Maturation medium compositions and methods for human heart organoid maturation

Abstract

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

Description

[0001] Cross-references to related patent applications

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

[0003] Government Rights

[0004] This invention was made with government support from the National Institute of Health and the National Heart, Lung and Blood Institute under Grants HL135464 and HL151505. The government has certain rights in this invention. Technical Field

[0005] The present disclosure relates to maturation media as well as methods and models for high-throughput generation of mature human cardiac organoids, such as fetal-like human cardiac organoids, which can be further matured into more adult-like human cardiac organoids. Background Art

[0006] Cardiovascular disease (CVD), which includes conditions of the heart and blood vessels, is the leading cause of death worldwide, causing an estimated 17.9 million deaths each year. 1 Laboratory models of the heart are used to better understand the etiology and mechanisms of CVD in more detail. Several model systems are 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, many of these systems fail to fully recapitulate the complex nature of the human heart for a variety of reasons, including the lack of endogenous extracellular matrix (ECM) and non-cardiomyocyte cardiac cell types, as well as the lack of physiological morphology and cellular organization. 8,9 Additionally, animal models have unique non-human physiological, metabolic, electrophysiological, and pharmacokinetic characteristics that often fail to accurately predict human-related responses. 8,9 Therefore, these systems and methods are not suitable for comprehensive study and modeling of human disease and physiology.

[0007] The introduction of human-relevant models is essential for discovering effective, clinically transferable solutions to CVD. Over the past decade, human induced pluripotent stem cells (hiPSCs) have been 10-12 and organoids 13,14 Technological advances have enabled advanced techniques to better simulate and study human systems with increasing precision. Recently, methods have been reported to generate human cardiac organoids from pluripotent stem cells. Due to their cellular complexity and physiological relevance, these methods have enabled the study of human cardiac development and disease in a dish. 15-19 However, these systems still fail to recapitulate the late embryonic human heart and important aspects of human heart development (e.g., anterior-posterior patterning, coronary vascularization) and lack important cell populations that contribute to cardiac architecture (e.g., neural crest).

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

[0009] Overall, there is a critical need to develop more mature and sophisticated in vitro model systems for studying human cardiac development and disease pathology. Summary of the invention

[0010] This section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features.

[0011] Provided herein is a maturation medium comprising a cell growth medium containing a medium supplement comprising one or more fatty acids, triiodothyronine (T3) growth hormone, insulin, one or more antioxidants, sugars and carnitine. The maturation medium also comprises one or more other fatty acids, other carnitine or creatine and other T3 growth hormone.

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

[0013] In another embodiment, provided are mature human cardiac organoids produced by the methods described herein.

[0014] Further embodiments, including particular aspects of the embodiments summarized above, will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figures 1A to 1H . Developmental induction methods for improved modeling of human cardiac organoid development. Figure 1A Schematic diagram describing the differentiation protocol used to generate human cardiac organoids and the culture medium conditions for the four maturation strategies (control, MM, EMM1, and EMM2 / 1). Figure 1B are bright field images of organoids throughout the 30-day culture period. Two representative organoids per condition are shown (data represent 23 to 24 organoids per condition). Scale bar = 400 μm. Figure 1C Quantification of long and short diameters of organoids at day 30 of culture for each maturation strategy (n=7 to 8 organoids per condition). Data are presented as mean±sem. Figure 1D Quantification of organoid area at day 30 of organoid culture for each maturation strategy (n=7 to 8 organoids / condition in two independent experiments). Data are presented as violin plots, including all points. Data are presented as violin plots, including all points. Figure 1E Quantification of the percentage of organoids visibly beating under bright field microscopy in each condition from 5 different organoid batches (n=22 to 24 organoids / condition in five independent experiments). Figure 1F TEM images showing sarcomeres, myofibrils (M), and I bands (arrows) in organoids at day 15 and organoids from each maturation condition at day 30 (n=4 organoids / each condition). Scale bar=1 μm. Figure 1G is quantification of sarcomere length in TEM images. Data are presented as mean ± sem (n = 4 organoids / each condition). One-way ANOVA with Brown-Forsythe and Welch multiple comparison tests. Figure 1HIs the mRNA expression of key sarcomeric genes involved in cardiomyocyte maturation at 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 to day 20. Values=mean±sem.

[0016] Figures 2A to 2D .Single-cell RNA sequencing of human cardiac organoids reveals distinct cardiac cell populations. Figure 2A UMAP projection of k-means clustering of single-cell RNA-seq data from organoids at day 34 for each condition. Cluster identifiers are in the legend below. Figure 2B is the quantification of the percentage of total cell count for each cluster. The color of the area corresponds to the Figure 2A The colors that exist in the legend. Figure 2C is a differential expression heat map showing the top 10 differentially expressed genes of all clusters. Figure 2D is a feature diagram showing the key marker genes for each cluster. The color intensity represents the relative value of gene expression.

[0017] Figures 3A to 3B Cluster identity and intercellular communication networks highlight the importance of self-organization in developing cardiac organoids. Figure 3A is a dot plot of differentially expressed genes in each cluster under each condition. The color indicates the average expression level in all cells, and the size of the circle indicates the percentage of cells in a particular cluster that express the corresponding gene. Figure 3B is a visualization of the intercellular ligand-receptor communication network under each condition. The color of the clusters (outside) matches the color of the UMAP projection. The ligands are shown in blue stripes and the receptors are shown by red stripes. The arrows in the figure show the pairing from the ligand to the receptor.

[0018] Figures 4A to 4K Human cardiac organoids develop increasingly mature metabolic profiles following developmental induction conditions. Figure 4A is the labeling of mitochondria in human cardiac organoids at day 30 in 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 is a quantification of the mitochondrial area surrounding each individual nucleus (n=6 organoids / each condition, n=50 to 70 measurements / each condition). Values=mean±sem, one-way ANOVA with Brown-Forsythe and Welch multiple comparison tests. Figure 4CTEM images showing mitochondria in day 15 organoids and day 30 organoids from each maturation condition (n=4 organoids / each condition). Yellow arrows indicate mitochondria, LD=lipid droplets, Gg=glycogen granules. Scale bar=1 μm. Figure 4D is quantification of mitochondrial area from TEM images. Values ​​= mean ± sem, one-way ANOVA with Brown-Forsythe and Welch multiple comparison tests (n = 4 organoids / each condition, n = 40 to 144 measured mitochondria / each condition). Figure 4E is the mRNA expression of metabolic genes PPARGC1A and CPT1B at day 20 to 30 of culture for each condition (n=8 organoids / each condition in three independent experiments). Data are shown as log2 fold change normalized to day 20. Values=mean±sem. Figure 4F are oxygen consumption rate measurements from the Agilent Seahorse XFe96 metabolic stress test assay for all conditions (n=8 organoids / condition in two independent experiments). Values=mean±sem. Figure 4G Quantification from oxygen consumption rate measurements for basal respiration (n=8 organoids / each condition in two independent experiments). Figure 4H Quantification from oxygen consumption rate measurements for maximal respiration (n=8 organoids / condition in two independent experiments). Fig. 4I Quantification from oxygen consumption rate measurements for spare respiratory capacity (n=8 organoids / condition in two independent experiments). Figure 4J is a feature graph showing key metabolic genes upregulated in VCM and ACM clusters. The color intensity represents the relative value of gene expression of each gene. Figure 4K is the expression heatmap of key metabolic genes in the VCM and ACM clusters under each condition. Data are shown as log2 fold change and normalized for each column (for each gene).

[0019] Figures 5A to 5I . Developmentally inductive conditions promote progressive electrophysiological maturation in human cardiac organoids. Figure 5A is a representative calcium transient trace from day 30 human cardiac organoids for each condition (n=12 organoids / condition in three independent experiments). Traces represent data from a single cardiomyocyte in a human cardiac organoid. Additional traces are in Fig. 10A Shown in. Figure 5BQuantification of peak amplitudes of calcium transient traces from each condition (n=12 organoids / each condition in three independent experiments). A minimum of 2 regions and 16 peaks per organoid were quantified and averaged. Values=mean±sem. Figure 5C Quantification of calcium transient peak frequency for each condition (n=12 organoids / each condition in three independent experiments). A minimum of 2 fields and 16 peaks per organoid were quantified and averaged. Values=mean±sem. Figure 5D is a feature graph showing key electrophysiological genes differentially expressed in VCM and ACM clusters under each condition. The color intensity represents the relative value of gene expression for each gene. Figure 5E is the mRNA expression of key electrophysiological genes at day 20 to day 30 of culture for each condition (n=8 organoids / day / per condition / per gene / in three independent experiments). Data are shown as log2 fold change normalized to day 20. Values=mean±sem. Fig. 5F Representative voltage traces of organoids in EMM2 / 1 and control conditions depicting atrial-like, nodal-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 Representative immunofluorescence images of caveolin-3 puncta within TNNT2+ regions in organoids for each condition (n=15 organoids / each condition in three independent experiments). Green=caveolin-3, red=TNNT2, blue=DAPI. Scale bar=20 μm. Figure 5H is for each condition, from Figure 5G Quantification of caveolin-3 positive area / per 400 μm2 of images presented in (n=15 organoids / each condition in three independent experiments). Data are shown as fold change normalized to control. Values=mean±sem, one-way ANOVA with Brown-Forsythe and Welch multiple comparison tests. Fig.5I Representative immunofluorescence images of KCNJ2+ puncta within TNNT2+ regions in organoids for each condition (n=14 organoids / each condition in 3 independent experiments). KCNJ2=green, TNNT2=red, DAPI=blue. Scale bar=20 μm. Figure 5J Is from Fig.5I Quantification of the total number of KCNJ2+ puncta per condition for images presented in (n=14 organoids / per condition in 3 independent experiments). Data are shown as fold change normalized to control. Values=mean±sem, one-way ANOVA with Dunnett's multiple comparison test.

[0020] Figures 6A to 6J . Developmental induction promotes the emergence of the proepicardial organ and the formation of distinct atrial and ventricular chambers through self-organization. Fig. 6A Representative surface and internal immunofluorescence images of single day 30 organoids in all conditions showing WT1 (green), TNNT2 (red) and DAPI (blue). Three organoids per condition are shown (n=12 to 15 organoids / per condition in two independent experiments). Scale bar=200 μm. Figure 6B Is from Fig. 6A Quantification of TNNT2+ chamber area in each condition (n=12 to 15 organoids / condition in two independent experiments). Values ​​are expressed as fold change normalized to control. Values=mean±sem, one-way ANOVA with Dunnett's multiple comparison test. Figure 6C Is from Fig. 6A Quantification of WT1+ chamber area in each condition in images presented in (n=12 to 15 organoids / condition in two independent experiments). Values ​​are expressed as fold change normalized to control. Values=mean±sem, one-way ANOVA with Dunnett's multiple comparison test. Fig.6D Representative surface and internal immunofluorescence images of single day 30 organoids in all conditions showing MYL2 (green), MYL7 (red) and DAPI (blue). Three organoids per condition are shown (n=13 organoids / per condition in three independent experiments). Scale bar=200 μm. Fig. 6E Is from Fig.6D Images presented in Quantification of MYL2+ area in each organoid under each condition (n=9 to 13 organoids / condition in three independent experiments). Values ​​are expressed as fold change normalized to control. Values=mean±sem, one-way ANOVA with Dunnett's multiple comparison test. Fig. 6F Representative immunofluorescence images of single day 30 organoids in all conditions showing NR2F2 (green), MYL7 (red) and DAPI (blue). Three organoids per condition are shown (n=12 organoids / per condition in three independent experiments). Scale bar=200 μm. Figure 6G Is from Fig. 6F Quantification of colocalization (Pearson's coefficient) between NR2F2 (green) and MYL3 (red) in the images presented in. Values ​​= mean ± sem, unpaired t-test. Figure 6His a feature map highlighting the VCM and ACM clusters for further use in 6I and 6J. Fig.6I is a feature graph showing the signature atrial chamber identity genes differentially expressed in the ACM cluster. The color intensity represents the relative value of gene expression for each gene. Figure 6J is a feature graph showing ventricular chamber identity genes differentially expressed in the VCM cluster. The color intensity represents the relative value of gene expression for each gene.

[0021] Figures 7A to 7J Endogenous retinoic acid gradients are responsible for the establishment of spontaneous anterior-posterior heart tube patterning. Fig. 7A Schematic diagram depicting the formation of the cardiac tube in utero, highlighting the localization and intensity of the retinoic acid gradient from the anterior (arterial pole) to the posterior (venous pole) of the primitive heart tube. Figure 7B Figure 2 is a plot of Raman spectral intensity of organoids from all four maturation conditions at day 30 of culture. Peaks of interest, such as DNA, cardiac troponin, and retinoic acid are labeled. Data presented are representative of n=3 organoids / each condition. Figure 7C is the mRNA expression of ALDH1A2 in all conditions at day 30 (n=7 organoids / each condition in two independent experiments). Data are presented as log2 fold change normalized to control. Values=mean±sem, one-way ANOVA with Dunnett's multiple comparison test. Fig.7D is a featured graph showing ALDH1A2 expression. The color intensity represents the relative value of gene expression. Fig. 7E Representative immunofluorescence images of individual day 30 organoids for all conditions showing ALDH1A2 (green), TBX18 (red), and DAPI (blue). Three organoids per condition are shown (representative of n=22 to 24 organoids / condition in three independent experiments). Scale bar=200 μm. Figure 7F yes Figure 7C High magnification images of EMM2 / 1 and control organoids shown in , showing ALDH1A2 (green), TBX18 (red) and DAPI (blue). The yellow squares in the top images (scale bar = 200 μm) represent the high magnification area. Scale bar = 50 μm. Figure 7G Is from Fig. 7E Images presented in Figure 2. ALDH1A2 expression in organoids under each condition. + TBX18 + Quantification of area (n=22 to 24 organoids / condition in three independent experiments). Data are shown as fold change normalized to control. Values=mean±sem, one-way ANOVA with Dunnett's multiple comparison test. Figure 7HIt is a representative immunofluorescence image of a single EMM2 / 1 organoid on day 30 after exposure to deoxyaminobenzaldehyde (DEAB), retinoic acid (RA) or no treatment (untreated). MYL3 (pink), NR2F2 (green) and DAPI (blue) staining were performed. Two organoids under each condition are shown (n=9 organoids / each condition in two independent experiments). Scale bar=200 μm. Fig.7I yes Figure 7H Quantification of NR2F2+ area of ​​organoids presented in (n=9 organoids / condition in two batches of organoids). Values=mean±sem, one-way ANOVA with Dunnett's multiple comparison test. Figure 7J yes Figure 7H Quantification of MYL3+ area of ​​organoids presented in (n=9 organoids / condition in two batches of organoids). Values=mean±sem, one-way ANOVA with Dunnett's multiple comparison test.

[0022] Figures 8A to 8J .Cardiac organoids treated with ondansetron mimic the morphological and electrophysiological phenotypes of congenital heart disease. Fig. 8A It is a representative immunofluorescence image of a single EMM2 / 1 organoid on the 30th day after exposure to different concentrations of ondansetron (1 μM, 10 μM or 100 μM) or no treatment (untreated) on the 9th to 30th day of culture. MYL2 (green), MYL7 (red) and DAPI (blue) staining were carried out. Three organoids of each condition are shown (in two independent experiments, n=12 organoids / each condition). Scale bar=200 μm. Figures 8B to 8C They are from Fig. 8A Quantification of MYL2+ area and MYL7+ area for each condition (n=12 organoids / each condition in two independent experiments) for images presented in . Data are shown as fold change normalized to untreated. Values=mean±sem, one-way ANOVA with Dunnett's multiple comparison test. Fig.8D is the mRNA expression of MYL2 in all conditions at day 30 (n=6 organoids / each condition in two independent experiments). Data are shown as log2 fold change normalized to control. Values=mean±sem, one-way ANOVA with Dunnett's multiple comparison test. Figures 8E to 6F Representative voltage traces of organoids, showing three voltage traces from independent organoids per condition (n=6 organoids per condition in two independent experiments). Figure 8G To J is from Figures 8E to 8F Traces presented in Quantification of voltage traces from a single organoid per condition (n=6 organoids / per condition in two independent experiments) showing frequency, amplitude, APD30 and APD90, respectively. Values=mean±sem, one-way ANOVA with Dunnett's multiple comparison test.

[0023] Figures 9A to 9C . Longitudinal assessment of apoptosis in all maturation conditions. Fig.9A Representative fluorescence images of day 20 and day 30 organoids from each condition showing FlipGFP fluorescence signal (n=12 organoids / each condition / day). Fig. 9B Representative fluorescence images of day 30 EMM2 / 1 organoids after 48 hours of exposure to doxorubicin showing FlipGFP fluorescence signal (n=6 organoids). Fig. 9C Is from Fig.9A Quantification of fluorescence intensity of images presented in (n=12 organoids / per condition / day). Data are shown as fold change normalized relative to day 20. Values=mean±sem, matched two-way ANOVA with Tukey's multiple comparison test.

[0024] Figures 10A to 10C .The transcriptomic organoid landscape reveals similarities to the developing human heart in vivo. Fig. 10A is a schematic diagram depicting the comparison of the timeline of embryonic heart development and human cardiac organoid development. Fig. 10B The UMAP projections show the human embryonic heart and human cardiac organoid scRNAseq datasets. The cluster naming of (Asp et al, Cell, 2019) is retained from the original text. The cluster identifiers and colors of the human cardiac organoid dataset are retained ( Figure 2A ) as shown. Fig. 10C yes Fig. 10B PCA plot of the dataset presented in .

[0025] Figures 11A to 11F Human cardiac organoids share key gene expression with the embryonic human heart in some cardiac cell types. Figures 11A to 11F Each feature graph shows the key marker genes for each cluster in the Asp 2019, Cui 2019, and human cardiac organoid datasets for the following corresponding clusters: atrial cardiomyocytes; ventricular cardiomyocytes; cells of proepicardial origin; epicardial cells; valvular cells; and conductance cells. The color intensity represents the relative value of gene expression.

[0026] Fig.12A. Calcium measurements show reproducibility in independent organoids. Fig.12 A is a representative calcium transient trace from day 30 human cardiac organoids per condition (n=12 organoids / per condition in three independent experiments). Data from 6 independent organs are shown. Traces represent data from single cardiomyocytes within human cardiac organoids.

[0027] Figures 13A to 13D .Formation of ventricular and atrial chambers was reproducible in three hPSC lines. Fig.13A Representative immunofluorescence images of single day 30 organoids from cell lines L1, BYS0111, and H9 in both control and EMM2 / 1 conditions showing NR2F2 (green), MYL3 (red), and DAPI (blue). Three organoids per condition for each cell line are shown (n=12 organoids / per condition in three independent experiments for L1 organoids; n=11 organoids / per condition in two independent experiments for BYS0111 organoids; n=12 organoids / per condition in two independent experiments for H9 organoids). Figures 13B to 13D They are from Fig.13A Quantification of colocalization (Pearson coefficient) between NR2F2 (green) and MYL3 (red) in the images of L1, BYS0111, and H9 organoids presented in. Values ​​= mean ± sem, unpaired t-test.

[0028] Figures 14A to 14C Real-time longitudinal imaging by optical coherence tomography reveals large, interconnected chambers within human cardiac organoids. Fig.14A Schematic diagram of the custom optical coherence tomography (OCT) system used for imaging human cardiac organoids. Fig. 14B are longitudinal OCT cross-sectional scans of human cardiac organoids at day 20 to 30 in each condition. Scale bar = 500 μm. Images shown are representative of 6 organoids per condition. Fig. 14C Is from Fig.14A The images presented are volumetric visualization of 3D segmentation of OCT scans, which reveal the temporal dynamics of chamber identity under each condition.

[0029] Figures 15A to 15D .Perturbing endothelial cell positioning and morphology through enhanced developmental maturation strategies. Fig.15A Representative immunofluorescence images using DAPI (blue), TNNT2 (red), and PECAM1 (green) from the surface and interior of day 30 organoids in each condition (n=7 to 8 organoids / each condition in two independent experiments). Scale bar=200 μm. Fig. 15B Is from Fig.15ARepresentative day 30 organoid immunofluorescence images with DAPI (blue), TNNT2 (red), and PECAM1 (green) of images presented in (n=7 to 8 organoids / condition in two independent experiments). Images are shown as maximum intensity projections. Scale bar=200 μm. Fig. 15C yes Fig. 15B Quantification of PECAM1+ area presented in (n=7 to 8 organoids / condition in two independent experiments). Data are shown as log fold change normalized to control. Values=mean±sem, one-way ANOVA with Dunnett's multiple comparison test. Fig.15D Representative high magnification immunofluorescence images of organoids under each condition using DAPI (blue), TNNT2 (red), and PECAM1 (green) (n=7 to 8 organoids / each condition in two independent experiments). Scale bar=50 μm. The top image is a representative low magnification organoid under each condition (scale bar=200 μm), where the yellow square represents the area of ​​high magnification. Images are shown as maximum intensity projections.

[0030] Figures 16A to 16D By using the EMM2 / 1 developmental maturation strategy, the emergence of the ALDH1A2+ anterior epicardial pole was reproducible in three hPSC lines. Fig.16A 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 per condition for each cell line are shown (n=22 to 24 organoids / condition in three independent experiments for L1 organoids; n=12 organoids / condition in two independent experiments for BYS0111 organoids; n=12 organoids / condition in two independent experiments for H9 organoids). Scale bar=200 μm. Figures 16B to 16D Each is from Fig.16A Images of L1, BYS0111, and H9 organoids presented in Figure 2. ALDH1A2 expression within the organoids in each condition + TBX18 + Quantification of area. Data are shown as fold change normalized to control. Values ​​= mean ± sem, unpaired t-test.

[0031] Figures 17A to 17G .Transcriptional profiles of key genes in human cardiac organoids are reproducible in three hPSC lines. Figures 17A to 17GFigure 5. mRNA expression of selected genes from L1, BYS0111, and H9 organoids at day 30 under control and EMM2 / 1 conditions, respectively (n=7 to 14 organoids / each condition in three independent experiments for L1 organoids; n=8 organoids / each condition in two independent experiments for BYS0111 organoids; n=8 organoids / each condition in two independent experiments for H9 organoids). Fig.17A It is the mRNA expression of MYL2 gene. Fig. 17B It is the mRNA expression of MYL7 gene. Fig. 17C It is the mRNA expression of MYH6 gene. Fig.17D It is the mRNA expression of MYH7 gene. Fig.17E It is the mRNA expression of the ALDH1A2 gene. Fig.17F It is the mRNA expression of PPARGC1A gene. Figure 17G is the mRNA expression of WT1 gene. For each cell line, data are shown as log2 fold change normalized to control. Values ​​= mean ± sem, unpaired t-test.

[0032] Figures 18A to 18C Apoptosis is not a contributing factor to ondansetron-induced cardiac organoid malformations. Fig.18A Is a representative fluorescence image of EMM2 / 1 organoids at day 30 from each condition after ondansetron treatment from day 9 to day 30 and doxorubicin treatment from day 28 to day 30, which shows FlipGFP fluorescence signal (in two independent experiments, each: for ondansetron group, n = 12 organoids / each condition; for doxorubicin group, n = 6 organoids). Scale bar = 200 μm. Fig.18B Is from Fig.18A Quantification of fluorescence intensity of images presented in . Data are shown as fold change normalized to untreated. Values ​​= mean ± sem, one-way ANOVA with Games-Howell multiple comparisons. Fig.18C is quantification of the percentage of beating organoids during the entire treatment period for each ondansetron condition from day 0 to day 30 (n=23 to 24 organoids per condition in two independent experiments). DETAILED DESCRIPTION

[0033] Exemplary embodiments are provided so that the present disclosure will be comprehensive, and the scope will be fully conveyed to those skilled in the art. Many specific details such as examples of specific compositions, components, devices and methods are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be adopted, and the exemplary embodiments can be embodied in many different forms, and both should not be construed as limiting the scope of the present disclosure. In some exemplary embodiments, known processes, known device structures and known technologies are not described in detail.

[0034] The terms used herein are only for the purpose of describing specific exemplary embodiments and are not intended to be restrictive. As used herein, unless the context clearly indicates otherwise, nouns modified by quantifiers may also be intended to include plural forms. The terms "include", "contain", "include" and "have" are inclusive, and therefore indicate the presence of the features, elements, compositions, steps, integers, operations and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or their groups. Although the open term "include / include" is understood as a non-limiting term for describing and claiming multiple embodiments set forth herein, in some aspects, the term may alternatively be understood as a more restrictive and constrained term, such as "consisting of" or "consisting essentially of". Thus, for any given embodiment in which compositions, materials, components, elements, features, integers, operations, and / or process steps are recited, the present disclosure also specifically includes embodiments consisting of or consisting essentially of such recited compositions, materials, components, elements, features, integers, operations, and / or process steps. In the case of "consisting of," alternative embodiments exclude any additional compositions, materials, components, elements, features, integers, operations, and / or process steps, while in the case of "consisting essentially of," any additional compositions, materials, components, elements, features, integers, operations, and / or process steps that materially affect the basic and novel properties are excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and / or process steps that do not materially affect the basic and novel properties may be included in such an embodiment.

[0035] Unless expressly identified as an order of performance, any method steps, processes, and operations described herein should not be construed as necessarily requiring their performance in the particular order discussed or illustrated. It should also be understood that additional or alternative steps may be employed unless otherwise specified.

[0036] Although the term first, second, third etc. can be used to describe multiple steps, elements, components, regions, layers and / or parts in this article, unless otherwise noted, these steps, elements, components, regions, layers and / or parts should not be limited by these terms. These terms can only be used to distinguish a step, element, component, region, layer or part from another step, element, component, region, layer or part. Unless the context clearly indicates, terms such as "first", "second" and other numerical terms do not mean order or sequence when used in this article. Therefore, without departing from the teaching of exemplary embodiments, the first step, element, component, region, layer or part discussed below can be referred to as the second step, element, component, region, layer or part.

[0037] For ease of description, spatially or temporally relative terms such as "before", "after", "inside", "outside", "below", "beneath", "lower", "above", "upper", etc. may be used herein to describe the relationship of one element or feature to another element or feature as illustrated in the accompanying drawings. The spatially or temporally relative terms may be intended to encompass different orientations of the device or system in use or operation in addition to the orientation depicted in the accompanying drawings.

[0038] Throughout this disclosure, numerical values ​​represent approximate measurements or limits to ranges to cover minor deviations from a given value and embodiments with approximately the values ​​mentioned and those with the exact values ​​mentioned. Except for the working examples provided at the end of the specific embodiments, all numerical values ​​of parameters (e.g., quantities or conditions) in this specification (including the appended claims) should be understood to be modified by the term "about" in all cases, regardless of whether "about" actually appears before the numerical value. "About" means that the numerical value allows some slight imprecision (some of which are close to the accuracy of the value; approximately or reasonably close to the value; almost). If the imprecision provided by "about" is not otherwise understood in this ordinary sense in the art, the "about" used herein at least represents the changes that can be caused by the common methods of measuring and using such parameters. For example, "about" may include the following changes: 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 aspects, optionally less than or equal to 0.1%.

[0039] Additionally, disclosure of ranges includes disclosure of all values ​​within the entire range and further divided ranges, including the endpoints and sub-ranges stated within the range.

[0040] Example 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 moving on to induced pluripotent stem cell-derived cardiac tissue (e.g., cardiomyocytes) and tissue engineering approaches (3D printing, biomaterials). The latest advance in human cardiac models is cardiac organoids generated from pluripotent stem cells15-17 ,115 However, these systems do not possess the true complexity of the in utero human heart due to a lack of maturity and fidelity to human physiology, morphology, cellular organization, and functionality. These shortcomings severely limit the scope of relevance of traditional model systems.

[0042] Therefore, it is necessary to develop and physiologically relevant models of adult hearts such as from hiPSC, which can be realized in an effective and high-throughput manner. Provided herein are maturation culture media and methods for producing mature human cardiac organoids using developmental induction strategies inspired by biological steps in utero in a repeatable and high-throughput manner, to produce human cardiac organoids with higher anatomical complexity and physiological relevance (accompanying early pregnancy fetal development). Maturation culture media and methods described herein can advantageously reproduce in vitro cardiac development, and by inducing progressive mitochondria and metabolic maturation, electrophysiological maturation, improved morphological complexity and cellular complexity, and by endogenous retinoic acid signaling and self-organization to reproduce the front-back heart tube pattern, so that organoids can obtain a high level of complexity and anatomical relevance.

[0043] A. Maturation medium

[0044] Provided herein is a maturation medium that can be used, for example, to induce early embryonic human cardiac organoids to develop into mature human cardiac organoids or to mature early embryonic human cardiac organoids into mature human cardiac organoids. The maturation medium comprises a cell growth medium and a medium supplement.

[0045] Some examples of suitable cell growth media include, but are not limited to: Roswell Park Memorial Institute (RPMI) medium, such as RPMI 1640, which includes its various formulations such as containing D-glucose, without D-glucose, containing L-glutamine, without L-glutamine, containing sodium bicarbonate, without sodium bicarbonate, HEPES modification, etc.; Dulbecco's Modified Eagle's Medium (DMEM), which includes its various formulations such as high glucose, low glucose, containing HEPES, etc.; derivatives of DMEM, such as Iscove's Modified Dulbecco's Medium (IMDM) or Advanced Dulbecco's Modified Eagle's Medium (ADMEM); or combinations thereof. These media are available from ThermoFisher Scientific, Sigma-Aldrich, Millipore Sigma, etc., and the same or same trade name of the culture medium indicates the same medium composition regardless of the manufacturer.

[0046] In any embodiments, the cell growth medium can be present in the maturation medium in an amount of 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%, based on the total volume of the maturation medium.

[0047] The culture medium supplement may include one or more fatty acids, triiodothyronine (T3) growth hormone, insulin, one or more antioxidants, sugars and carnitine. For example, the culture medium supplement may include one or more of the following: biotin, L-carnitine, corticosterone, ethanolamine, D (+) -galactose, glutathione (reduced), linoleic acid, linolenic acid, oleic acid, pipecolic acid, progesterone, putrescine, retinol 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 TM Supplement preparations (available from Thermo Fisher Scientific), such as B-27 TM Supplement (50×), serum-free; B-27 TM Supplements, Insulin Removed; B-27 TM Plus Supplement (50×); B-27 TM Supplement (50×), minus vitamin A; B-27 TM Supplement (50×), antioxidant removed; etc.

[0048] In any embodiment, the culture medium supplement may be present in the maturation medium in an amount of 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 from 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%, based on the total volume of the maturation medium.

[0049] Additionally or alternatively, the maturation culture medium may also include antibiotics. Any suitable antibiotics for cell culture may be included. For example, antibiotics may include amphotericin B, ampicillin, cephalosporin, dihydrostreptomycin, gentamicin sulfate, penicillin-streptomycin, kanamycin sulfate, lincomycin hydrochloride, neomycin sulfate, nystatin, paromomycin sulfate, penicillin-G, phenoxymethylpenicillin acid, polymyxin B sulfate, spectinomycin, streptomycin, tetracycline hydrochloride, tylosin tartrate or a combination thereof. It is expected that antibiotics may be optional herein, and do not need to be present in the maturation culture medium. When present in the maturation medium, the antibiotic can be present in an amount of 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 from 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%, based on the total volume of the maturation medium.

[0050] In any embodiment, the maturation medium further comprises 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 components" refers to components that are present in a certain amount in the medium supplement in addition to the components or types of components already present in the medium supplement. For example, "additional T3 growth hormone" refers to T3 growth hormone present in the maturation medium in addition to the T3 growth hormone present in the medium supplement.

[0051] Suitable fatty acid includes but is not limited to palmitic acid, oleic acid, linoleic acid, stearic acid or its combination.For example, the maturation medium may include palmitic acid, oleic acid and linoleic acid.In any embodiment, oleic acid and linoleic acid may be present in a culture medium supplement, and in a further amount 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 negligible amounts.

[0052] In any embodiment, one or more additional fatty acids, individually or in combination, can be present in the maturation medium in an amount of 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 the oleic acid present in the medium supplement, the maturation medium may also include about 20 to 60 μM of oleic acid, and in addition to the linoleic acid present in the medium supplement, the maturation medium may also include about 10 to 40 μM of linoleic acid.

[0053] Additionally or alternatively, one or more fatty acids, alone or in combination, may be present in the maturation medium in an amount of about 10 μM or more, about 20 μM or more, about 40 μM or more, about 50 μM or more, about 100 μM or less, about 90 μM or less, about 80 μM or less, about 70 μM or less, or about 60 μM or less; 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 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.

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

[0055] In any embodiment, the additional carnitine and / or creatine can each be present in the maturation medium in an amount of 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 medium supplement, the maturation medium can also include about 60 μM to about 160 μM carnitine.

[0056] Additionally or alternatively, carnitine and / or creatine may each be present in the maturation medium in an amount of 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 carnitine and / or creatine.

[0057] In any embodiment, the additional T3 growth hormone can be present in the maturation medium in an amount of 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 culture medium supplement, the maturation medium may also include about 10 nM to about 50 nM of T3 growth hormone.

[0058] Additionally or alternatively, the T3 growth hormone can be present in the maturation medium in a total amount of about 10 nM or more, about 15 nM or more, about 20 nM or more, about 25 nM or more, about 50 nM or less, about 45 nM or less, about 40 nM or less, about 35 nM or less, or about 30 nM or less; 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 T3 growth hormone.

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

[0060] In any embodiment, additional sugar (e.g., glucose) can be present in the maturation medium in an amount of 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 amount of the aforementioned additional sugar may correspond to the total amount of the sugar (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, the maturation medium may contain ascorbic acid (vitamin C), which may be a supplement to other antioxidants present in the medium supplement.

[0062] In any embodiment, the antioxidant (e.g., ascorbic acid (vitamin C)) can be present in the maturation medium in an amount of 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 nM; or about 0.1 mM to about 1 mM, about 0.1 mM to about 0.8 mM, about 0.1 mM to about 0.6 mM, about 0.1 mM to about 0.5 mM, about 0.2 mM to about 0.8 mM, or about 0.2 mM to about 0.6 mM. It is contemplated herein that the amount of the aforementioned additional antioxidant may correspond to the total amount of the antioxidant (e.g., ascorbic acid (vitamin C)) present in the maturation medium.

[0063] Additionally or alternatively, the maturation medium may further comprise a growth factor, such as IFG-1, IFG-2, or a combination thereof. In any embodiment, the growth factor may be present in the maturation medium in an amount of 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 various aspects, the maturation medium can comprise a cell growth medium as described herein (e.g., RPMI1640), a medium supplement 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 carnitines as described herein (e.g., L-carnitine) or creatine, additional T3 growth hormone, and optionally antibiotics as described herein (e.g., penicillin streptomycin). For example, the maturation medium can comprise about 97% RPMI 1640 medium; about 2% medium supplements (e.g., B-27 TM supplement); about 1% penicillin-streptomycin; about 52.5 μM palmitic acid in total; about 43.95 μM oleic acid in total; about 26 μM linoleic acid in total; about 132.2 μM L-carnitine in total, and about 33.01 nM T3 hormone in total.

[0065] In another aspect, the maturation medium can comprise a cell growth medium as described herein (e.g., RPMI1640), a medium supplement 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 carnitines as described herein (e.g., L-carnitine) or creatine, 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 can comprise about 97% RPMI 1640 medium (without D-glucose); about 2% medium supplements (e.g., B-27TM supplement); about 1% penicillin-streptomycin; about 52.5 μM palmitic acid in total; about 43.95 μM oleic acid in total; about 26 μM linoleic acid in total; about 132.2 μM L-carnitine in total, about 33.01 nM T3 hormone in total, about 0.4 mM ascorbic acid in total; and about 4 mM glucose in total.

[0066] In another aspect, the maturation medium can comprise a cell growth medium as described herein (e.g., RPMI1640), a medium supplement 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 carnitines as described herein (e.g., L-carnitine) or creatine, additional T3 growth hormones, 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 comprise about 97% RPMI1640 medium (without D-glucose); about 2% medium supplements (e.g., B-27 TM supplement); about 1% penicillin-streptomycin; about 52.5 μM palmitic acid in total; about 43.95 μM oleic acid in total; about 26 μM linoleic acid in total; about 132.2 μM L-carnitine in total, about 33.01 nM T3 hormone in total, about 0.4 mM ascorbic acid in total; about 4 mM glucose in total and about 50 ng / mL IGF-1.

[0067] In any embodiment, the maturation medium may not include exogenous retinoic acid and / or extracellular matrix materials, such as hydrogels (e.g. As used herein, "exogenous retinoic acid" refers to retinoic acid that is not naturally present in or produced by human cardiac organoids.

[0068] B. Maturation Method

[0069] Also provided herein is a method for maturing early embryonic human cardiac organoids into mature human cardiac organoids. These methods may also be referred to as developmental induction strategies. The method includes contacting early embryonic human cardiac organoids with a maturation culture medium described herein. "Early embryonic human cardiac organoids" used herein refers to a three-dimensional body having an inner surface containing myocardial tissue and an outer surface containing epicardial tissue, and exhibits both the first and second heart regions and cardiac chambers. Early embryonic cardiac organoids may also include at least one chamber or microchamber defined by myocardial tissue, and the at least one chamber or microchamber may be lined with endocardial cells. Epicardial tissue (including epicardial cells) may be configured on at least a portion of the surface. Early embryonic cardiac organoids may also include cardiac fibroblasts and endothelial vasculature, and may beat. As used herein, "mature human cardiac organoids" encompass fetal-like human cardiac organoids and adult human cardiac organoids, which may be obtained by a longer culture period. "Fetal-like human cardiac organoids" refer to organoids with clearly defined atrial chambers and ventricular chambers. For example, a fetal-like human cardiac organoid can be considered comparable to a fetal human heart from about gestational day 45 to about gestational day 90. "Adult human cardiac organoid" refers to a cardiac organoid with well-defined atrial and ventricular chambers and a metabolic and electrophysiological profile characteristic of an adult heart (e.g., fatty acid metabolism, presence of atria, ventricles, and conduction of action potentials).

[0070] In any embodiment, early embryonic human cardiac organoids can be formed by methods known in the art. For example, early embryonic human cardiac organoids can be formed from the differentiation of human induced pluripotent stem cells (hiPSCs), as described in International Patent Publication No. WO2021 / 257812, which is hereby incorporated by reference in its entirety.

[0071] After hiPSC differentiation begins, early embryonic human cardiac organoids can be contacted with a maturation medium described herein. The differentiation of hiPCS can begin at day zero (0). After the zeroth day of hiPSC differentiation, early embryonic human cardiac organoids can be contacted with a maturation medium described herein. For example, on any day from the 15th day to the 25th day after the zeroth day of hiPSC differentiation, early embryonic human cardiac organoids can be contacted with a maturation medium described herein. In various aspects, on the 20th day after the zeroth day of hiPSC differentiation, embryonic human cardiac organoids can be contacted with a maturation medium described herein.

[0072] In any embodiment, the early embryonic human cardiac organoids can be contacted with the maturation medium described herein for a suitable amount of time to mature into mature human cardiac organoids. For example, the early embryonic human cardiac organoids can be contacted with 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, the early embryonic human cardiac organoids can be contacted with the maturation medium described herein for about 10 days, for example, from about the 20th day to about the 30th day after the zeroth day of hiPSC differentiation. As needed, for example, every 24 hours to 72 hours (e.g., every 24 hours, every 48 hours, every 72 hours), at least a portion of the maturation medium contacting the early embryonic human cardiac organoids can be replaced with fresh maturation medium. Fresh maturation culture medium may have a composition identical or different to the maturation culture medium replaced. It is also contemplated herein that a portion of the maturation culture medium replaced remains in contact with early embryonic human heart organoids. Alternatively, substantially all maturation culture mediums contacting early embryonic human heart organoids may be replaced with fresh maturation culture medium.

[0073] In various aspects, the early embryonic human cardiac organoid can be contacted with a maturation medium at any time, such as from about day 20 to about day 30, wherein the maturation medium comprises a cell growth medium as described herein (e.g., RPMI1640), a medium supplement 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 carnitines as described herein (e.g., L-carnitine) or creatine, additional T3 growth hormone, and optionally antibiotics as described herein (e.g., penicillin-streptomycin). For example, the maturation medium may comprise about 97% RPMI 1640 medium; about 2% medium supplements (e.g., B-27 TM supplement); about 1% penicillin-streptomycin; about 52.5 μM palmitic acid in total; about 43.95 μM oleic acid in total; about 26 μM linoleic acid in total; about 132.2 μM L-carnitine in total, and about 33.01 nM T3 hormone in total.

[0074] In another aspect, the early embryonic human cardiac organoid can be contacted with a maturation medium at any time, such as from about day 20 to about day 30, comprising a cell growth medium as described herein (e.g., RPMI1640), a medium supplement as described herein (e.g., B-27 TMSupplements), one or more additional fatty acids as described herein (e.g., palmitic acid, oleic acid, linoleic acid), additional carnitines as described herein (e.g., L-carnitine) or creatine, additional T3 growth hormones, 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 can comprise about 97% RPMI 1640 medium (without D-glucose); about 2% medium supplements (e.g., B-27 TM supplement); about 1% penicillin-streptomycin; about 52.5 μM palmitic acid in total; about 43.95 μM oleic acid in total; about 26 μM linoleic acid in total; about 132.2 μM L-carnitine in total, about 33.01 nM T3 hormone in total, about 0.4 mM ascorbic acid in total; and about 4 mM glucose in total.

[0075] In another aspect, the early embryonic human cardiac organoids can be contacted with a maturation medium at any time, e.g., day 20 to about day 30, comprising a cell growth medium as described herein (e.g., RPMI1640), a medium supplement 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 carnitines as described herein (e.g., L-carnitine) or creatine, additional T3 growth hormones, 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 comprise about 97% RPMI1640 medium (without D-glucose); about 2% medium supplements (e.g., B-27 TM supplement); about 1% penicillin-streptomycin; about 52.5 μM palmitic acid in total; about 43.95 μM oleic acid in total; about 26 μM linoleic acid in total; about 132.2 μM L-carnitine in total, about 33.01 nM T3 hormone in total, about 0.4 mM ascorbic acid in total; about 4 mM glucose in total, and about 50 ng / mL IGF-1.

[0076] It is also contemplated herein that the early embryonic human cardiac organoids may be contacted with more than one maturation medium as described herein, e.g., during day 20 to day 30. In any embodiment, the early embryonic human cardiac organoids may be contacted with a maturation medium (a first maturation medium) e.g., during day 20 to day 26, comprising a cell growth medium as described herein (e.g., RPMI 1640), a medium supplement as described herein (e.g., B-27 TM Supplement), one or more additional fatty acids as described herein (e.g., palmitic acid, oleic acid, linoleic acid), additional carnitines as described herein (e.g., L-carnitine) or creatine, additional T3 growth hormone, and further comprising 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). In addition, the embryonic human cardiac organoid can be contacted with a maturation medium (second maturation medium), for example, on day 26 to day 30, the maturation medium (second maturation medium) comprising a cell growth medium as described herein (e.g., RPMI1640), a medium supplement 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 carnitines as described herein (e.g., L-carnitine) or creatine, additional T3 growth hormone, and further comprising additional antioxidants as described herein (e.g., ascorbic acid), additional sugars as described herein (e.g., glucose), wherein the maturation medium does not comprise a growth factor as described herein, such as IGF-1. In any embodiment, a portion of the maturation medium from day 20 to day 26 (the first maturation medium) can be contacted with the early embryonic human cardiac organoid 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, instead of removing all of the first maturation medium used from day 20 to day 26, only a portion of the first maturation medium is removed and replaced with the second maturation medium.

[0077] In any embodiment, during the methods described herein, no exogenous retinoic acid and / or extracellular matrix material may be added.

[0078] Also provided are mature human cardiac organoids produced by the methods described herein.

[0079] It was found that the methods described herein utilizing the maturation culture medium described herein can advantageously produce human cardiac organoids having several unique and key features representative of the early fetal human heart, including the presence of anterior-posterior patterning with a retinoic acid gradient at the posterior pole and polar separation of the atrioventricular chambers with an epicardial layer at the anterior end. In addition, the disclosed methods can also produce human cardiac organoids having valvular cells, conduction cells, proepicardial cells, etc., as well as large hollow chambers, functional electrophysiology, and increased mitochondrial density and metabolic transcriptional profiles similar to those of a gestational human heart. For example, single-cell gene expression of key genes associated with multiple cell clusters revealed that human cardiac organoids produced by contacting early embryonic human cardiac organoids with two different maturation culture media on days 20 to 30 as described above can produce the highest similarity to a human heart developing at 6.5 weeks after conception (GD45) in vivo. 41 In addition, the strategies described herein lead to the expansion and reduction of certain cardiac cell type populations, such as atrial and ventricular cardiomyocytes and mesenchymal cell types (stromal cells), which is considered a process of fine-tuning and remodeling. Interestingly, the emergence of valvular and conducting cell types of the human cardiac organoids generated herein was observed.

[0080] The mature human cardiac organoids generated herein are significantly responsive to developmental maturation stimuli and are metabolically mature with increased mitochondrial growth, density, respiratory rate, and gene expression. These significant responses compared to traditional approaches may be the result of synergistic effects between 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 progressive electrophysiological maturation throughout the cardiac syncytium, including the complex interplay between multiple ion channels and their subtypes and depolarization through the t-tubule, comprises key aspects of cardiac development and functionality. 103-106,126,127,145 . It was found that human cardiac organoids produced by the methods described herein (e.g., by contacting early embryonic human cardiac organoids with two different maturation media at days 20 to 30 as described above) produced significant calcium transients with improved physiological mimicry (due to their increased amplitude and reduced frequency). In addition, these organoids can produce higher levels of transverse tubules, inwardly rectifying potassium ion channels, and hERG channels compared to other maturation strategies. In fact, many efforts to mature cardiomyocytes in vitro have difficulty or fail to induce transverse tubules. 146,147 and inwardly rectifying potassium channels that are still essential for establishing a low resting membrane potential 148,149Furthermore, the cardiac hERG channel represents a critical channel important for pharmacological screening because it has a high potential for arrhythmogenicity if perturbed. 116,117 Nonetheless, the summation of multiple ionic transients generates the cardiac action potential, the ultimate driver of contraction and functionality of the human heart. Furthermore, these generated organoids possess ventricular-, atrial-, and nodal-like 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 signaling events to pattern along the anterior-posterior axis, loop, and eventually form a four-chambered heart. 150,151 . It was unexpectedly found that human cardiac organoids generated by the maturation methods described herein, e.g., when early embryonic human cardiac organoids as described above were contacted with two different maturation media at day 20 to day 30, formed a two-chamber structure with cardiomyocytes, forming one chamber with an atrial identity and another chamber with a ventricular fate. The dense epicardial layer at the atrial chamber defines the anterior epicardial organoid as the posterior pole of the heart tube 152 And it was revealed that these organoids spontaneously patterned along the above-mentioned anterior-posterior axis. It was also found that this self-organization and patterning in these organoids was driven by the endogenous retinoic acid signaling gradient. It was observed that the enzyme ALDH1A2 required for retinoic acid synthesis was spatially restricted to the posterior end of the organoid and co-localized with TBX18, which is a transcription factor that determines that the anterior epicardial organoid is a functional epicardium. Therefore, in various aspects, the methods described herein can produce mature human cardiac organoids comprising one or more of the following characteristics: (i) endogenous retinoic acid; (ii) at least two chambers of the heart (e.g., the atrial chamber and the ventricular chamber); (iii) the posterior anterior epicardial pole (see Fig. 7A ); and (iv) anterior-posterior heart tube patterning (e.g., anterior-posterior patterning of the ventricular (anterior pole) and atrial (posterior) chambers) (see Fig. 7A). In addition, the method described herein can produce human heart organoids with valve cells, conduction cells, proepicardial cells, etc. similar to those of the human heart during pregnancy, as well as large hollow chambers, functional electrophysiology, and improved mitochondrial density and metabolic transcriptional profiles. 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 the 30th day of the method. Endogenous retinoic acid can be present in mature human heart organoids in a gradient. As discussed above, retinoic acid is a powerful morphogen involved in cardiac development and provides instructions for cell development and pattern building to the heart. The retinoic acid gradient can originate from and / or be located at the posterior pole (proepicardial pole / atrial pole) of the obtained human heart organoids. In addition, mature human heart organoids can be able to beat, for example, 60 to 80 times / minute. In the case of such characteristics, it can be considered that the mature human heart organoids described herein are comparable to the fetal human heart from about the 45th day of gestation to about the 90th day of gestation.

[0083] In summary, it is believed that human cardiac organoids generated by the methods described herein can recapitulate the events that occur during in utero gestation, where the anterior epicardial organoid surrounds the posterior pole of the patterned heart tube, where posterior atrial cardiomyocytes and anterior epicardial cells produce retinoic acid to form a signaling gradient that further instructs the rest of the heart tube with information for patterning and specification 41,56,131,134,152 .

[0084] Example

[0085] Example 1

[0086] Methods and Operations

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

[0088] Self-assembled human cardiac organoid differentiation. Provides a step-by-step, detailed protocol describing the generation and differentiation of human cardiac organoids 14. Briefly, hiPSCs were grown to 60% confluence in 6-well plates and dissociated using Accutase (InnovativeCell Technologies) to obtain single-cell solutions. HiPSCs were collected and centrifuged at 300g for 5 minutes and resuspended in Essential 8Flex medium containing 2μM ROCK inhibitor (Thiazovivin) (Millipore Sigma). HiPSCs were counted using a Moxi cell counter (Orflo Technologies) and seeded in round-bottom 96-well ultra-low attachment plates (Costar) at a concentration of 10,000 cells / well in a volume of 100μL on day -2. The plates were then centrifuged at 100g for 3 minutes and subsequently placed in a 37°C and 5% CO2 incubator. After 24 hours (day -1), 50μL was removed from each well and 200μL of fresh Essential 8Flex medium was added to each well to obtain a final volume of 250μL per well. Then plate is placed in 37 ℃ and 5% CO Incubator.After 24 hours (the 0th day), from each well, remove 166 μ L culture medium.Then, 166 μ L of RPMI (Gibco) (hereinafter referred to as "RPMI / B27 removing insulin") containing B27 supplement (removing insulin) supplemented with 1% penicillin-streptomycin (Gibco) containing CHIR99021, BMP4 and activin A is added to each well, to obtain the final concentration of 4 μ M CHIR99021, 36pM (1.25ng / mL) BMP4 and 8pM (1.00ng / mL) activin A. Subsequently plate is placed in 37 ℃ and 5% CO Incubator.After 24 hours (the 1st day), from each well, remove 166 μ L culture medium, and replace with 166 μ L fresh RPMI / B27 removing insulin. On the second day, 166 μL of culture medium was removed from each well and 166 μL of RPMI / B27 with Wnt-C59 (Selleck) removed from insulin was added to obtain a final concentration of 2 μM Wnt-C59 in each well. The plate was then incubated for 48 hours. On the fourth day, 166 μL was removed and replaced with fresh RPMI / B27 with insulin removed and incubated for 48 hours. On the sixth day, 166 μL was removed and replaced with 166 μL of RPMI (hereinafter referred to as "RPMI / B27") containing B27 supplement (containing insulin) and 1% penicillin-streptomycin. The plate was incubated for 24 hours. On the seventh day, 166 μL of culture medium was removed from each well and 166 μL of RPMI / B27 containing CHIR99021 was added to obtain a final concentration of 2 μM CHIR99021 in each well. The plate was incubated for 1 hour.After 1 hour, 166 μL of medium was removed from each well and 166 μL of fresh RPMI / B27 was added to each well. The plates were incubated for 48 hours. From day 9 to day 19, medium was replaced 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. Organoids were subjected to various maturation media conditions starting at day 20. The control strategy was to continue culturing in RPMI / B27 from day 20 to day 30 with standard media changes every 48 hours.

[0090] From day 20 to day 30, a maturation medium (MM) strategy was used, with MM medium used for medium replacement every 48 hours, consisting of stock 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). The preparation details of MM medium are provided in Table 1 below.

[0091] Table 1. MM medium

[0092]

[0093] From the 20th day to the 30th day, an enhanced maturation medium 1 (EMM1) strategy was used, and EMM1 medium was used for medium replacement every 48 hours. The EMM1 medium was composed of the following stock RPMI 1640 medium (Gibco) supplemented with glucose-free: 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). The preparation details of EMM1 medium are provided in Table 2 below.

[0094] Table 2. EMM1 medium

[0095]

[0096]

[0097] Adopt enhanced maturation medium 2 / 1 (enhanced maturation medium 2 / 1, EMM2 / 1) strategy from the 20th day to the 30th day, use the combination of two kinds of culture medium to carry out culture medium replacement every 48 hours.From the 20th day to the 26th day, use EMM2 culture medium, it is by being supplemented with the following storage liquid RPMI 1640 culture medium (Gibco) composition that does not contain glucose: 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), 30nM T3 hormone (Sigma), 0.4mM ascorbic acid (Thermo Fisher Scientific), 4mM glucose (Gibco) and 50ng / mL IGF-1.Continue EMM2 / 1 strategy, from the 26th day to the 30th day, use EMM1 culture medium. The formulation details of EMM2 medium are provided in Table 3 below.

[0098] Table 3. EMM2 medium

[0099]

[0100]

[0101] Organoids were collected on day 30 for analysis.

[0102] Immunofluorescence. Human cardiac organoids were transferred from round-bottom ultra-low attachment 96-well plates to 1.5 mL microcentrifuge tubes (Eppendorf) using cut 200 μL pipette tips (in order to increase the tip inner diameter without damaging the organoids). Organoids were fixed in 4% paraformaldehyde (VWR) in PBS for 30 minutes. Subsequently, the organoids were washed three times with PBS-glycine (1.5 g / L) for 5 minutes each. Then, the organoids were blocked and permeabilized overnight at 4 ° C on a thermal mixer at 300 rpm using a solution containing 0.5% BSA (Thermo Fisher Scientific), 0.5% Triton X-100 (Sigma), and 10% donkey normal serum (Sigma). Organoids were then washed three times with PBS and incubated with primary antibodies (Table 4) in a solution containing 0.5% BSA, 0.5% Triton X-100, 1% donkey normal serum in PBS (hereinafter referred to as "antibody solution") at 300 rpm at 4°C for 24 hours on a thermomixer.

[0103] Table 4. Antibodies used for immunofluorescence.

[0104]

[0105] The organoids were then washed three times with PBS for 5 minutes each. The organoids were then incubated with secondary antibodies in antibody solution (Table 4) on a thermomixer at 300 rpm at 4°C in the dark for 24 hours. The organoids were then washed three times with PBS for 5 minutes each and mounted on glass microscope slides (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 so that the organoids could retain their three-dimensionality. The organoids were transferred to a glass microscope slide using a cut 200 μL pipette tip and mounted using the clearing solution previously described. 153 FITC-conjugated wheat germ agglutinin (WGA) lectin (Sigma) was used for transverse tubule staining.

[0106] Confocal microscopy and image analysis. Immunofluorescence images were obtained using a confocal laser scanning microscope (Nikon Instruments A1 confocal laser microscope). Images were analyzed using Fiji. When comparing images in or between conditions, for each channel of the measured image, where appropriate, the pixel intensity value of the image was balanced with the pixel intensity value of the control or EMM2 / 1 condition. In order to measure organoid diameter and area, straight lines and hand-drawn tools were used respectively. In order to measure mitochondrial (MitoTracker) area, Cav-3+ area, KCNJ2+ spots, MYL2+ area, MYL7+ area, PECAM1+ area and ALDH1A2+TBX18+ area, an automatic threshold function was used. In order to measure TNNT 2+ and WT1+ chamber size, an elliptical selection tool was used, and the wall of the organoid was used as the boundary area of ​​the corresponding area to be drawn. For area measurement (low magnification image) using the entire organoid, data points were normalized to the organoid area. In order to measure FlipGFP fluorescence intensity, mean gray value was calculated. To measure the Pearson coefficient, the JaCOP colocalization plugin (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) was used. A threshold of the equalized image intensity values ​​was generated. A spatial resolution of 1.243 microns per pixel was used.

[0107] Heart organoids dissociate.Collect the organoids from each maturation strategy (control, MM, EMM1, EMM2 / 1) at the 30th day.Organoids are placed in separate 1.5mL microcentrifuge tubes (Eppendorf) respectively, dissociate and merge.Organoids are dissociated into single cell suspensions using the modified scheme of STEMdiff cardiomyocyte dissociation kit (STEMCELL Technologies).After being transferred to microcentrifuge tubes, organoids are washed with PBS, immersed in 200 μL warm dissociation medium (37 ° C), and placed on a thermomixer at 37 ° C and 300rpm, for 5 minutes.Then, collect supernatant and transfer to 15mL falcon tubes (Corning) containing 5mL corresponding culture medium (control, MM, EMM1 etc.) containing 2% BSA (Thermo Fisher Scientific).Then the dissociation medium (37 ° C) of other 200 μL warm is added back to the organoid on a thermomixer (37 ° C). The organoid dissociation culture medium solution is then gently pipetted up and down 3 to 5 times. The organoid is placed on a thermal mixer for another 5 minutes. If the organoid is still visible, the process is repeated. Once the organoid is no longer visible, the microcentrifuge tube solution is gently pipetted up and down 3 to 5 times, and its entire contents are transferred to a 15mL falcon tube containing the corresponding culture medium + 2% BSA and cells. These tubes are then centrifuged at 300g for 5 minutes. The supernatant is aspirated, and the cell pellet is resuspended in the corresponding culture medium + 2% BSA. Using a hemocytometer, viability, cell count and aggregation percentage are obtained.

[0108] Single-cell RNA sequencing. Libraries were prepared using the 10× Chromium Next GEM Single Cell 3' Kit, v3.1 and associated components. The complete library was QC'd and quantified using a combination of Qubit dsDNA HS, Agilent 4200 TapeStation HSDNA1000, and Invitrogen Collibri Library Quantification qPCR Assay. The libraries were pooled in equimolar proportions and the pool was quantified again using the Invitrogen Collibri qPCR assay. The pool was loaded onto two lanes of the Illumina NovaSeq6000SP flow cell (v1.5) and sequenced in a customized paired-end format with 28 cycles for read 1, 2 10 cycle index reads and 90 cycles for read 2. Sequencing was performed using the v1.5100 cycle NovaSeq kit. The 28 bp read 1 contains the 10× cell barcode and UMI, and read 2 is a cDNA read. The output of Real Time Analysis (RTA) was demultiplexed and converted to FastQ format using Illumina Bcl2fastq v2.20.0. After demultiplexing, the reads 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 with k-means clustering of 8 clusters and UMAP visualization. 154-156 Used to evaluate gene ontology. Pathview Web was used to generate biological pathway diagrams 101,102 Using Liana 157 and Celltalker 158 Perform cell-cell communication analysis (https: / / github.com / arc85 / celltalker). To accomplish this task, import count and cluster data from Loupe Browser into Seurat 159 Both programs use the standard Seurat findmarkers differential expression function to perform differential expression between clusters and then rank ligand and receptor pairs by significant p-value and log2 fold change. To identify ligand and receptor pairs, Liana uses the OmniPath database 160 And Celltalker uses the Ramilowski-pairs database of ligand-receptor interactions 161 .

[0109] Transmission Electron Microscopy (TEM). On the 15th and 30th days, human cardiac 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 100mM phosphate buffer and post-fixed with 1% osmium tetroxide in 100mM phosphate buffer, dehydrated in a gradient series of acetone, and infiltrated and embedded in Spurr (Electron Microscopy Sciences). 70nm thin sections were obtained with a Power Tome ultramicrotome (RMC, Boeckeler Instruments. Tucson, AZ) and post-stained with uranyl acetate and lead citrate. Images were acquired using a JEOL 1400Flash transmission electron microscope (Japan Electron Optics Laboratory, Japan) at an accelerating voltage of 100k.

[0110] Calcium imaging. Fluo-4 AM (Thermo Fisher Scientific) was used to assess calcium transient activity in human cardiac organoids. Fluo-4 AM was dissolved in DMSO according to the manufacturer's instructions. 1.5 μM Fluo-4 solution was prepared in the corresponding culture medium (control, MM, EMM1, etc.). Organoids were washed twice with RPMI 1640 basal medium, and then Fluo-4 AM was added, with a final concentration of 1 μM, and incubated at 37 ° C and 5% CO2 for 30 minutes. The organoids were then washed twice with their corresponding culture medium (control, MM, EMM1, etc.) and transferred to the chamber coverslip (Cellvis) using a cut 200 μL pipette tip. Cellvivo microscope (Olympus) was used to acquire images as image stacks at 100 frames per second in a total of 10 seconds. The sample was excited at 494 nm excitation, and 506 nm emission was collected. Fiji and Microsoft Excel were used to process data. The baseline F0 of the fluorescence intensity F was calculated using the average of the lowest 50 intensity values ​​in the obtained data set. The fluorescence change ΔF / F0 was calculated using the following equation:

[0111]

[0112] Voltage imaging. Voltage 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 corresponding culture 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 minutes. The organoids were then washed twice with their corresponding culture medium (control, MM, EMM1, etc.) and transferred to the chamber coverslip (Cellvis) using a cut 200 μL pipette tip. Images were acquired as image stacks at 100 frames per second in a total of 10 seconds using a Cellvivo microscope (Olympus) at 20 × magnification (ondansetron) or 100 × magnification (mature electrophysiology). The samples were excited at 465 nm and the emission at 630 nm was collected. Data were processed using Fiji and Microsoft Excel. The baseline F0 of the fluorescence intensity F was calculated using the average of the lowest 50 intensity values ​​in the obtained data set. The fluorescence change ΔF / F0 was calculated using the same method as for calcium imaging above. APD30 and APD90 were measured from the midpoint of the upstroke until 30% or 90% repolarization, respectively. 162 .

[0113] Real-time RT-PCR. Organoids were collected on days 20, 21, 23, 25, and 30 and stored in RNAprotect (Qiagen) at -20°C. RNA was extracted using the Qiagen RNEasy Mini kit primarily according to the manufacturer's instructions. Organoids were lysed using a bead mill 4 homogenizer (Fisher Scientific) at speed 2 for 30 seconds. 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 (ThermoFisher Scientific) was used as an amplifier for DNA intercalating dye and reaction vessels. Real-time qPCR was performed using a QuantStudio 5 real-time PCR system (Applied Biosystems) using a total reaction volume of 20 μL. Gene expression levels were normalized to HPRT1 expression in each independent sample. The double delta CT method was used to obtain log fold changes with a base of 2. At least 4 independent samples were run for each gene expression assay at each time point under each condition. The mRNA expression data are shown as log fold changes with a base of 2 relative to the control.

[0114] Mitochondrial imaging.The presence of intracellular mitochondria in human cardiac organoids is visualized using Mitotracker Deep Red FM (Thermo Fisher Scientific).Mitotracker is prepared according to the manufacturer's instructions.150nM Mitotracker is prepared in the corresponding culture medium (control, MM, EMM1, etc.).In addition, NucBlue (ThermoFisher Scientific) is used to visualize the nucleus.NucBlue (as described above) is prepared by adding 2 drops in the Mitotracker solution of 150nM per ml.Organoids are washed twice using 166 μL of RPMI 1640 basal culture medium, and then 166 μL Mitotracker is added to achieve a final concentration of 100nM, and incubated for 30 minutes at 37°C and 5% CO2.Organoids are incubated for 30 minutes at 37°C and 5% CO2.Then the organoids are washed twice using their corresponding culture medium (control, MM, EMM1, etc.), and are transferred to the chamber coverslip (Cellvis) using a cut 200 μL pipette tip. Images were acquired using a Cellvivo microscope (Olympus). Data were processed using Fiji.

[0115] Raman microscopy. Raman spectra of organoids were acquired by 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 milliseconds, and an average of 100 accumulated times were used to acquire data for each scanning position of the organoid. In order to avoid strong background signals, quartz slides (Chemglass Life Sciences, NJ, USA) were used as substrates for Raman spectra acquisition. Organoids were collected on day 30 for analysis. Optical coherence tomography. A spectral-Domain Optical Coherence Tomography (SD-OCT) system similar to previous work was used. 128,129Label-free longitudinal imaging for cardiac organoids. A superluminescent diode (EXALOS, EXC250023-00) is used as a light source, wherein the central wavelength is about 1300nm, and the 3dB spectral range is about 180nm. A spectrometer (Wasatch Photonics, Cobra 1300) based on a 2048-pixel InGaAs line scan camera (SensorsUnlimited, GL2048) is used to provide a maximum A scan rate of 147kHz. A 5× objective lens is used, and the lateral and axial resolutions measured in the tissue are about 2.83μm and about 3.04μm, respectively. From the 20th day to the 30th day, longitudinal 3D OCT imaging is performed every other day. Each 3D OCT scan includes 600 A scans / each B scan and 600 B scans. Using an exposure time of about 40 microseconds for each A scan, each organoid requires about 22 seconds for image acquisition. Eight organoids from each group are imaged and used for analysis. The medium level in each well was adjusted during imaging to reduce image artifacts and minimize light absorption. The acquired OCT images were rescaled using ImageJ to obtain isotropic pixel sizes in the xyz dimension (Schneider et al., 2012). Registration, cavity segmentation, and 3D rendering of the same organoids on different days were performed using Amira software (Thermo Fisher Scientific). The cavity and total volume inside the organoids were quantified from the segmentation data.

[0116] Ondansetron treatment.Ondansetron hydrochloride (Sigma) is prepared in DMSO at 200 μM, and further diluted in DMEM / F12 (Dulbecco's modified Eagle culture medium / nutrient mixture F-12), then aseptically filtered by 0.22 μM PVDF filter (Sigma).Ondansetron is applied to cardiac organoids at a final concentration of 1 μM, 10 μM and 100 μM in EMM2 / 1 culture medium, and applied on the 9th to 20th day of cultivation.Organoids are collected for analysis at the 30th day.

[0117] Lentiviral transduction. HEK293T (Horizon Inspired Cell Solutions) cells were transfected with Flip-GFP plasmid (VectorBuilder) and packaging plasmids pMD2 and psPAX2 using lipofectamine and Plus reagent (Thermo) to produce lentivirus. 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 cells lacking lentivirus disappeared from the wells. Surviving clones were selected, collected, re-plated and further amplified to produce FlipGFP lines.

[0118] Doxorubicin treatment. Doxorubicin hydrochloride (Sigma) was diluted to 1 mM in DMEM / F12 and applied to cardiac organoids at a final concentration of 10 μM for 48 hours on day 28 to day 30 of culture.

[0119] DEAB and retinoic acid treatment. 4-diethylaminobenzaldehyde (DEAB) (Sigma) was prepared in DMSO with 1M, further diluted to 10mM using DMSO, and then finally diluted to 1mM in DMEM / F12. Retinoic acid (RA) (Sigma) was prepared in DMSO with 1M and diluted to 100μM in DMEM / F12. The diluted solution of DEAB and RA was aseptically filtered through a 0.22μm PVDF filter (Sigma). DEAB was applied to cardiac organoids at a final concentration of 10μM. RA was applied to cardiac organoids at a final concentration of 1μM. DEAB, RA and DEAB+RA were applied to cardiac organoids cultured from the 20th to the 30th day using the EMM2 / 1 strategy. Organoids were collected for analysis at the 30th day.

[0120] Agilent Seahorse metabolic assay. Real-time extracellular flux assays were performed using the Agilent Seahorse XFe96 (Agilent). One day before the assay, 200 μL of XF calibrants were loaded into each well of the 96-well utility plate contained in the sensor box, and the sensor was immersed in a 37°C incubator without CO2 overnight. Also one day before the assay, polylysine (Sigma) was used to coat the XFe96 spheroid microplate. In brief, 100 μg / mL of polylysine was prepared in water, and 30 μL of the 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 a minimum of 30 minutes. The plate was then warmed in a 37°C incubator without CO2 for 30 minutes for 30 minutes. Finally, 100 μ L of 37° C. DMEM / F12 is added to each well of the microplate, and the microplate is returned to 37° C. without CO2 in an incubator overnight. The following steps describe the operation performed on the day of the assay in order. XFRPMI (without phenol red) (Agilent) is prepared as the basal medium for assay, and the basal medium is supplemented with 1 mM pyruvate (Agilent), 2 mM glutamine (Agilent), 11.1 mM glucose (Gibco) and 12.2 μM L-carnitine (Sigma). Using the XF RPMI prepared in this way, the drug solution from the Cell Mito Stress Test kit (Agilent) is resuspended, vortexed for 1 minute, and left to stand for 1 hour at room temperature. At this point, the poly-lysine coated XFe96 spheroid microplate was removed from the incubator and the DMEM / F12 was removed from the plate, washed once with 166 μL of prepared XF RPMI, and finally, 175 μL of prepared XF RPMI was added to each well. Day 30 organoids from each condition were then washed twice with 166 μL of prepared XF RPMI and transferred to the poly-lysine coated XFe96 spheroid microplate. The organoids were transferred to the wells using a cut p200 pipette tip. Ensure that the organoid is in the center of the well. The plate was then placed at 37°C without CO. 2 Incubator, continue 1 hour.Drug solution (oligomycin, FCCP and Rot / AA) is loaded into channel (port) A, B and C respectively.The channel concentration of oligomycin, FCCP and Rot / AA is respectively 25 μM, 20 μM and 20 μM, so that their final concentration in solution is respectively 2.5 μM, 2 μM and 2 μM.Configure this mensuration so that baseline phase runs 6 cycles, and oligomycin, FCCP and Rot / AA stage respectively run 10 cycles.Each cycle includes 3 minutes mixing, 0 minute waiting and 3 minutes measurement stage.Data are normalized to organoid area.

[0121] Statistics and reproducibility. Raw data were collected using Microsoft Excel. Graphpad Prism 9 software was used for all analyses. Data are shown as normal distributions. Statistical significance (p<0.05) was evaluated using one-way ANOVA with Dunnett or Brown-Forsyth and Welch post-test correction, or, where appropriate, unpaired t-test. All data are shown as mean ± sem. Statistical methods are specifically shown in the legends. The number of independent organoids used for each quantification and each statistical test is shown in the legends. When more than one independent experiment (plate / batch of organoids) was performed (most of the data in this manuscript), this was appropriately shown in the legends.

[0122] result

[0123] Expanded cardiac development modeling through improved developmental induction strategies

[0124] A detailed protocol for the generation of self-organized early embryonic human cardiac organoids has been described previously. 15 In brief, cardiac organoids were differentiated from hiPSC embryoid bodies to the cardiac lineage from day 0 to day 7 by a timewise 3-step Wnt pathway modulation strategy and then cultured in RPMI until day 20. 15 To examine the effects of more advanced organoid culture strategies that mimic intrauterine conditions on cardiac organoid development, day 20 early embryonic-like cardiac organoids were subjected to four different developmental induction strategies from day 20 to day 30 ( Figure 1A ). This is consistent with previous human and animal development studies. 20-24 In comparison, these strategies represent steps of increasing complexity relative to in utero 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 medium supplemented with fatty acids (oleic acid, linoleic acid, and palmitic acid at embryo-relevant concentrations). 23,24 and L-Carnitine 25 RPMI / B27 to promote the developmentally relevant shift from glucose utilization to fatty acid metabolism characteristic of the fetal human heart 26-30 The MM strategy also uses the T3 hormone, a potent activator of organ growth during embryonic development and metabolic maturation, which has been shown to stimulate cardiovascular growth. 31,32The Enhanced Maturation Medium 1 (EMM1) strategy uses the same basal 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 shift toward 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 Medium Strategy 2 / 1 (EMM2 / 1)" utilizes a combination of two different medium formulations. From day 20 to 26, EMM2 medium is used and the EMM2 medium is identical to the base composition of EMM1 supplemented with IGF-1. IGF-1 plays an important role in tissue growth and maturation during embryonic and fetal development, especially in the heart, as demonstrated in mouse and human studies. 38-40 Starting from day 26, EMM1 medium was utilized in the EMM2 / 1 strategy. The EMM2 / 1 strategy represents the most advanced conditions and mimics in utero cardiac development to the greatest extent. A more detailed description of all developmental induction strategies and the concentrations of the corresponding media preparations can be found in the Materials and Methods section above.

[0125] Cardiac organoids treated with different developmental induction strategies continued to grow and develop, with morphology changing dramatically depending on the conditions ( Figures 1B to 1D From day 0 to day 10, organoids underwent a period of rapid growth, increasing in diameter while maintaining their spheroid structure ( Figure 1B ) and continued to grow until day 30. After day 20, the organoids developed a distinct oval morphology, as observed by bright-field microscopy, elongation and deformation, and grew to a long diameter of 1000 to 1600 μm and a short diameter of 600 μm to about 1000 μm on day 30 ( Figures 1B to 1C ). Organoid area measured by bright-field microscopy revealed a similar trend in each condition, at 0.6 mm 2 Up to 0.9mm 2 ( Figure 1D By day 30 of culture, nearly 100% of organoids were observed to be beating in each condition in five independent experiments (n = 22 to 24 organoids / condition / experiment) ( Figure 1E Transmission electron microscopy (TEM) images demonstrated the presence of well-developed myofibrils and sarcomere formation within organoids under all conditions ( Figure 1F ), where the sarcomere under EMM1 conditions showed a significantly increased sarcomere length (1.58±0.323μm) relative to the control ( Figure 1GqRT-PCR revealed expression of hallmark cardiomyocyte sarcomere genes from day 20 to day 30, as expected. Interestingly, different conditions showed differential expression of MYL2, MYL7, MYH7, and MYH6 at different developmental time points, suggesting that the developmental maturation strategy of the present invention triggers distinct transcriptomic effects on cardiac organoid growth ( Figure 1H Single-cell RNA sequencing (scRNA-seq) of developmentally induced human cardiac organoids reveals cell type complexity and differences in cellular composition

[0126] To characterize the cellular and transcriptome composition of cardiac organoids under each developmental induction condition, scRNA-seq was performed on day 34 of organoid culture. UMAP projections show unsupervised K-means clustering analysis for each condition ( Figure 2A ). 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), conductance cells (CC), and endothelial cells (EC) were revealed in all conditions of cardiac organoids. The abundance of several important cell groups varied depending on the developmental media conditions. Control organoids were composed of 17% VCM, 17% ACM, 3% VC, 17% PEDC, 1% EPC, 18% SC, 10% CPC, 5% CC, and 1% EC ( Figure 2B). Relative 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%). Relative to the control, EMM1 organoids contained 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%). Relative 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 signature genes for identification of clusters ( Figures 2C to 2D ). ACM has high expression of MYH6, MYL7, NPPA and GJA5 4,41-43 VCM showed high expression of MYL3, MYH7, TNNC1 and HSPB7 41,42,44-47 PEDCs showed high expression of PDGFRB, SEMA3D, POSTN, and TCF21 48-52 EPCs and PEDCs share slight similarities, but also differentially expressed genes, including WT1, TBX18, ITLN1, and TNNT1 41,53-56 CC showed high expression of STMN2, CHGA, SCG2, and INSM1; these genes are involved in neuronal growth, development, and neuroendocrine signaling 57-62 and shared similarities with the human embryonic heart dataset in neural crest cell and Schwann cell clusters 41 ECs have high expression of PECAM1, ESAM, SOX18, and FLT 41,63-66 SCs were identified by the expression of SOX2, ANXA4, SOX9, and CD24 41,67-71 VC is determined by the expression of SOX9, UGDH, ID2, and FLRT2 42,69,72-78 Taken together, these results demonstrate that cardiac organoids have cell types similar to those present in the developing human heart and suggest that, consistent with previous studies of cardiac development, 41,42 , i.e., by day 20, the major cardiac cell lineages have been identified and developmentally inductive conditions can exert significant effects on the expansion and maturation of these cell types to better reflect in vivo cardiac development.

[0127] To investigate whether the differences in the proportions of cell types in organoids between conditions were due to apoptosis, a genetic reporter iPSC line named FlipGFP was created that fluoresces when the active form of caspase 3, a master regulator of apoptosis, is present. 79 We found that from day 20 to day 30, apoptosis levels in cardiac organoids were very low ( Fig.9A ), and no difference in apoptosis levels between conditions ( Fig. 9B ). 48 h doxorubicin treatment was used as a positive control and showed a high level of fluorescence ( Fig. 9C ). This data suggests that the ratio of cell types in organoids from different conditions is not driven by apoptosis.

[0128] Several additional specific marker gene sets were identified in the cardiac organoid dataset. Cardiac fibroblasts were identified in the PEDC cluster, which showed expression of DCN, LUM, OGN, and POSTN as well as COL1A1. 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). In addition, organoids showed high upregulation of proliferation markers such as MKI67, PCNA, AURKB, and CDK1 in all conditions, suggesting that important growth and remodeling is still ongoing at day 34 of differentiation. 85-87 (Data not shown). Cells of the first heart field (FHF) and second heart field contribute to linear heart tube expansion and subsequent chamber formation and are important for proper cardiac morphogenesis. 88 Multiple FHF and SHF markers were observed in organoids under all conditions 89,90 (Data not shown). For all conditions, HAND1, HAND2, TBX5, and HCN4 were upregulated in the VCM and ACM clusters. For each condition, ISL1 was upregulated in the VCM and ACM clusters as well as the CC cluster. In addition, for all conditions, outflow tract markers such as RSPO3 were upregulated. 91 and WNT5A 92,93 Upregulated in PEDC, ACM, VCM, and SC clusters (data not shown).

[0129] These analyses were extended to begin at gestational day 45 (GD45) and from gestational weeks 5 to 13. 42 Starting with the human embryonic heart, we used publicly available data from the Human Cell Atlas project. 41, to compare human cardiac organoids to the developing human heart ( Figures 10A to 10C Based on their time in culture, human cardiac organoids should most closely resemble the human fetal heart at GD45 or 6 to 7 weeks of gestation ( Fig. 10A ). These scRNAseq datasets were integrated and a high degree of overlap between cell type annotations and those presented by the Human Cell Atlas Project was found ( Fig. 10B ), with atrial and ventricular cardiomyocytes, cells of proepicardial origin (named fibroblast-like, smooth muscle cells, and cells of epicardial origin in the Human Cell Atlas Project dataset), endothelial cells, and epicardial cells showing high clustering between datasets. Interestingly, valvular cells mapped closely to capillary endothelial cells, stromal cells of the present invention mapped closely to immune cells, and conduction cells had no clear mapping correlation, even though the conduction cell cluster showed a similar gene expression profile to the cardiac neural crest cluster in the Human Cell Atlas Project dataset 41 These datasets were then used to compare gene expression profiles at the single-cell level ( Fig. 10C and Figures 11A to 11F Using the top 1000 differentially expressed genes in each dataset from the VCM, ACM, PEDC, and EPC mapping regions, high similarities between organoids from each condition and those from embryonic hearts were shown ( Fig. 10C ), where control organoids and EMM2 / 1 organoids clustered tightly into embryonic hearts of week 6, while MM and EMM1 organoids clustered more tightly into embryonic hearts of weeks 7 to 9, which could indicate that the MM and EMM1 maturation strategies accelerated the developmental transcriptome in organoids at a rate exceeding that of traditional developmental paradigms compared to control and EMM2 / 1 organoids (which are consistent with expected developmental stages). Individual gene expression levels in clusters of embryonic hearts and human heart organoids were also assessed and showed high similarity in all major clusters ( Figures 11A to 11F ).

[0130] To complement the above scRNA-seq analysis, dot plots depicting the mean and percentage expression of key lineage-defined differentially expressed genes for each cluster are depicted for each developmental induction condition, illustrating the cellular complexity of cardiac organoids obtained at 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. The intercellular communication networks of key genes found in organoids were computationally analyzed. Multiple complex receptor-ligand communication pathways were identified in human cardiac organoids obtained under each condition ( Figure 3B). The receptor-ligand network includes JAG1-NOTCH1, PDGFR, IGF2-IGF2R, INSR and VEGF, among others. Gene Ontology (GO) analysis was also performed for biological process terms corresponding to the highest differentially expressed genes contributing to the ontology of each cluster, as well as the highest shared genes for each cluster between all four conditions (data not shown). To further investigate the intercellular communication network, scRNA-seq data were used to highlight key receptor-ligand pairs in organoids from each maturation condition (data not shown). The data highlight the ability and sensitivity of the obtained organoids to respond to a variety of developmental maturation stimuli surrounding the intercellular communication paradigm.

[0131] Mitochondrial maturation and oxidative metabolism in human cardiac organoids under developmental induction conditions

[0132] The early developing human heart relies heavily on glycolysis for energy consumption. As it continues to grow, it reduces its reliance on glycolysis and switches to fatty acid oxidation for most of its energy consumption. 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 within cardiac organoids. Real-time mitochondrial content was visualized within cardiac organoids at day 30 of culture by adding mitochondria-permeable fluorescent MitoTracker ( Figure 4A ). Control organoids displayed few and scattered mitochondria, whereas EMM2 / 1 organoids had the best developed mitochondrial content (abundance, morphology) of all conditions ( Figures 4A to 4B Quantification of the increased mitochondrial content in MM, EMM1, and EMM2 / 1 organoids relative to controls (fold changes of 1.73 ± 0.10, 2.60 ± 0.11, and 3.10 ± 0.18, respectively) suggests that maturing organoids have increasing aerobic capacity and actively respond to maturation stimuli ( Figure 4B TEM reveals high-magnification details of the presence of mitochondria in an organoid at day 30 in culture ( Figure 4C ). The mitochondrial size of control organoids was similar to that of mitochondria at day 15 ( Figure 4D ). However, mitochondrial size was significantly increased in MM, EMM1, and EMM2 / 1 organoids relative to that in control organoids. qRT-PCR was used to investigate differential gene expression of two key OXPHOS genes in cardiac metabolic maturation at different time points from day 20 to day 30 of organoid culture: PPARGC1A (master regulator of mitochondrial biogenesis) 98 ) and CPT1B (a key rate-limiting fatty acid transport element 99,100 )( Figure 4E ). Relative to the control, CPT1B expression increased 1.5 times under EMM2 / 1 conditions at the 30th day, while the expression in MM and EMM1 organoids decreased by about 1 to 2 times. From the 21st to the 25th day, the PPARGC1A level in the EMM2 / 1 organoid was as high as 2.5 times relative to the control, and by the 30th day, it ended with a 1.5-fold high fold change. From the 21st to the 25th day, the expression of MM and EMM1 organoids also showed improvement relative to the control, but not as high as EMM2 / 1. By the 30th day, the expression in the EMM1 organoid remained similar to the control, while MM and EMM2 / 1 organoids showed 1.2-fold and 1.7-fold high levels, respectively. At the 30th day under EMM2 / 1 conditions, the expression of CPT1B increased 1.5 times relative to the control, but for the EMM1 organoid, the expression in MM remained similar or decreased.

[0133] To investigate real-time metabolic parameters, organoids in each condition were subjected to the Agilent Seahorse MitoStress Test ( Figure 4F ). Compared to controls, organoids grown in EMM2 / 1 conditions exhibited basal respiration ( Figure 4G ), maximum breathing( Figure 4H ) and percent spare respiratory capacity (%) Fig. 4I ), which is closely related to the metabolic enhancement present in EMM2 / 1 organoids shown in previous mitochondrial and metabolic data.

[0134] These findings were supported using scRNAseq data, showing 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, a gene encoding mitochondrial creatine kinase and important for metabolic efficiency and involved in cardiac maturation; NMRK2, involved in cardiac maturation and lipid metabolism, and activated in high-energy states; and KLF9, a gene associated with lipogenesis 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 find a broader set of metabolic markers as organoids developed under different conditions. It was found that organoids under EMM2 / 1 conditions expressed key metabolic genes at much higher levels than controls, including those involved in fatty acid metabolism, amino acid metabolism, TCA cycle, and mitochondrial dynamics ( Figure 4K ). In addition, computational transcriptome analysis and mapping of KEGG metabolic pathways were performed using Pathview. 101,102(Data not shown). Consistent with other metabolic data, EMM2 / 1 organoids showed reduced activity of glycolytic complexes (data not shown) and increased activity of mitochondrial respiratory complexes (data not shown), suggesting progressive developmental maturation. Collectively, these results suggest that EMM2 / 1 organoids recapitulate important aspects of cardiac metabolism in vitro, reminiscent of fetal heart development at similar stages.

[0135] Developmentally inductive conditions promote progressive electrophysiological maturation of human cardiac organoids.

[0136] The presence and development of the cardiac conduction system (including specific ion channels and membrane receptors, such as those surrounding calcium, potassium, and sodium currents) represents the myocyte action potential. 103-105 and key elements of fetal heart development 106,107 We sought to characterize the functionality of cardiac organoids under developmentally induced conditions by electrophysiology and immunofluorescence of key markers. The membrane permeable dye Fluo-4 was used to assess calcium transient activity in individual cardiomyocytes within day 30 human cardiac organoids ( Figure 5A ). Organoids in all conditions showed distinct and regular calcium transient activity, with different peak amplitudes and action potential frequencies ( Figures 5B to 5C ). Control and MM organoids exhibited smaller peak amplitudes compared to EMM1 and EMM2 / 1 organoids, suggesting that contractions were less robust ( Figure 5B ) and also exhibited a similar beating frequency of about 1.5 Hz. At this stage, the EMM1 organoids exhibited an abnormally high beating frequency of the heart (about 2.5 Hz), while the EMM2 / 1 organoids exhibited a beating frequency of about 1 to 1.5 Hz ( Figure 5C Overall, and with the exception of the EMM1 organoids, the beating rates of the developmentally induced organoids appeared consistent with the beating rate of the early human embryo at GD45108 ,109 (60 to 80 beats / min). Calcium traces from organoids in all conditions were reproducible ( Fig.12 A).

[0137] The overall electrophysiological activity encompassing the cardiomyocyte action potential involves a complex coordination of multiple ionic currents (e.g., calcium, potassium, and sodium) and supporting channels (e.g., ryanodine receptors). The expression levels of multiple electrophysiological genes (including RYR, ATP2A2, SCN5A, KCNJ2, and KCNH2) were investigated in cardiac organoids and robust expression patterns were found in both ACM and VCM clusters under all conditions ( Figure 5D). The expression levels of all genes in the EMM2 / 1 condition showed slight to moderate increases relative to the control. It is noteworthy that for all maturation conditions, especially in the EMM2 / 1 condition, the expression of KCNJ2 was significantly increased relative to the control. Another very important ion channel is the hERG channel encoded by the gene KCNH2 110-112 Mutations and perturbations of this channel can lead to shortening or prolonging of the QT interval. 111,113-115 , and drug interactions with this channel can lead to arrhythmias, representing a key bottleneck associated with drug discovery and development 116,117 KCNH2 expression within organoids showed high expression levels in ACM and VCM clusters under all conditions.

[0138] In addition, autonomic control of the cardiac conduction system via adrenergic signaling plays a large role in physiological functionality. 118-120 , and underlie a range of CVDs from heart failure to hypertension to arrhythmias 121,122 The presence of key β-adrenergic receptor genes ADRB1 and ADRB2, encoding β-adrenergic receptors 1 and 2, was identified in organoids under each condition (data not shown). While ADRB2 was expressed in both ACM and VCM clusters under each condition, ADRB1 showed expression in both ACM and VCM clusters under MM, EMM1, and EMM2 / 1 conditions, but only in the ACM cluster under control conditions. Expression of ADRB3 was sparse relative to ADRB1 and ADRB2, consistent with cardiac physiology. 41,123-125 .

[0139] To investigate the temporal dynamics of key ion channels by applying our maturation strategy, levels of calcium (ATP2A2), sodium (SCN5A), and potassium (KCNJ2) transporters were assessed by qRT-PCR from day 20 to day 30 of organoid culture ( Figure 5E ). ATP2A2 expression was increased in all conditions relative to the control, with EMM2 / 1 showing the most significant 4-fold upregulation at days 25 and 30. SCN5A expression was upregulated from day 21 to day 30 for all conditions. Notably, MM and EMM2 / 1 organoids showed a 3-fold increase at day 30 relative to control organoids that showed only a 2-fold increase. KCNJ2 expression was steadily reduced in EMM1 conditions relative to the control, with MM organoids showing upregulation at day 30 and EMM2 / 1 showing upregulation (compared to the control) throughout the culture period.

[0140] Voltage activity within control and EMM2 / 1 cardiac organoids was investigated using the potentiometric dye di-8-ANEPPS and distinct action potentials were identified within single cardiomyocytes, indicative of the presence of specialized atrial-like and nodal-like cells, but interestingly, ventricular-like action potentials were only observed in EMM2 / 1 organoids ( Fig. 5F ).

[0141] Furthermore, proper excitation-contraction coupling and depolarization and repolarization of cardiomyocytes depend on specialized invaginations (transverse tubules) of the sarcolemma, which indicate that cardiomyocyte maturation 126,127 The presence of transverse tubules in human cardiac organoids at day 30 was assessed by caveolin-3 immunofluorescence imaging ( Figure 5G ), and in each condition within the organoids the myotome (TNNT2 + ), where the increased transverse tubule density under EMM2 / 1 conditions was quantified ( Figure 5H Fluorescently labeled wheat germ agglutinin (WGA) was also used to assess transverse tubules in human cardiac organoids at day 30 (data not shown), and similar results were found indicating that EMM2 / 1 organoids had significantly increased transverse tubule density. The presence of the potassium channel KCNJ2 was also assessed by confocal microscopy ( Fig.5I ). KCNJ2 was observed in each condition + Spots where there is a 2-fold increase in EMM2 / 1 conditions relative to the control ( Figure 5J ), which supports previous data showing elevated levels of KCNJ2 transcripts in EMM2 / 1 organoids. To investigate the temporal dynamics of key ion channels by applying a developmental maturation strategy, levels of calcium (ATP2A2), potassium (KCNJ2), and sodium (SCN5A) transporters were assessed by qRT-PCR from day 20 to day 30 of organoid culture ( Fig.11A ). Relative to the control, ATP2A2 expression is increased under all conditions, wherein EMM2 / 1 shows the most significant 7-fold increase. Relative to the control, KCNJ2 expression is steadily reduced under EMM1 conditions, wherein MM organoids show upregulation at the 30th day, and EMM2 / 1 shows upregulation during the entire culture period. SCN5A expression shows upregulation at the 25th and 30th day for MM conditions, and EMM2 / 1 also shows upregulation at the 30th day (relative to the control). Until the 25th day, EMM1 expression remains consistent with the control and is downregulated at the 30th day. In short, these data show that the mature organoid platform (particularly the EMM2 / 1 strategy) produces organoids that reproduce the important electrophysiological aspects of cardiac development, physiology and disease.

[0142] Developmental induction promotes the emergence of the proepicardial organ and the formation of the atrial and ventricular chambers through self-organization.

[0143] Developmentally induced cardiac organoids have been shown to exhibit improved cellular, biochemical, and functional properties when compared to their control counterparts and to display multiple features similar to the GD45 human fetal heart. However, previous attempts at cardiac organoids, including previous work 15-7 The generation of anatomically relevant cardiac structures and morphology was largely lacking. Given the dramatic changes observed by applying the EMM2 / 1 strategy, it was decided to characterize the morphological changes that occurred under this improved condition. Organoids were harvested on day 30 of culture and stained for WT1 (proepicardial and epicardial cells) and TNNT2 (cardiomyocytes) ( Fig. 6A ). Organoids in each developmental induction condition showed TNNT2 + and WT1 + cells, consistent with previous observations 15 , indicating the presence of epicardial and cardiomyocyte populations that are widely distributed in the organoids. Evaluation of both the surface and interior planes of the organoids revealed that organoids in all conditions had + and TNT2 + Two distinct "compartments" of cells labeled. + The cells are densely packed in the lower chambers and form a thick myocardial wall, and are also present in a less dense arrangement in the WT1 + In the upper region just below the cell, WT1 was found in EMM2 / 1. + Cells densely covered the outer surface of the budding region, while present in dispersed, sparse groups on the surface of the lower region. These staining patterns were not observed in control, MM, or EMM1 culture conditions. + 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 EMM2 / 1 organoids were found to show an increase of 1.54 (fold change) relative to the control. + The chamber area did not differ relative to controls, while EMM1 and EMM2 / 1 organoids showed increases in area of ​​1.77 and 1.98 (fold changes), respectively. The data indicate that organoids under all conditions underwent significant morphological changes that resulted in highly specific and reproducible cellular organization, including the appearance of organoids with advanced myocardial dual-chamber morphology and anterior epicardial poles.

[0144] Upon further examination, it was determined that ventricular myosin (MYL2) and atrial myosin (MYL7), which indicate ventricular and atrial cardiomyocyte subpopulations, respectively, were largely spatially restricted, particularly in EMM2 / 1 organoids. Fig.6D ). All organoids expressed MYL7 throughout the majority of the organoid, but expression was stronger in the upper chamber in EMM2 / 1, suggesting an atrial-like chamber. In both control and MM conditions, organoids had MYL2 in a variety of locations that were not limited to the polar ends of the organoid or to any one chamber in particular. On the other hand, organoids in EMM1 and EMM2 / 1 conditions showed MYL2 + The marked increase in staining and organization indicated that MYL2 was restricted to one polar end of the organoids, and that EMM2 / 1 organoids displayed MYL2 + The area has increased by 5.5 times ( Fig. 6E ); indicating the formation of a ventricle-like chamber. These findings are particularly interesting because in these EMM2 / 1 organoids, the anterior epicardial region is directly above the atrial chamber, and the ventricular chamber is located on the opposite side of the organoid. Overall, this organization is comparable to the anterior-posterior axial patterning that occurs in utero in the forming heart tube (see schematic for Fig. 7A ).

[0145] To further investigate the identity of ventricle-like and atrial-like chambers in human cardiac organoids, staining for additional atrial and ventricular chamber markers NR2F2 (atria) and MYL3 (ventricles) was performed ( Fig. 6F ). EMM2 / 1 organoids showed a clear, increased degree of separation between the two compartments, whereas control organoids showed a greater overlap of both proteins ( Figure 6G ); This suggests that EMM2 / 1 organoids have a higher degree of specialization and maturity of chamber development. Notably, these results were reproduced in two additional PSC lines: BYS0111 (iPSC) and H9 (ESC) ( Fig.13A ). Although for reproducibility and comparison purposes, L1 control and EMM2 / 1 organoids are shown again ( Fig. 13B ), but control BYS0111 organoids showed similar overlap of NR2F2 and MYL3, while EMM2 / 1BYS0111 organoids showed clear separation of NR2F2 and MYL3 ( Fig.13A and 13C ), among which MYL3 + Cells highlight the thick myocardial wall in EMM2 / 1 conditions. Control H9 organoids showed significantly reduced expression of both NR2F2 and MYL3 compared to EMM2 / 1 H9 organoids, where EMM2 / 1 H9 organoids showed NR2F2 +and MYL3 + Clear separation of chambers ( Fig.13A and 13D To support these immunofluorescence results describing the potential identity of the atrial and ventricular chambers in the cardiac organoids of the present invention, gene expression patterns were investigated using scRNAseq data in the ACM and VCM clusters ( Figures 6H to 6J ). Compared with VCM, ACM showed increased gene expression of hallmark atrial chamber identity markers such as NR2F2, TBX5, NPPA, and NR2F1 (source) ( Fig.6I ). Meanwhile, VCMs showed elevated gene expression of hallmark ventricular chamber identity markers such as MYL3, HEY2, IRX4, and HAND1 (source) compared to ACMs ( Figure 6J These results highlight not only the ability of existing cardiac organoid platforms to recapitulate important structural events in cardiac development, but also the capacity of the EMM2 / 1 strategy to elicit distinct cell types, morphological features, and chamber identities in multiple PSC lineages.

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

[0147] Vasculature formation under developmentally inductive conditions was also assessed. On day 30 of culture, endothelial cell (PECAM1+) vasculature formation was examined by immunofluorescence and confocal microscopy ( Figures 15A to 15D ). Evaluation of organoids on both the surface and internal planes revealed the presence of endothelial cells in the myocardial region of all organoids ( Fig.15A). Organoids in EMM1 and EMM2 / 1 conditions exhibited fewer PECAM1+ cells than control and MM organoids. Control and MM organoids displayed a robust, interconnected endothelial cell network throughout the myocardial (TNNT2+) tissue ( Fig. 15B ). The 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 controls ( Fig. 15C ). High-magnification images of the organoids further revealed the morphological transition state of endothelial cells within the cardiomyocyte-rich regions ( Fig. 15B Overall, this data suggests that vascularization of organoids under EMM1 and EMM2 / 1 conditions may be partially outweighed by some factors, perhaps due to the timing or concentration of growth factors, and further studies will be needed to fine-tune the culture medium conditions.

[0148] Endogenous retinoic acid gradients are responsible for spontaneous anterior-posterior heart tube patterning in EMM2 / 1 organoids.

[0149] The emergence of a spatially restricted retinoic acid gradient originating from the posterior pole of the heart tube (generated by the epicardium and primitive atria) is a key developmental step in mammalian heart development. This gradient establishes the anterior-posterior axis that cues the formation of the ventricle and the inflow and outflow tracts, while also helping to define the cardiac progenitors and possibly other structures. 130,131 ( Fig. 7A To determine whether the tubular structures observed in the heart under EMM2 / 1 conditions (Figure 6) were indeed reminiscent of retinoic acid-mediated cardiac patterning, Raman microscopy was performed using a microscope designed for this purpose to detect their molecular signatures (data not shown). The presence of myosin, troponin T, tropomyosin, collagen I, and other relevant molecular signatures were identified in organoids under all conditions, as expected, and was particularly evident in the presence of retinoic acid in EMM2 / 1 ( Figure 7B Retinoic acid synthesis is mainly carried out by retinal dehydrogenase 2 (ALDH1A2) during embryogenesis. 131-133 ALDH1A2 levels were assessed in all conditions using qRT-PCR starting at day 30 of organoid culture ( Figure 7C ). ALDH1A2 expression increased approximately 2.2-fold under EMM2 / 1 conditions relative to the control, whereas expression showed no significant changes under MM or EMM1 conditions.

[0150] To assess the cell-specific dynamics of retinoic acid production in organoids, scRNAseq data were used to show that ALDH1A2 is expressed in EPCs, PEDCs, and ACMs in organoids ( Fig.7D ), consistent with the reported in vivo expression pattern. To complement this analysis and further investigate the localization of retinoic acid production in organoids, organoids from all conditions at day 30 were quantified for ALDH1A2 and TBX18 (epicardial transcription factor that marks the proepicardial organoid / atrial pole). 56 ,134 ,135 We performed immunostaining with antibodies against EMM2 / 1 and found that EMM2 / 1 conditioned organoids had a similar expression profile to TBX18 + The localized, polarized expression of ALDH1A2 in co-localized cells confirmed that the retinoic acid gradient that enabled the organoid model was derived from the anterior epicardiac pole / atrial pole (the posterior pole of the in utero heart tube) ( Fig. 7A , 7E and 7F). Control, MM, and EMM1 organoids showed no ALDH1A2 expression. The area of ​​colocalization between ALDH1A2 and TBX18 was quantified and showed that organoids in EMM2 / 1 conditions were significantly more responsive to the induction of retinoic acid synthesis ( Figure 7G ). Notably, these results were reproduced in two additional PSC lines, BYS0111 and H9 ( Fig.16A ). When L1 control and EMM2 / 1 organoids are shown again for reproducibility and comparison purposes ( Fig. 16B ), control and EMM2 / 1BYS0111 organoids showed similar ALDH1A2 and TBX18 expression patterns as L1 organoids; EMM2 / 1BYS0111 organoids showed polarized ALDH1A2 compared with control BYS0111 organoids. + TBX18 + The amount of cells increased significantly ( Fig.16A and 16C ). H9 organoids showed a similar degree of reproducibility, with EMM2 / 1 H9 organoids showing ALDH1A2 expression compared to control H9 organoids. + TBX18 + A significant increase in cells ( Fig.16A and 16D Control and EMM2 / 1 organoids from all three cell lines also showed similarly robust and reproducible transcriptomes for ALDH1A2 and other important genes such as MYL2, MYL7, WT1, and PPARGC1A ( Figures 17A to 17G ), as determined by qRT-PCR.

[0151] To more strongly demonstrate that retinoic acid contributes to patterning in EMM2 / 1 organoids, ALDH1A2 was inhibited using DEAB (source), and by immunostaining for NR2F2 and MYL3, it was shown that ALDH1A2 inhibition resulted in reduced patterning in cardiac organoids ( Figures 7H to 7J ). ALDH1A2 knockdown organoids showed MYL3 expression relative to untreated organoids. + and NR2F2 + The areas were reduced by 0.35-fold and 0.42-fold, respectively. It also showed that the addition of exogenous retinoic acid did not lead to differences in patterning, suggesting that EMM2 / 1 organoids already endogenously produce optimal amounts of retinoic acid.

[0152] Together, these data demonstrate the ability of EMM2 / 1 organoids to endogenously synthesize retinoic acid in a spatially restricted manner that colocalizes with the epicardium (TBX18), a phenomenon that closely mimics the processes observed during in utero heart development and cardiac tube patterning.

[0153] Ondansetron treatment during cardiac organoid development captures congenital heart disease phenotypes.

[0154] Organoids have the unique ability to better simulate and study human development, organogenesis, and disease modeling at an unprecedented scale and precision. However, in the context of organogenesis and disease modeling, human cardiac organoids have so far only been used to model developmental perturbations in diabetes-induced cardiomyopathy during pregnancy (Yoni), gene knockout studies (Drakhlis), developmental cryoinjury (Hofbaur), and hypertrophic and fibrotic remodeling (Meier Epicardioid). Therefore, while cardiac organoids show promise for unraveling unanswered questions surrounding cardiac organoid development and pathology, key areas such as studying developmental drug toxicity and more extensive morphological perturbations in cardiac pathology remain to be discovered. During pregnancy, women are commonly prescribed the antiemetic ondansetron, also known as Zofran, for off-label use, with X% of pregnant women reported to take ondansetron at some point during pregnancy. Despite this, ondansetron has been associated with causing congenital heart defects and orofacial defects, but consensus in the field is divided and well-designed studies investigating its safety are largely lacking. The difficult and unethical nature of studying human cardiac development and human congenital heart defects represents a critical bottleneck to inquiry surrounding many aspects of cardiac research. In this way, attempts to study the effects of ondansetron during development of human cardiac organoids ( Figures 8A to 8J ). Clinical data on ondansetron plasma blood levels were used to determine relevant concentrations for t shetudies 136Three concentrations of ondansetron were applied to cardiac organoids from day 9 until day 30, and the EMM2 / 1 strategy was applied, and then on day 30, their morphology against MYL7 and MYL2 was evaluated ( Fig. 8A ), two key myosins that are heavily involved in heart development (source). Untreated organoids displayed the previously shown morphology and pattern building of MYL7 staining throughout the organoid and MYL2 staining localized to one end of the organoid, reminiscent of ventricle-like chambers. MYL2 is also a protein involved in ventricular septal defects, the primary heart defect associated with ondansetron use. Strikingly, MYL2 increased with increasing ondansetron concentrations. + The number of cells began to decrease, especially at 10 μM and 100 μM ( Fig. 8A ). These results were quantified and shown to be relative to untreated MYL2 + The area is reduced to 0.55 times and 0.18 times ( Figure 8B ), while MYL7 + The area remains constant under all conditions ( Figure 8C ). Organoids in the 100 μM condition also appeared less organized structurally, with less defined chamber walls and loose chamber septa compared to untreated. In support of these results, qRT-PCR was performed on organoids from all conditions and showed that MYL2 expression was reduced to 0.58-fold and 0.40-fold at 10 μM and 100 μM, respectively, relative to untreated ( Fig.8D ). Taken together, these data suggest that ondansetron can interfere with key steps in cardiac development by inhibiting MYL2 at both the protein and transcriptional levels.

[0155] Ondansetron has been associated with a prolongation of the QT interval, a potentially lethal phenomenon. The electrophysiological effects of ondansetron on cardiac organoid development have also been investigated using the potentiometric dye di-8-ANEPPS ( Figures 8E to 8J ). The action potentials of organoids treated with 10 μM and 100 μM were significantly different compared to untreated ( Figures 8E to 8F ), showing a decrease in frequency ( Figure 8G ), amplitude reduction ( Figure 8H ) and APD30 / 90 improvement ( Figures 8I to 8J ), suggesting that ondansetron induces a progressive electrophysiological pathological phenotype during cardiac development. However, it is interesting to show that although ondansetron did not induce apoptosis in human cardiac organoids ( Figures 18A to 18B ), but ondansetron contributed to a progressive loss of beating activity in cardiac organoids over time, with the 100 μM condition showing the most significant loss of cardiac activity ( Fig.18C ).

[0156] Collectively, the data provide unprecedented insights into the morphological and electrophysiological effects of ondansetron during human development and provide a framework for future studies targeting the safety and efficacy of the drug during pregnancy and the pathology of congenital heart disease.

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

[0158] Additional embodiments

[0159] Embodiment 1. A maturation medium comprising: a cell growth medium containing a medium supplement, wherein the medium supplement comprises 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.

[0160] Embodiment 2. The maturation medium described in Embodiment 1, wherein the cell growth medium comprises Roswell Park Memorial Institute (RPMI) medium; Dulbecco's modified Eagle's medium (DMEM); a derivative of DMEM such as Iscove's modified Dulbecco's medium (IMDM) or Advanced Dulbecco's modified Eagle's medium (ADMEM); or a combination thereof.

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

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

[0163] Embodiment 5. The maturation medium of any one of Embodiments 1 to 4, wherein the one or more additional fatty acids comprise palmitic acid, oleic acid, linoleic acid, stearic acid, or a combination thereof.

[0164] Embodiment 6. The maturation medium of embodiment 5, wherein the maturation 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] Embodiment 7. The maturation medium of any one of Embodiments 1 to 6, wherein the maturation medium comprises additional carnitine comprising L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, or a combination thereof.

[0166] Embodiment 8. The maturation medium of any one of embodiments 1 to 7, further comprising an additional sugar, such as fructose, galactose or glucose.

[0167] Embodiment 9. The maturation medium of embodiment 8, wherein the additional sugar comprises glucose, such as about 2 to 6 mM glucose.

[0168] Embodiment 10. The maturation medium of any one of embodiments 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] Embodiment 11. The maturation medium of any one of Embodiments 1 to 10, further comprising ascorbic acid (vitamin C), such as about 0.1 to 1 mM ascorbic acid (vitamin C).

[0170] Embodiment 12. The maturation medium of any one of embodiments 1 to 11, further comprising a growth factor, such as IGF-1 or IGF-2.

[0171] Embodiment 13. The maturation medium of any one of Embodiments 1 to 12, further comprising IGF-1 or IGF-2, such as about 10 to 100 ng / mL.

[0172] Embodiment 14. The maturation medium of any one of Embodiments 1 to 13, wherein the maturation medium does not comprise extracellular matrix material and / or exogenous retinoic acid.

[0173] Embodiment 15. A method for maturing early embryonic human cardiac organoids into mature human cardiac organoids, the method comprising contacting the early embryonic human cardiac organoids with the maturation medium, the maturation 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, a sugar and carnitine; one or more additional fatty acids; additional carnitine or creatine; and additional T3 growth hormone.

[0174] Embodiment 16. The method described in Embodiment 15, wherein the cell growth medium comprises Roswell Park Memorial Institute (RPMI) medium; Dulbecco's modified Eagle's medium (DMEM); a derivative of DMEM such as Iscove's modified Dulbecco's medium (IMDM) or Advanced Dulbecco's modified Eagle's medium (ADMEM); or a combination thereof.

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

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

[0177] Embodiment 19. The method of any one of Embodiments 15 to 18, wherein the one or more additional fatty acids comprise palmitic acid, oleic acid, linoleic acid, stearic acid, or a combination thereof.

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

[0179] Embodiment 21. The method of any one of Embodiments 15 to 20, wherein the maturation medium comprises additional carnitine comprising L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, or a combination thereof.

[0180] Embodiment 22. The method of any one of Embodiments 15 to 21, wherein the maturation medium further comprises an additional sugar, such as fructose, galactose or glucose.

[0181] Embodiment 23. The method of embodiment 22, wherein the additional sugar comprises glucose, such as about 2 to 6 mM glucose.

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

[0183] Embodiment 25. The method of any one of Embodiments 15 to 24, wherein the maturation medium further comprises ascorbic acid (vitamin C), such as about 0.1 to 1 mM ascorbic acid (vitamin C).

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

[0185] Embodiment 27. The method of any one of Embodiments 15 to 26, wherein the maturation medium further comprises IGF-1 or IGF-2, e.g., about 10 to 100 ng / mL.

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

[0187] Embodiment 29. The method of embodiment 15, wherein

[0188] - from day 20 to day 26, contacting the early embryonic human cardiac organoids with the maturation medium, wherein the maturation medium further comprises an additional antioxidant, such as ascorbic acid; an additional sugar, such as glucose; and a growth factor, such as IGF-1, and

[0189] - from day 26 to day 30, contacting the early embryonic cardiac organoids with said maturation medium, wherein said maturation medium further comprises an additional antioxidant, such as ascorbic acid; an additional sugar, such as glucose, and does not comprise IGF-1; and

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

[0191] Embodiment 30. The method of Embodiment 29, wherein a portion of the maturation medium from day 20 to day 26 is contacted with the early embryonic cardiac organoid from day 26 to day 30.

[0192] Embodiment 31. The method of Embodiment 15, wherein the contacting occurs for 9, 10, 11, 12 or more days, preferably 10 days.

[0193] Embodiment 32. The method of any one of Embodiments 15 to 31, wherein the maturation medium contacting the early embryonic cardiac organoid is replaced with fresh maturation medium approximately every 48 hours.

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

[0195] Embodiment 34. The method of any one of embodiments 15 to 33, wherein the mature human cardiac organoid comprises one or more of:

[0196] (i) Endogenous retinoic acid;

[0197] (ii) at least two cardiac chambers, one atrial and one ventricular;

[0198] (iii) proepicardial organs; and

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

[0200] Embodiment 35. The method of any one of Embodiments 15 to 34, wherein the mature human cardiac organoid is capable of beating.

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

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

[0203] (2) about 97% RPMI 1640 medium without glucose; about 2% medium supplement; about 1% penicillin-streptomycin; about 52.5 μM palmitic acid in total; about 43.95 μM oleic acid in total; about 26 μM linoleic acid in total; about 132.2 μM L-carnitine in total; about 33.01 nM T3 hormone in total; about 0.4 mM ascorbic acid in total; and about 4 mM glucose in total; or

[0204] (3) about 97% RPMI 1640 medium without glucose; about 2% medium supplement; about 1% penicillin-streptomycin; a total of about 52.5 μM palmitic acid; a total of about 43.95 μM oleic acid; a total of about 26 μM linoleic acid; a total of about 132.2 μM L-carnitine; a total of about 33.01 nM T3 hormone; a total of about 0.4 mM ascorbic acid; a total of about 4 mM glucose, and about 50 ng / mL IGF-1.

[0205] Embodiment 37. A mature human cardiac organoid produced by the method of any one of Embodiments 15 to 36.

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

[0207] (i) Endogenous retinoic acid;

[0208] (ii) at least two cardiac chambers, one atrial and one ventricular;

[0209] (iii) proepicardial organs; and

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

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

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Claims

1. A maturation medium comprising: a cell growth medium containing a medium supplement, wherein the medium supplement comprises 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.

2. The maturation medium of claim 1, wherein the cell growth medium comprises Roswell Park Memorial Institute (RPMI) medium; Dulbecco's modified Eagle's medium (DMEM); a derivative of DMEM such as Iscove's modified Dulbecco's medium (IMDM) or Advanced Dulbecco's modified Eagle's medium (ADMEM); or a combination thereof.

3. The maturation medium of claim 1, wherein the additional carnitine or creatine is present in an amount of about 60 to 160 μM, and / or wherein the total amount of carnitine or creatine present in the maturation medium is about 60 to 200 μM.

4. The maturation medium of claim 1, wherein the additional T3 growth hormone is present in an amount of about 10 to 50 nM, and / or wherein the total amount of T3 growth hormone present in the maturation medium is about 10 to 60 nM.

5. The maturation medium of claim 1, wherein the one or more additional fatty acids comprise palmitic acid, oleic acid, linoleic acid, stearic acid, or a combination thereof.

6. The maturation medium of claim 5, wherein the maturation 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.

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

8. The maturation medium of claim 1, further comprising an additional sugar, such as fructose, galactose or glucose.

9. The maturation medium of claim 8, wherein the additional sugar comprises glucose, such as about 2 to 6 mM glucose.

10. The maturation medium of claim 1, further comprising additional antioxidants such as ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid, carotene, tocopherol (vitamin E) and panthenol.

11. The maturation medium of claim 1, further comprising ascorbic acid (vitamin C), such as about 0.1 to 1 mM ascorbic acid (vitamin C).

12. The maturation medium of claim 1, further comprising a growth factor, such as IGF-1 or IGF-2.

13. The maturation medium of claim 1, further comprising IGF-1 or IGF-2, e.g., about 10 to 100 ng / mL.

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

15. A method for maturing early embryonic human cardiac organoids into mature human cardiac organoids, the method comprising contacting the early embryonic human cardiac organoids with a maturation medium comprising: a cell growth medium containing a medium supplement, wherein the medium supplement comprises 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.

16. The method of claim 15, wherein the cell growth medium comprises Roswell Park Memorial Institute (RPMI) medium; Dulbecco's modified Eagle's medium (DMEM); a derivative of DMEM, such as Iscove's modified Dulbecco's medium (IMDM) or Advanced Dulbecco's modified Eagle's medium (ADMEM); or a combination thereof.

17. The method of claim 15, wherein the additional carnitine or creatine is present in an amount of about 60 to 160 μM, and / or wherein the total amount of carnitine or creatine present in the maturation medium is about 60 to 200 μM.

18. The method of claim 15, wherein the additional T3 growth hormone is present in an amount of about 10 to 50 nM, and / or wherein the total amount of T3 growth hormone present in the maturation medium is about 10 to 60 nM.

19. The method of claim 15, wherein the one or more additional fatty acids comprise palmitic acid, oleic acid, linoleic acid, stearic acid, or a combination thereof.

20. The method of claim 19, wherein the maturation medium comprises about 20 to 80 μM palmitic acid, about 20 to 80 μM oleic acid, and 10 to 60 μM linoleic acid.

21. The method of claim 15, wherein the maturation medium comprises additional carnitine comprising L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, or a combination thereof.

22. The method of claim 15, wherein the maturation medium further comprises an additional sugar, such as fructose, galactose or glucose.

23. The method of claim 22, wherein the additional sugar comprises glucose, such as about 2 to 6 mM glucose.

24. The method of claim 15, wherein the maturation medium further comprises additional antioxidants, such as ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid, carotene, tocopherol (vitamin E), and panthenol.

25. The method of claim 15, wherein the maturation medium further comprises ascorbic acid (vitamin C), such as about 0.1 to 1 mM ascorbic acid (vitamin C).

26. The method of claim 15, wherein the maturation medium further comprises a growth factor, such as IGF-1 or IGF-2.

27. The method of claim 15, wherein the maturation medium further comprises IGF-1 or IGF-2, e.g., about 10 to 100 ng / mL.

28. The method of claim 15, wherein the early embryonic human cardiac organoid is formed by differentiation of human induced pluripotent stem cells (hiPSCs) and is contacted with the maturation medium on day 20 after day zero of initiation of differentiation of the hiPSCs.

29. The method of claim 15, wherein - from day 20 to day 26, contacting the early embryonic human cardiac organoids with the maturation medium, wherein the maturation medium further comprises an additional antioxidant, such as ascorbic acid; an additional sugar, such as glucose; and a growth factor, such as IGF-1, and - from day 26 to day 30, contacting the early embryonic cardiac organoids with the maturation medium, wherein the maturation medium further comprises an additional antioxidant, such as ascorbic acid; an additional sugar, such as glucose, and does not comprise IGF-1; and The maturation medium was replaced on day 26.

30. The method of claim 29, wherein a portion of the maturation medium from day 20 to day 26 is contacted with the early embryonic cardiac organoid from day 26 to day 30.

31. The method of claim 15, wherein the contacting occurs for 9, 10, 11, 12 or more than 12 days, preferably 10 days.

32. The method of claim 15, wherein the maturation medium contacting the early embryonic cardiac organoid is replaced with fresh maturation medium approximately every 48 hours.

33. The method of claim 15, wherein no exogenous retinoic acid and / or extracellular matrix material is added.

34. The method of claim 15, wherein the mature human cardiac organoid comprises one or more of: (v) endogenous retinoic acid; (vi) at least two heart chambers, one atrial and one ventricular; (vii) proepicardial organs; and (viii) The anterior-posterior heart tube pattern was constructed.

35. The method of claim 15, wherein the mature human cardiac organoid is capable of beating.

36. A method for maturing an early embryonic human cardiac organoid into a mature human cardiac organoid, the method comprising contacting the early embryonic human cardiac organoid with one or more maturation media, the maturation media comprising: (1) about 97% RPMI 1640 medium; about 2% medium supplement; about 1% penicillin-streptomycin; about 52.5 μM palmitic acid in total; about 43.95 μM oleic acid in total; about 26 μM linoleic acid in total; about 132.2 μM L-carnitine in total, and about 33.01 nM T3 hormone in total; or (2) about 97% RPMI 1640 medium without glucose; about 2% medium supplement; about 1% penicillin-streptomycin; about 52.5 μM palmitic acid in total; about 43.95 μM oleic acid in total; about 26 μM linoleic acid in total; about 132.2 μM L-carnitine in total; about 33.01 nM T3 hormone in total; about 0.4 mM ascorbic acid in total; and about 4 mM glucose in total; or (3) about 97% RPMI 1640 medium without glucose; about 2% medium supplement; about 1% penicillin-streptomycin; a total of about 52.5 μM palmitic acid; a total of about 43.95 μM oleic acid; a total of about 26 μM linoleic acid; a total of about 132.2 μM L-carnitine; a total of about 33.01 nM T3 hormone; a total of about 0.4 mM ascorbic acid; a total of about 4 mM glucose, and about 50 ng / mL IGF-1.

37. Mature human cardiac organoids produced by the method of claim 15.

38. The mature human cardiac organoid of claim 37, wherein the mature human cardiac organoid comprises one or more of: (v) endogenous retinoic acid; (vi) at least two heart chambers, one atrial and one ventricular; (vii) proepicardial organs; and (viii) The anterior-posterior heart tube pattern was constructed.

39. The mature human heart organoid of claim 37, wherein the mature human heart is capable of beating.

Images