were then used to identify a broader set of metabolic biomarkers as the organoids developed under different conditions. Key metabolic genes were found to be expressed at significantly higher levels in organoids under EMM2 / 1 conditions than in controls, including those involved in fatty acid metabolism, amino acid metabolism, TCA cycle, and mitochondrial dynamics. Figure 4K Furthermore, Pathview was used to perform computational transcriptome analysis and mapping of the KEGG metabolic pathway. 101,102 (Data not shown). Consistent with other metabolic data, EMM2 / 1 organoids showed decreased glycolytic complex activity (data not shown) and increased mitochondrial respiratory complex activity (data not shown), indicating progressive developmental maturation. Overall, these results demonstrate that EMM2 / 1 organoids reproduce important aspects of cardiac metabolism in vitro, reminiscent of fetal heart development at similar stages.

[0135] Developmental induction conditions promote the progressive electrophysiological maturation of human heart organoids.

[0136] The presence and activity of the cardiac conduction system (including specific ion channels and membrane receptors, such as those surrounding calcium, potassium, and sodium currents) represents the action potential of cardiomyocytes. 103-105 Key elements of fetal heart development 106,107 This study aimed to characterize the functionality of cardiac organoids under developmental induction conditions using electrophysiological and immunofluorescence markers. The calcium transient activity of individual cardiomyocytes within human cardiac organoids on day 30 was assessed using the membrane-permeable dye Fluo-4. Figure 5A Organoids under all conditions exhibited different and regular calcium transient activities, with varying peak amplitudes and action potential frequencies. Figures 5B to 5C Compared to EMM1 and EMM2 / 1 organoids, control and MM organoids exhibited smaller peak amplitudes, suggesting less robust contraction. Figure 5B Furthermore, they also exhibited a similar pulsating frequency of approximately 1.5 Hz. At this stage, EMM1 organoids exhibited an abnormally high pulsating frequency of the heart (approximately 2.5 Hz), while EMM2 / 1 organoids exhibited a pulsating frequency of approximately 1 to 1.5 Hz. Figure 5C In general, and except for the EMM1 organoid, the pulsation rates of development-induced organoids showed consistency with those of early human embryos at GD45. 108,109 (60 to 80 beats / minute). Calcium trajectories from organoids under all conditions were reproducible. Figure 12 A).

[0137] The overall electrophysiological activity encompassing cardiomyocyte action potentials involves the complex coordination of various ion currents (e.g., calcium, potassium, and sodium) and supporting channels (e.g., ranotide receptors). Multiple electrophysiological genes in cardiac organoids (including...) were investigated. RYR , ATP2A2 , SCN5A , KCNJ2 and KCNH2 The expression levels of ) were analyzed, and robust expression patterns were found in the ACM and VCM clusters under all conditions. Figure 5D Compared to the control, the expression levels of all genes under the EMM2 / 1 condition showed a slight to moderate increase. Notably, for all maturation conditions, especially under the EMM2 / 1 condition, KCNJ2 The expression of [specific component] was significantly increased compared to the control. Another very important ion channel is generated by the gene [specific component]. KCNH2 Encoded hERG channel 110–112 Mutations and perturbations in this channel can lead to a shortening or prolongation of the QT interval. 111,113-115 Furthermore, the interaction between drugs and this channel can lead to arrhythmias, representing a key bottleneck related to drug discovery and development. 116,117 Within organoids KCNH2 The expression level was high in both the ACM and VCM clusters under all conditions.

[0138] Furthermore, the voluntary control of the cardiac conduction system through adrenergic signal transduction plays a significant role in physiological function. 118-120 Furthermore, this forms the basis for a series of CVD conditions, from heart failure and hypertension to arrhythmias. 121,122 Key β-adrenergic receptor genes encoding β-adrenergic receptors 1 and 2. ADRB1 and ADRB2 The presence of [a specific substance] was identified in organoids under every condition (data not shown). Although ADRB2 It is expressed in both the ACM and VCM clusters under each condition, but ADRB1It was shown to be expressed in the ACM and VCM clusters under MM, EMM1, and EMM2 / 1 conditions, but only in the ACM cluster under control conditions. (Compared to...) ADRB1 and ADRB2 , ADRB3 The expression of this is rare, which is consistent with cardiac physiology. 41,123-125 .

[0139] To investigate the temporal dynamics of key ion channels using the inventors' developmental maturation strategy, calcium ( ) was assessed using qRT-PCR from day 20 to day 30 of organoid culture. ATP2A2 ),sodium( SCN5A ) and potassium ( KCNJ2 ) level of transport proteins ( Figure 5E Compared to the control, ATP2A2 expression was increased under all conditions, with EMM2 / 1 showing the most significant 4-fold upregulation on days 25 and 30. From day 21 to day 30, for all conditions, SCN5A Expression was upregulated in all organoids. Notably, MM and EMM2 / 1 organoids showed a 3-fold increase at day 30, compared to the control organoids which showed only a 2-fold increase. Compared to the control, KCNJ2 Expression steadily decreased under EMM1 conditions, with MM organoids showing upregulation at day 30 and EMM2 / 1 showing upregulation throughout culture (compared to control).

[0140] Voltage activity in control and EMM2 / 1 cardiac organoids was investigated using the potentiometric dye di-8-ANEPPS, and unique action potentials indicating the presence of specialized atrial-like and nodular-like cells were identified within individual cardiomyocytes. Interestingly, ventricular-like action potentials were observed only in EMM2 / 1 organoids. Figure 5F ).

[0141] Furthermore, the appropriate excitation-contraction coupling, as well as depolarization and repolarization, of cardiomyocytes depend on specialized invaginations (transverse tubules) of the sarcolemma, indicating cardiomyocyte maturation. 126,127 The presence of transverse tubules in human heart organoids on day 30 was assessed by caveolin-3 immunofluorescence imaging. Figure 5G ), and under each condition, the myosarcoma (TNNT2) within the organoid + Transverse tubes were found between and around the [unclear], and the increased transverse tube density under EMM2 / 1 conditions was quantified. Figure 5H Fluorescently labeled wheat germ lectin (WGA) was also used to evaluate transverse tubes in human heart organoids on day 30 (data not shown), and similar results were found indicating a significantly increased transverse tube density in EMM2 / 1 organoids. The presence of the potassium channel KCNJ2 was also assessed by confocal microscopy. Figure 5IKCNJ2 was observed under each condition. + Spots, which showed a 2-fold increase compared to the control under EMM2 / 1 conditions ( Figure 5J This supports previous data showing increased levels of KCNJ2 transcripts in EMM2 / 1 organoids. To investigate the temporal dynamics of key ion channels by applying a developmental maturation strategy, calcium ( ) was assessed using qRT-PCR from day 20 to day 30 of organoid culture. ATP2A2 ), potassium ( KCNJ2 ) and sodium ( SCN5A ) level of transport proteins ( Figure 11A Compared to the control group, ATP2A2 Expression was increased under all conditions, with EMM2 / 1 showing the most significant 7-fold upregulation. Compared to the control, KCNJ2 Expression steadily decreased under EMM1 conditions, with MM organoids showing upregulation at day 30, and EMM2 / 1 showing upregulation throughout culture. SCN5A Expression was upregulated under MM conditions on days 25 and 30, while EMM2 / 1 was also upregulated on day 30 (relative to control). EMM1 expression remained consistent with control until day 25 and was downregulated on day 30. In summary, these data demonstrate that well-developed organoid platforms (particularly the EMM2 / 1 strategy) produce organoids that reproduce important electrophysiological aspects of cardiac development, physiology, and disease.

[0142] Developmental induction promotes the emergence of anterior epicardial organs and the formation of atrial and ventricular chambers through self-organization. become.

[0143] It has been shown that developmentally induced heart organoids exhibit improved cellular, biochemical, and functional properties compared to their control counterparts, and display a variety of features similar to the GD45 human fetal heart. However, previous attempts at heart organoids (including previous work) have been hampered by limitations. 15-7 The production of anatomically relevant cardiac structures and morphology was largely lacking. Given the significant changes observed by applying the EMM2 / 1 strategy, it was decided to characterize the morphological changes occurring under these improved conditions. Organoids were harvested on day 30 of culture, and WT1 (anterior epicardial and epicardial cells) and TNNT2 (cardiomyocytes) were stained. Figure 6A Organoids under each developmental induction condition showed TNNT2. + and WT1 + The cells were consistent with previous observations. 15 This indicates the presence of a widespread population of epicardial and cardiomyocytes within the organoids. Evaluation of both the surface and internal planes of the organoids revealed that, under all conditions, the organoids exhibited [something] via WT1. + and TNT2+ Two distinct "compartments" of cell labeling. TNT2 + Cells densely accumulate in the lower compartments, forming a thick myocardial wall, while also existing in a less dense arrangement in WT1. + In the upper region directly beneath the cell. WT1 was found in EMM2 / 1. + Cells densely covered the outer surface of the budding region, while present in dispersed, sparsely distributed clusters on the surface of the lower region. These staining patterns were not observed under control, MM, or EMM1 culture conditions. All WT1 cells under maturation conditions were... + and TNT2 + The area of ​​the chamber was quantified. Figures 6B to 6C TNT2 was found in MM and EMM1 organoids. + The chamber area was not different from the control, but the EMM2 / 1 organoids showed a 1.54-fold increase in area compared to the control. Additionally, WT1 was found in the MM organoids. + The chamber area was not different from the control, while the EMM1 and EMM2 / 1 organoids showed an increase of 1.77 and 1.98 (fold change), respectively. These data indicate that the organoids underwent significant morphological changes leading to highly specific and reproducible cellular organization under all conditions, including the emergence of organoids with advanced myocardial dual-chamber morphology and anterior epicardial poles.

[0144] Further examination revealed that ventricular myosin (MYL2) and atrial myosin (MYL7), which respectively indicate ventricular and atrial cardiomyocyte subsets, are largely spatially restricted, particularly in EMM2 / 1 organoids. Figure 6D All organoids expressed MYL7 in most parts of the organoid, but expression was stronger in the upper ventricle of EMM2 / 1, indicating an atrial-like ventricle. In control and MM conditions, the organoids exhibited MYL2 in multiple locations, not limited to the polar ends of the organoid or any particular ventricle. In another aspect, organoids under EMM1 and EMM2 / 1 conditions showed MYL2. + The significantly enhanced staining and histification levels indicate that MYL2 is confined to a polar terminal of the organoid, and that the EMM2 / 1 organoid displays MYL2. + The area increased by 5.5 times. Figure 6E This indicates the formation of ventricular-like chambers. These findings are particularly interesting because in these EMM2 / 1 organoids, the anterior epicardial region lies directly above the atrial chambers, and the ventricular chambers are located on the opposite side of the organoid. Overall, this organization in the formation of cardiac tubes is comparable to the anterior-posterior axis pattern present in the uterus (see schematic diagram). Figure 7A ).

[0145] To further investigate the ventricular and atrial-like chamber identities in human heart organoids, additional atrial and ventricular chamber markers NR2F2 (atrium) and MYL3 (ventricle) were stained. Figure 6F EMM2 / 1 organoids showed a marked, enhanced separation between the two chambers, while control organoids showed greater overlap between the two proteins. Figure 6G This indicates that EMM2 / 1 organoids possess a higher degree of specialization and maturity in compartmental development. Notably, these results were reproduced in two other PSC lines: BYS0111 (iPSC) and H9 (ESC). Figure 13A Although for reproducibility and comparison purposes, L1 control and EMM2 / 1 organoids are shown again (). Figure 13B However, the control group BYS0111 organoids showed similar overlap between NR2F2 and MYL3, while the EMM2 / 1 BYS0111 organoids showed clear separation between NR2F2 and MYL3. Figure 13A and 13C ), of which MYL3 + Cells protruded from the thick myocardial wall under EMM2 / 1 conditions. Compared with EMM2 / 1 H9 organoids, control H9 organoids showed significantly reduced expression of both NR2F2 and MYL3, with EMM2 / 1 H9 organoids showing significantly reduced expression of NR2F2. + and MYL3 + Obvious separation of chambers ( Figure 13A and 13D To support these immunofluorescence results describing the potential identities of the atrial and ventricular chambers in the cardiac organoids of the present invention, gene expression patterns were investigated using scRNAseq data from the ACM and VCM clusters. Figures 6H to 6J Compared to the VCM, the ACM displays iconic atrial and ventricular identity markers such as... NR2F2 , TBX5 , NPPA and NR2F1 Increased gene expression (from source) Figure 6I Meanwhile, compared to the ACM, the VCM exhibits distinctive ventricular chamber identity markers (e.g., MYL3 , HEY2 , IRX4 and HAND1 Increased gene expression (from source) Figure 6J These results not only highlight the ability of existing cardiac organoid platforms to reproduce important structural events in cardiac development, but also underscore the ability of the EMM2 / 1 strategy to elicit different cell types, morphological features, and compartment identities across various PSC lineages.

[0146] To further investigate the morphology and contours of cardiac organoid chambers, optical coherence tomography (OCT) was used to image the organoids in real time over time. A custom-designed OCT microscopy system was employed to measure growth under developmental induction conditions and monitor the dynamics of chamber development. This system is suitable for high-content screening. 128,129 ( Figure 14A The chambers were found to initially exhibit highly dynamic behavior and merge into larger structures over time. Between days 20 and 30 of culture, the EMM2 / 1 conditions resulted in the largest internal chambers within the human cardiac organoids of this invention, typically comprising two large internal chambers, as previously observed by confocal microscopy. Figures 14B to 14C While MM organoids exhibited a single internal chamber, organoids grown under control, EMM1, and EMM2 / 1 conditions possessed multiple smaller, interconnected chambers. Control and EMM2 / 1 organoids had chambers throughout most of the organoid, while the EMM1 organoid showed chambers primarily facing one side of the organoid. These data confirm the formation of well-established cardiac chambers and further support observations regarding the role of developmental induction conditions.

[0147] The formation of the vascular system under developmental induction conditions was also evaluated. On day 30 of culture, the formation of the vascular system in endothelial cells (PECAM1+) was examined by immunofluorescence and confocal microscopy. Figures 15A to 15D Evaluation of organoids on both surface and internal planes revealed the presence of endothelial cells in the myocardial region of all organoids. Figure 15A Organoids under EMM1 and EMM2 / 1 conditions showed fewer PECAM1+ cells than control and MM organoids. Control and MM organoids exhibited robust, interconnected endothelial cell networks and were distributed throughout myocardial (TNNT2+) tissue. Figure 15B Total PECAM1+ area was quantified, and MM organoids showed no significant difference compared to control organoids, while EMM1 and EMM2 / 1 organoids had only 52% and 61% of the PECAM1+ area, respectively, compared to the control. Figure 15C High-magnification images of the organoids further revealed the morphological transition of endothelial cells in cardiomyocyte-rich regions. Figure 15B In summary, these data suggest that angiogenesis in organoids may be partially outpaced by several factors, possibly due to the timing or concentration of growth factors, under EMM1 and EMM2 / 1 conditions, and further investigation is needed to fine-tune the culture medium conditions.

[0148] The endogenous retinoic acid gradient is responsible for the spontaneous anterior-posterior heart tube pattern formation of EMM2 / 1 organoids.

[0149] The emergence of a spatially confined retinoic acid gradient originating from the posterior pole of the cardiac tube (derived from the epicardium and primitive atria) is a key developmental step in mammalian heart development. This gradient establishes the anterior-posterior axis, which provides clues to the formation of the ventricles, as well as the inflow and outflow tracts, and also helps to clearly define cardiac progenitor cells and potentially other structures. 130,131 ( Figure 7A To determine whether the cardiac tubular structures observed under EMM2 / 1 conditions (Fig. 6) truly suggest retinoic acid-mediated cardiac pattern building, Raman microscopy was performed using a microscope designed for this purpose to detect their molecular tags (data not shown). The presence of myosin, troponin T, tropomyosin, collagen I, and other relevant molecular tags was identified in the organoids under all conditions, as expected, and particularly in EMM2 / 1, due to the presence of retinoic acid. Figure 7B Retinic acid synthesis is mainly carried out by retinal dehydrogenase 2 (ALDH1A2) during embryogenesis. 131-133 Starting from day 30 of organoid culture, qRT-PCR was used to evaluate the organoids under all conditions. ALDH1A2 level ( Figure 7C Compared to the control, under EMM2 / 1 conditions... ALDH1A2 Expression was increased by approximately 2.2-fold, with no significant changes observed under MM or EMM1 conditions.

[0150] To assess the cell-specific dynamics of retinoic acid production in organoids, scRNA-seq data were used to reveal... ALDH1A2 Expression in organoids EPC, PEDC, and ACM ( Figure 7D This was consistent with the reported in vivo expression pattern. To supplement this analysis and further investigate the localization of retinoic acid production in organoids, ALDH1A2 and TBX18 (epidermal transcription factors used to label anterior epicardial organs / atrial poles) were analyzed in organoids under all conditions at day 30. 56,134,135 Immunostaining with antibodies was performed. Organoids under EMM2 / 1 conditions were found to have similar characteristics to TBX18. + The localized and polarized expression of ALDH1A2 colocalized in cells confirms that the retinoic acid gradient enabling organoid modeling originates from the anterior lateral pole / atrial pole (posterior pole of the intrauterine cardiac tube). Figure 7A , 7E And 7F). Control, MM, and EMM1 organoids did not show ALDH1A2 expression. The co-localization region between ALDH1A2 and TBX18 was quantified, and it was shown that organoids under EMM2 / 1 conditions were significantly more responsive to the induction of retinoic acid synthesis ( ). Figure 7GIt is worth noting that these results were reproduced in two other PSC series, BYS0111 and H9. Figure 16A When L1 control and EMM2 / 1 organoids were again shown for reproducibility and comparative purposes ( Figure 16B The control and EMM2 / 1 BYS0111 organoids showed similar ALDH1A2 and TBX18 expression patterns to the L1 organoids; compared with the control BYS0111 organoid, the EMM2 / 1 BYS0111 organoids showed polarized ALDH1A2. + TBX18 + The number of cells increased significantly ( Figure 16A and 16C H9 organoids showed a similar degree of reproducibility, with EMM2 / 1H9 organoids exhibiting ALDH1A2 compared to control H9 organoids. + TBX18 + Significant increase in cells ( Figure 16A and 16D Controls and EMM2 / 1 organoids from all three cell lines also showed ALDH1A2 and other important genes (e.g., MYL2 , MYL7 , WT1 and PPARGC1A Similar robust and reproducible transcriptomes ( ) Figures 17A to 17G (e.g., as determined by qRT-PCR).

[0151] To more strongly demonstrate that retinoic acid contributes to pattern building in EMM2 / 1 organoids, ALDH1A2 was inhibited using DEAB (derived from retinoic acid), and immunostaining with NR2F2 and MYL3 showed that ALDH1A2 inhibition resulted in reduced pattern building in cardiac organoids. Figures 7H to 7J Compared to untreated organoids, ALDH1A2-inhibited organoids showed MYL3... + and NR2F2 + The area decreased by 0.35-fold and 0.42-fold, respectively. This also indicates that the addition of exogenous retinoic acid did not lead to differences in pattern building, suggesting that the EMM2 / 1 organoids have endogenously produced the optimal amount of retinoic acid.

[0152] In summary, these data demonstrate the ability of EMM2 / 1 organoids to endogenously synthesize retinoic acid in a spatially restricted manner co-located with the epicardium (TBX18), a phenomenon that closely mimics the process observed in intrauterine heart development and cardiac tube pattern formation.

[0153] Ondansetron treatment during cardiac organoid development captured the congenital heart disease phenotype.

[0154] Organoids possess a unique ability to better model and study human development, organogenesis, and disease modeling on an unprecedented scale and with unprecedented precision. However, in the context of organogenesis and disease modeling, human cardiac organoids have so far been used only to model developmental perturbations in gestational diabetes-induced cardiomyopathy (Yoni), gene knockout studies (Drakhlis), developmental cryo-injury (Hofbaur), and hypertrophic and fibrotic remodeling (Meier Epicardioid). Therefore, while cardiac organoids show promise for unraveling unanswered questions surrounding cardiac organogenesis and pathology, key areas such as the study of developmental drug toxicity and broader morphological perturbations in cardiopathology remain to be explored. During pregnancy, women are commonly prescribed the antiemetic ondansetron, also known as Zofran, for off-label use; it has been reported that X% of pregnant women take ondansetron at some point during pregnancy. Despite this, ondansetron is associated with congenital heart defects and orofacial defects, but consensus in this field is divided, and well-designed studies investigating its safety are largely lacking. The difficult and unethical nature of studying human heart development and congenital heart defects represents a critical bottleneck in many aspects of research surrounding the heart. In this way, attempts are being made to investigate the role of ondansetron during the development of human heart organoids. Figures 8A to 8J Clinical data on ondansetron plasma blood levels were used to determine the relevant concentrations for t studies. 136 From day 9 to day 30, three concentrations of ondansetron were applied to cardiac organoids using the EMM2 / 1 strategy, and then on day 30, their morphology against MYL7 and MYL2 was evaluated. Figure 8A These two key myosin proteins are largely involved in heart development (source). Untreated organoids showed the morphological and pattern building of MYL7 staining throughout the entire organoid and MYL2 staining confined to one end of the organoid, reminiscent of ventricular-like chambers, as previously illustrated. MYL2 is also a protein involved in ventricular septal defects, a major cardiac defect associated with ondansetron use. Notably, MYL2 increases with increasing ondansetron concentration. + Cell counts began to decrease, especially under 10 μM and 100 μM conditions ( Figure 8A These results were quantified and shown relative to untreated MYL2. + The area was reduced to 0.55 times and 0.18 times ( Figure 8B ), while MYL7 + The area remains constant under all conditions. Figure 8CCompared to untreated organoids, those at 100 μM also showed weaker structural organization, with less defined chamber walls and looser chamber septa. To support these results, qRT-PCR was performed on organoids under all conditions, showing that MYL2 expression was reduced by 0.58-fold and 0.40-fold, respectively, at 10 μM and 100 μM compared to untreated organoids. Figure 8D In summary, these data suggest that ondansetron can interfere with key steps in heart development by inhibiting MYL2 at both the protein and transcriptional levels.

[0155] Ondansetron is associated with QT interval prolongation, a potentially fatal phenomenon. The electrophysiological effects of ondansetron on cardiac organoid development were also investigated using the potentiometric dye di-8-ANEPPS. Figures 8E to 8J The action potentials of organoids were significantly different at 10 μM and 100 μM compared to untreated conditions. Figures 8E to 8F ), indicating a decrease in frequency ( Figure 8G ), amplitude reduction ( Figure 8H ) and APD30 / 90 improved ( Figures 8I to 8J This indicates that ondansetron induces a progressive electrophysiological pathotype during heart development. However, interestingly, it shows that while ondansetron does not promote apoptosis in human heart organoids (…),… Figures 18A to 18B However, over time, ondansetron contributed to a progressive loss of cardiac activity in the cardiac organoids, with the most significant loss of cardiac activity observed at 100 μM. Figure 18C ).

[0156] In summary, this data provides unprecedented insights into the morphological and electrophysiological effects of ondansetron during human development and provides a framework for future research on the safety and efficacy of the drug during pregnancy and the pathology of congenital heart disease.

[0157] All publications, patent applications, granted patents, and other documents mentioned in this specification are incorporated herein by reference as if each individual publication, patent application, granted patent, or other document were specifically and individually indicated as incorporated herein by reference in its entirety. Definitions contained in the text incorporated by reference are excluded to the extent that they conflict with those in this disclosure.

[0158] Other implementation plans

[0159] Implementation Scheme 1. A maturation culture medium comprising: a cell growth medium containing a culture medium supplement, wherein the culture medium supplement contains one or more fatty acids, triiodothyronine (T3) growth hormone, insulin, one or more antioxidants, sugars and carnitine; one or more additional fatty acids; additional carnitine or creatine; and additional T3 growth hormone.

[0160] Implementation Scheme 2. The mature culture medium described in Implementation Scheme 1, wherein the cell growth medium comprises Roswell Park Memorial Institute (RPMI) medium; Dulbecco modified Eagle medium (DMEM); derivatives of DMEM such as Iscove modified Dulbecco medium (IMDM) or Advanced Dulbecco modified Eagle medium (ADMEM); or combinations thereof.

[0161] Implementation Scheme 3. The maturation culture medium of Implementation Scheme 1 or 2, wherein the additional carnitine or creatine is present in an amount of about 60 to 160 μM, and / or the total amount of carnitine or creatine present in the maturation culture medium is about 60 to 200 μM.

[0162] Implementation Scheme 4. The maturation culture medium of any one of Implementation Schemes 1 to 3, wherein the additional T3 growth hormone is present in an amount of about 10 to 50 nM, and / or the total amount of T3 growth hormone present in the maturation culture medium is about 10 to 60 nM.

[0163] Implementation Scheme 5. The maturation culture medium of any one of Implementation Schemes 1 to 4, wherein the one or more additional fatty acids include palmitic acid, oleic acid, linoleic acid, stearic acid, or combinations thereof.

[0164] Implementation Scheme 6. The maturation culture medium of Implementation Scheme 5, wherein the maturation culture medium comprises a total amount of about 20 to 80 μM palmitic acid, about 20 to 80 μM oleic acid and about 10 to 60 μM linoleic acid.

[0165] Implementation Scheme 7. The maturation culture medium of any one of Implementation Schemes 1 to 6, wherein the maturation culture medium contains additional carnitine, the additional carnitine including L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, or combinations thereof.

[0166] Implementation Scheme 8. The maturation culture medium of any one of Implementation Schemes 1 to 7, further comprising additional sugars, such as fructose, galactose or glucose.

[0167] Implementation Scheme 9. The maturation culture medium described in Implementation Scheme 8, wherein the additional sugar comprises glucose, for example, about 2 to 6 mM glucose.

[0168] Implementation Scheme 10. The maturation culture medium of any one of Implementation Schemes 1 to 9, further comprising additional antioxidants, such as ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid, carotene, tocopherol (vitamin E) and panthenol.

[0169] Implementation Scheme 11. The maturation culture medium of any one of Implementation Schemes 1 to 10, further comprising ascorbic acid (vitamin C), for example about 0.1 to 1 mM ascorbic acid (vitamin C).

[0170] Implementation Scheme 12. The maturation culture medium of any one of Implementation Schemes 1 to 11, further comprising growth factors, such as IGF-1 or IGF-2.

[0171] Implementation Scheme 13. The maturation culture medium of any one of Implementation Schemes 1 to 12, further comprising IGF-1 or IGF-2, for example, about 10 to 100 ng / mL.

[0172] Implementation Scheme 14. The maturation culture medium of any one of Implementation Schemes 1 to 13, wherein the maturation culture medium does not contain extracellular matrix material and / or exogenous retinoic acid.

[0173] Implementation Scheme 15. A method for maturing early embryonic human heart organoids into mature human heart organoids, the method comprising contacting the early embryonic human heart organoids with a maturation culture medium comprising: a cell growth medium containing a culture medium supplement, wherein the culture medium supplement comprises one or more fatty acids, triiodothyronine (T3) growth hormone, insulin, one or more antioxidants, sugars and carnitine; one or more additional fatty acids; additional carnitine or creatine; and additional T3 growth hormone.

[0174] Implementation Scheme 16. The method of Implementation Scheme 15, wherein the cell growth medium comprises Roswell Park Memorial Institute (RPMI) medium; Dulbecco modified Eagle medium (DMEM); derivatives of DMEM such as Iscove modified Dulbecco medium (IMDM) or Advanced Dulbecco modified Eagle medium (ADMEM); or combinations thereof.

[0175] Implementation Scheme 17. The method of Implementation Scheme 15 or 16, wherein the additional carnitine or creatine is present in an amount of about 60 to 160 μM, and / or the total amount of carnitine or creatine present in the maturation culture medium is about 60 to 200 μM.

[0176] Implementation Scheme 18. The method of any one of Implementation Schemes 15 to 17, wherein the additional T3 growth hormone is present in an amount of about 10 to 50 nM, and / or the total amount of T3 growth hormone present in the maturation medium is about 10 to 60 nM.

[0177] Implementation Scheme 19. The method of any one of Implementation Schemes 15 to 18, wherein one or more additional fatty acids include palmitic acid, oleic acid, linoleic acid, stearic acid, or combinations thereof.

[0178] Implementation Scheme 20. The method of Implementation Scheme 19, wherein the maturation culture medium comprises about 20 to 80 μM palmitic acid, about 20 to 80 μM oleic acid and 10 to 60 μM linoleic acid.

[0179] Implementation Scheme 21. The method of any one of Implementation Schemes 15 to 20, wherein the maturation culture medium comprises additional carnitine, said additional carnitine including L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, or combinations thereof.

[0180] Implementation Scheme 22. The method of any one of Implementation Schemes 15 to 21, wherein the maturation culture medium further comprises additional sugars, such as fructose, galactose or glucose.

[0181] Implementation Scheme 23. The method of Implementation Scheme 22, wherein the additional sugar comprises glucose, for example, about 2 to 6 mM glucose.

[0182] Implementation Scheme 24. The method of any one of Implementation Schemes 15 to 23, wherein the maturation culture medium further comprises additional antioxidants, such as ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid, carotene, tocopherol (vitamin E) and panthenol.

[0183] Implementation Scheme 25. The method of any one of Implementation Schemes 15 to 24, wherein the maturation culture medium further comprises ascorbic acid (vitamin C), for example about 0.1 to 1 mM ascorbic acid (vitamin C).

[0184] Implementation Scheme 26. The method of any one of Implementation Schemes 15 to 25, wherein the maturation culture medium further comprises a growth factor, such as IGF-1 or IGF-2.

[0185] Implementation Scheme 27. The method of any one of Implementation Schemes 15 to 26, wherein the maturation culture medium further comprises IGF-1 or IGF-2, for example, about 10 to 100 ng / mL.

[0186] Implementation Scheme 28. The method of Implementation Scheme 15 or Implementation Scheme 25, wherein the early embryonic human heart organoid is formed by differentiation of human induced pluripotent stem cells (hiPSCs) and is contacted with the maturation culture medium on day 20 after day 0 of the start of the differentiation of the hiPSCs.

[0187] Implementation Scheme 29. The method described in Implementation Scheme 15, wherein

[0188] - From day 20 to day 26, the early embryonic human heart organoids were contacted with the maturation culture medium, which also contained additional antioxidants, such as ascorbic acid; additional sugars, such as glucose; and growth factors, such as IGF-1.

[0189] - From day 26 to day 30, early embryonic human heart organoids were contacted with the maturation culture medium, which also contained additional antioxidants, such as ascorbic acid; additional sugars, such as glucose, and did not contain IGF-1; and

[0190] The maturation culture medium was replaced on day 26.

[0191] Implementation Scheme 30. The method of Implementation Scheme 29, wherein a portion of the maturation culture medium from day 20 to day 26 is contacted with an early embryonic human heart organoid from day 26 to day 30.

[0192] Implementation Scheme 31. The method of Implementation Scheme 15, wherein the contact occurs for 9, 10, 11, 12 or more than 12 days, preferably 10 days.

[0193] Implementation Scheme 32. The method of any one of Implementation Schemes 15 to 31, wherein the maturation culture medium in contact with the early embryonic human heart organoid is replaced with fresh maturation culture medium approximately every 48 hours.

[0194] Implementation Scheme 33. The method of any one of Implementation Schemes 15 to 32, wherein no exogenous retinoic acid and / or extracellular matrix material is added.

[0195] Implementation Scheme 34. The method of any one of Implementation Schemes 15 to 33, wherein the mature human heart organoid comprises one or more of the following:

[0196] (i) Endogenous retinoic acid;

[0197] (ii) At least two cardiac chambers, one atrium and one ventricle;

[0198] (iii) Anterior epicardial organs; and

[0199] (iv) The anterior-posterior heart tube pattern was established.

[0200] Implementation Scheme 35. The method of any one of Implementation Schemes 15 to 34, wherein the mature human heart organoid is capable of beating.

[0201] Implementation Scheme 36. A method for maturing early embryonic human heart organoids into mature human heart organoids, the method comprising contacting the early embryonic human heart organoids with one or more maturation culture media, the maturation culture media comprising:

[0202] (1) Approximately 97% RPMI 1640 medium; approximately 2% medium supplement; approximately 1% penicillin and streptomycin; approximately 52.5 μM palmitic acid; approximately 43.95 μM oleic acid; approximately 26 μM linoleic acid; approximately 132.2 μM L-carnitine; and approximately 33.01 nM T3 hormone; or

[0203] (2) Approximately 97% glucose-free RPMI 1640 medium; approximately 2% medium supplement; approximately 1% penicillin-streptomycin; approximately 52.5 μM palmitic acid; approximately 43.95 μM oleic acid; approximately 26 μM linoleic acid; approximately 132.2 μM L-carnitine; approximately 33.01 nM T3 hormone; approximately 0.4 mM ascorbic acid; and approximately 4 mM glucose; or

[0204] (3) Approximately 97% glucose-free RPMI 1640 medium; approximately 2% medium supplement; approximately 1% penicillin and streptomycin; approximately 52.5 μM palmitic acid; approximately 43.95 μM oleic acid; approximately 26 μM linoleic acid; approximately 132.2 μM L-carnitine; approximately 33.01 nM T3 hormone; approximately 0.4 mM ascorbic acid; approximately 4 mM glucose; and approximately 50 ng / mL IGF-1.

[0205] Implementation Scheme 37. Mature human heart organoids, which are produced by any one of Implementation Schemes 15 to 36.

[0206] Implementation Scheme 38. The mature human heart organoid of Implementation Scheme 37, wherein the mature human heart organoid comprises one or more of the following:

[0207] (i) Endogenous retinoic acid;

[0208] (ii) At least two cardiac chambers, one atrium and one ventricle;

[0209] (iii) Anterior epicardial organs; and

[0210] (iv) The anterior-posterior heart tube pattern was established.

[0211] Implementation Scheme 39. The mature human heart organoid described in Implementation Scheme 37 or 38, wherein the mature human heart is capable of beating.

[0212] References

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Claims

1. Maturation culture medium, which includes: Cell growth medium; B-27™ Culture Medium Supplement; One or more additional fatty acids, wherein the maturation medium contains a total amount of 20 μM to 80 μM palmitic acid, 20 μM to 80 μM oleic acid and 10 μM to 60 μM linoleic acid; Additional carnitine or creatine, wherein the additional carnitine or creatine is present in an amount of 60 µM to 200 µM, and / or wherein the total amount of carnitine or creatine present in the maturation culture medium is 60 µM to 200 µM. The additional triiodothyronine hormone, wherein the additional triiodothyronine hormone is present in an amount of 10 nM to 60 nM, and / or wherein the total amount of triiodothyronine hormone present in the maturation culture medium is 10 nM to 60 nM. Additional sugars, wherein the additional sugars are present in an amount of 1 mM to 10 mM, and / or wherein the total amount of sugars present in the maturation medium is 1 mM to 10 mM; and The additional antioxidants, wherein the additional antioxidants are present in an amount of 0.1 mM to 1 mM, and / or wherein the total amount of antioxidants present in the maturation medium is 0.1 mM to 1 mM, the additional antioxidants include ascorbic acid, glutathione, lipoic acid, uric acid, carotene, tocopherol or panthenol.

2. The mature culture medium of claim 1, wherein the cell growth medium comprises RPMI medium, DMEM, a derivative of DMEM, or a combination thereof, wherein the derivative is selected from IMDM and ADMEM.

3. The maturation culture medium of claim 1, wherein the additional carnitine comprises L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, or a combination thereof.

4. The maturation culture medium of claim 1, wherein the additional sugar comprises fructose, galactose or glucose.

5. The maturation culture medium of claim 4, wherein the additional sugar comprises 2 mM to 6 mM glucose.

6. The maturation culture medium of claim 1, wherein the additional antioxidant comprises 0.1 mM to 1 mM ascorbic acid.

7. The maturation culture medium of claim 1, further comprising growth factors selected from IGF-1 and IGF-2.

8. The maturation culture medium of claim 7, comprising 10 ng / mL to 100 ng / mL of IGF-1 or IGF-2.

9. The maturation culture medium of claim 1, wherein the maturation culture medium does not contain extracellular matrix material and / or exogenous retinoic acid.

10. A method for maturing early embryonic human heart organoids into mature human heart organoids, the method comprising contacting the early embryonic human heart organoids with a maturation culture medium, the maturation culture medium comprising: Cell growth medium; B-27™ Culture Medium Supplement; One or more additional fatty acids, wherein the maturation medium contains 20 μM to 80 μM palmitic acid, 20 μM to 80 μM oleic acid and 10 μM to 60 μM linoleic acid; Additional carnitine or creatine, wherein the additional carnitine or creatine is present in an amount of 60 µM to 200 µM, and / or wherein the total amount of carnitine or creatine present in the maturation culture medium is 60 µM to 200 µM. The additional triiodothyronine hormone, wherein the additional triiodothyronine hormone is present in an amount of 10 nM to 60 nM, and / or wherein the total amount of triiodothyronine hormone present in the maturation culture medium is 10 nM to 60 nM. Additional sugars, wherein the additional sugars are present in an amount of 1 mM to 10 mM, and / or wherein the total amount of sugars present in the maturation medium is 1 mM to 10 mM; and The additional antioxidants, wherein the additional antioxidants are present in an amount of 0.1 mM to 1 mM, and / or wherein the total amount of antioxidants present in the maturation medium is 0.1 mM to 1 mM, the additional antioxidants include ascorbic acid, glutathione, lipoic acid, uric acid, carotene, tocopherol or panthenol.

11. The method of claim 10, wherein the cell growth medium comprises RPMI medium, DMEM, a derivative of DMEM, or a combination thereof, wherein the derivative is selected from IMDM and ADMEM.

12. The method of claim 10, wherein the additional carnitine comprises L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, or a combination thereof.

13. The method of claim 10, wherein the additional sugar comprises fructose, galactose, or glucose.

14. The method of claim 13, wherein the additional sugar comprises 2 mM to 6 mM of glucose.

15. The method of claim 10, wherein the additional antioxidant comprises 0.1 mM to 1 mM ascorbic acid.

16. The method of claim 10, wherein the maturation culture medium further comprises growth factors selected from IGF-1 and IGF-2.

17. The method of claim 16, wherein the maturation culture medium further comprises 10 ng / mL to 100 ng / mL of IGF-1 or IGF-2.

18. The method of claim 10, wherein the early embryonic human heart organoid is formed by the differentiation of hiPSCs and is contacted with the maturation culture medium on day 20 after day 0 of the onset of hiPSC differentiation.

19. The method of claim 10, wherein - From day 20 to day 26, the early embryonic human heart organoid was brought into contact with the maturation culture medium, wherein the maturation culture medium also contained growth factors, and - From day 26 to day 30, the early embryonic human heart organoid was contacted with the maturation culture medium, wherein the maturation culture medium did not contain growth factors; and The maturation culture medium was replaced on day 26.

20. The method of claim 19, wherein the growth factor is IGF-1.

21. The method of claim 19 or 20, wherein a portion of the maturation culture medium from day 20 to day 26 is contacted with the early embryonic human heart organoid from day 26 to day 30.

22. The method of claim 10, wherein the contact occurs for 9, 10, 11, 12 or more days.

23. The method of claim 10, wherein every 48 hours, the maturation culture medium that has been in contact with the early embryonic human heart organoid is replaced with fresh maturation culture medium.

24. The method of claim 10, wherein no exogenous retinoic acid and / or extracellular matrix material are added.

25. The method of claim 10, wherein the mature human heart organoid comprises: at least two heart chambers, one atrium and one ventricle; and one or more of the following: (i) Endogenous retinoic acid; (ii) Anterior epicardial organs; and (iii) The anterior-posterior heart tube pattern was established.

26. The method of claim 10, wherein the mature human heart organoid is capable of beating.

27. A method for maturing early embryonic human heart organoids into mature human heart organoids, the method comprising contacting the early embryonic human heart organoids with one or more maturation culture media, the maturation culture media comprising: (1) 97% RPMI 1640 medium; 2% medium supplement; 1% penicillin and streptomycin; 52.5 μM palmitic acid; 43.95 μM oleic acid; 26 μM linoleic acid; 132.2 μM L-carnitine; and 33.01 nM triiodothyronine; or (2) 97% glucose-free RPMI 1640 medium; 2% medium supplement; 1% penicillin-streptomycin; 52.5 μM palmitic acid; 43.95 μM oleic acid; 26 μM linoleic acid; 132.2 μM L-carnitine; 33.01 nM triiodothyronine; 0.4 mM ascorbic acid; and 4 mM glucose; or (3) 97% glucose-free RPMI 1640 medium; 2% medium supplement; 1% penicillin and streptomycin; 52.5 μM palmitic acid; 43.95 μM oleic acid; 26 μM linoleic acid; 132.2 μM L-carnitine; 33.01 nM triiodothyronine; 0.4 mM ascorbic acid; 4 mM glucose; and 50 ng / mL IGF-1.

Images