Method of making human heart organoids with integrated macrophages

Abstract

A method is provided herein for generating a human heart organoid (hHO) with integrated macrophages (MPs), including adding monocytes to a first medium containing an hHO, where the hHO has differentiated for at least 5 days, and agitating the hHO, monocytes, and first or fresh medium to generate an hHO with integrated MPs. Also provided is an hHO with integrated MPs prepared by the disclosed method.

Description

METHOD OF MAKING HUMAN HEART ORGANOIDS WITH INTEGRATED MACROPHAGESCROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Application No. 63 / 605,646 filed on 4 December 2023 and U.S. Provisional Application No. 63 / 570,955 filed on 28 March 2024. The entire contents of each application recited above is hereby incorporated by reference.FIELD

[0002] This disclosure generally relates to a method of generating a human heart organoid (hHO) with integrated macrophages (MPs).GOVERNMENT SUPPORT

[0003] This invention was made with government support under HL151505 and HL125464 awarded by the U.S. National Institute of Health, and National Heart, Lung, and Blood Institute. The government has certain rights in the invention.BACKGROUND

[0004] This section provides background information related to the present disclosure which is not necessarily prior art.

[0005] Cardiovascular disease, including congenital heart diseases, is the leading cause of death in the developed world. However, most of our knowledge about the development and disease pathology of the human heart is based on animal models. Animal models, while practical for studying general physiology in cardiac development and disease pathology, do not recapitulate many aspects of the human heart. For example, mice, a common animal model to study cardiovascular physiology, have only two heart chambers, have significantly less cardiac output, develop much more quickly than humans, and live shorter life spans. Furthermore, 90% of pharmacotherapies tested in mice do not go on to pass clinical trials for cardiovascular disease. Hence, the human heart likely possesses unique characteristics on a tissue, cellular, and molecular level during human heart development and disease. Thus, there remains a need for more accurate models that function more similarly to the human heart.

[0006] Since the advent of human pluripotent stem cells (hPSCs), groups have developed protocols to differentiate hPSCs to specific human cell types and tissues. For example, cardiomyocytes can be sustained in culture for long periods of time, unlike ex vivo tissues, and closely resemble human heart cardiomyocytes structurally, transcriptomically, phenotypically, and functionally. However, the human heart is three dimensional, which is crucial to its function, and is made-up of multiple cell types, not just cardiomyocytes. An advanced, three-dimensional (3D) human heart organoid (hHO) system derived from human pluripotent stem cells (hPSCs) was recently developed. See International Patent Publication No. WO 2021 / 257812. The hHOs physically beat, have detectable calcium signaling, have measurable electrophysiology, contain chambers, and are composed of appropriate cell types: cardiomyocytes, epicardial cells, endocardial cells, endothelial cells, and cardiac fibroblasts. Although the current hHO system mimics many aspects of the developing human heart, it still lacks some key cell types, such as tissue-resident macrophages. Thus, there is a need for developing a method to make a hHO with integrated MPs.SUMMARY

[0007] This section provides a general summary of the disclosure and is not a comprehensive disclosure of its full scope or all of its features.

[0008] In certain aspects, the present disclosure provides a method of generating a human heart organoid (hHO) with integrated macrophages (MPs), also referred to herein as a human heart-macrophage assembloid (hHMA). The method may include adding monocytes to a first medium comprising an hHO, where the hHO has differentiated for at least 5 days, and agitating the hHO, monocytes, and first or freshly replaced medium to generate an hHO with integrated MPs. In some embodiments, agitating the hHO, monocytes, and medium may include shaking the hHO, centrifuging the hHO, resuspending the hHO, or a combination thereof. In certain aspects, resuspending may involve one or more rounds of pipette resuspension.

[0009] In further aspects, the monocytes may be added in an amount and frequency to achieve an amount of integrated MPs of about 1% to about 5% of the total cell population in the hHO. For example, monocytes may be added one or more times, and about 10,000 to about 40,000 monocytes may be included in each addition. Additionally or alternatively, in some embodiments, a majority of the integrated MPs may have migrated inside of the hHO. In some embodiments, the integrated macrophages may have a tissue-resident phenotype. In yet further aspects, the integrated MPs may express CD45, CD163, CD14, CD163, CD68, CSFR1, AIF1, CD74, CD206, or a combination thereof, and may form gap junctions with cardiomyocytes.

[0010] The present disclosure also provides an hHO with integrated MPs prepared according to the method described herein. In some embodiments, the hHO may express one or more protein that promotes monocyte adhesion, monocyte-macrophage differentiation, macrophage migration, and / or macrophage viability, such as VCAM1 and / or CSF1. In some embodiments, the hHO with integrated MPs may beat similar to a human heart.

[0011] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0013] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

[0014] FIG. 1 is a heat map of normalized expression of genes involved in Leukocyte Adhesion to Vascular Endothelial Cell, Positive Regulation of Monocyte Differentiation, and Positive Regulation of Macrophage of Chemotaxis in hHOs from bulk RNA-seq data from Day 0 to Day 19 of hHO differentiation.

[0015] FIG. 2 is a high magnification confocal immunofluorescence (IF) image for DAPI (blue), VCAM1 (green), and TNNT2 (red), indicated by the white box in a. Scale = 200 pm. n = 8.

[0016] FIG. 3 is a flowchart depicting the human heart-macrophage assembloid (hHMA) generation protocol.

[0017] FIG. 4 shows phase contrast microscopy images depicting hHMAs at day 13, 15, 17, and 20 with incremental additions of monocytes (10,000, 20,000, and 30,000) at day 8, 12, and 16. After each monocyte addition, the hHO and monocytes were subject to two pipet resuspensions. Scale = 200 pm. n = 8.

[0018] FIG. 5 shows representative confocal IF images displaying the exterior (left panel) and interior (right panel) of day 20 hHMAs stained for DAPI (blue), CD45 (green) and TNNT2 (red). Top row Scale = 200 pm. n > 6.

[0019] FIG. 6 is a bar graph showing the quantification of immune cell (CD45+) integration using z-planes from IF images depicting the interior (three images per hHMA) and exterior of individual hHMAs with incremental monocyte additions of 0 (blue), 10,000 (red), 20,000 (green), and 30,000 (purple) monocytes, represented as an I / E ratio: I / E Ratio = (%CD45+ cells located on the interior of the hHMAs / %CD45+ cells located on the exterior of the hHMAs). n > 5. Value = mean ± s.e.m., 1-way ANOVA multiple comparison test.

[0020] FIG. 7 is a bar graph showing cell quantification of immune cells using flow cytometry data for CD45+ cells as a percentage of total cells at day 12, 16, and 20 with incremental monocyte additions of 0 (blue), 10,000 (red), 20,000 (green), and 30,000 (purple) monocytes (n > 7). The gray shaded area indicates the expected physiologic range of macrophages in the developing human heart (1-4%). n > 5. Value = mean ± s.e.m., 1-way ANOVA multiple comparison test.

[0021] FIG. 8 shows representative flow cytometry data showed day 20 hHMAs (left to right) with incremental monocyte additions of 0, 10,000, 20,000, and 30,000 cells. The black box represents a gate for CD45+ cells.

[0022] FIG. 9 is a bar graph representing the quantification of % CD45+ cells as determined by flow cytometry in three cell lines: H9-hESCs, Hl-hESCs, and Ll-hiPSCs. The gray shaded area indicates the typical physiologic range of macrophages in the developing human heart (1- 3%). n > 7. Value = mean ± s.e.m., 1-way ANOVA multiple comparison test.

[0023] FIG. 10 includes representative confocal IF images for DAPI (blue), CD45 (green), CD163 (cyan), and TNNT2 (red), in day 20 hHMAs at low (top row) and high magnification (bottom row). The white box indicates the location where high-magnification images were taken from the low-magnification image, n = 8. Top row scale = 200pm. Bottom row scale = 20pm.

[0024] FIG. 11 is a bar graph representing RT-qPCR analyses on day 20 hHMAs for classical proteins markers expressed in cardiac embryonic macrophages, PTPRC (CD45), CD 14, CD68, CSF1R, AIF1, CD74, MRC1 (CD206), and CCR2. n > 6. Value = mean ± s.e.m., Two-way ANOVA with multiple comparison test.

[0025] FIG. 12 includes UMAP feature plots displaying relative expression for cardiac embryonic tissue-resident macrophages (MPs) markers, PTPRC (CD45), CD14, CD68, MRC1, CSF1R, AIF1, and CD74 in hHMAs (day 15 and day 26 hHMA overlay). CCR2 is included and is not classically expressed in cardiac embryonic tissue-resident MPs. Color intensity represents the relative value of gene expression per gene. The MP cluster is labeled on each UMAP plot.

[0026] FIG. 13 includes phase-contrast microscopy images depicting individual control hHOs and hHMAs at day 20, 28, 36, 44, 52, and 60. hHMAs had 20,000 monocytes added to them on days 8, 12, and 16. Scale = 200pm.

[0027] FIG. 14 is a bar graph showing the quantification of control hHOs (blue) and hHMAs (red) area under phase-contrast microscopy at day 20, 28, 36, 44, 52, and 60. n = 8. Value = mean ± s.e.m. One-way ANOVA with Turkey’s correction with multiple comparisons.

[0028] FIG. 15 is a bar graph showing the quantification of control hHOs (blue) and hHMAs (red) circularity under phase-contrast microscopy at days 20, 28, 36, 44, 52, and 60. n = 8. For all graphs: Value = mean ± s.e.m. One-way ANOVA with Turkey’s correction with multiple comparisons.

[0029] FIG. 16 includes representative IF images of day 20, 28, 36, 44, 52, and 60 hHMAs stained for TNNT2 (red), CD45 (green), CD163 (cyan), and DAPI (blue), n = 8. Scale = 200pm.

[0030] FIG. 17 is a line graph representing the cell quantification of CD45+ cells using flow cytometry for CD45+ cells as a percentage of total cells in day 20, 28, 36, 44, 52, and 60 hHMAs. The dashed line represents the MP population on day 20. The gray shaded area indicates the expected physiologic range of macrophages in the developing human heart (1-4%). n > 7. Value = mean ± s.e.m.

[0031] FIG. 18 includes high magnification IF images of a day 60 hHMAs stained for TNNT2 (red), CD45 (green), CD 163 (cyan), and DAPI (blue), n = 8. Scale = 20pm.

[0032] FIG. 19 includes UMAP dimensional reduction plots of integrated scRNA-seq data for each condition (from left to right): Day 26 hHOs and Day 26 hHMAs. Cluster identity labels are on the UMAP plots.

[0033] FIG. 20 represents the quantification of total cell count percentages per cluster for Day26 hHOs and Day 26 hHMAs. Colors of regions correspond to the adjacent legend.

[0034] FIG. 21 is a graph representing Log2 fold differences (log2FD) in the proportion of cells across clusters between Day 26 hHOs and Day 26 hHMAs. Clusters highlighted in red indicate an average |log2 fold difference! greater than 0.38 compared to hHOs (permutation test; n = 10,000).

[0035] FIG. 22 includes UMAP dimensional reduction plots of integrated scRNA-seq data of embryonic human hearts from Asp, M., et al. (2019). A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart. Cell 179, 1647-1660. (left) and Day26 hHMAs (right). Cluster labels from Asp et al. 2019 are preserved from the original text (left) and hHMA labels from FIG. 10 (right).

[0036] FIG. 23 is a dot plot of differentially expressed genes in each cluster for each condition. The color indicates the average expression level across all cells, and the circle's size means the percentage of cells within a particular cluster that expresses the respective gene.

[0037] FIG. 24A and FIG. 24B include gene set enrichment plots of differential gene expression between hHMAs and hHOs for Innate Immune Response Gene Ontology 2023 (FIG. 24A) and Leukocyte Migration Gene Ontology 2023 (FIG. 24B).

[0038] FIG. 25 includes circularized Ligand-Receptor plots for day 15 hHOs, day 26 hHOs, day 15 hHMAs, and day 26 hHMAs representing the top 10 ligand-receptor interactions for each specific cell type.

[0039] FIG. 26 is a ligand-Receptor dot plot displaying the top 20 ligand-receptor interactions between MPs and specific cell types in day 26 hHMAs.

[0040] FIG. 27 shows the top 10 upregulated ligand-receptor interactions from the differential ligand-receptor analysis between day 26 hHMAs vs day 26 hHOs.

[0041] FIG. 28 shows hallmark gene ontology biological processes that MP ligands contribute to in hHMAs.

[0042] FIG. 29 is a schematic of EV isolation and proteomic analysis from hHOs and hHMAs.

[0043] FIG. 30 is a Venn diagram displaying the distribution of proteins found in EVs unique to hHO culture (blue), hHMA culture (red), and both cultures (light grey).

[0044] FIG. 31 is a dot plot displaying differential expression of the most prevalent proteins in EVs from only hHMAs by cluster. Color is indicative of the normalized average gene expression between each cluster, and the size of the circle is indicative of the percentage of cells within the cluster that express the respective gene.

[0045] FIG. 32 is a string plot of proteins found in hHMA EVs involved in the GO Biological Processes in FIG. 33. Colors correlate to the color of bars found in FIG. 33.

[0046] FIG. 33 shows 10 significantly upregulated GO Biological Processes using the proteins found in hHMA EVs.

[0047] FIG. 34 includes gene set enrichment plots of differential gene expression between hHMAs and hHOs for Cardiac Muscle Contraction, GO Biological Process 2023.

[0048] FIG. 35 includes representative low-magnification confocal IF images for DAPI (blue), MYL3 (red), and NR2F2 (green) in hHOs (top row) and hHMAs (bottom row), n = 7. Scale Bar = 200pm.

[0049] FIG. 36 is a bar graph representing the quantification of MYL3+ area as a percentage of total area in confocal IF images of day 26 hHOs and hHMAs averaged across 5 z-slices per organoid, n = 7. Value = mean ± s.e.m., Student’s t-test.

[0050] FIG. 37 includes representative high-magnification confocal IF images of sarcomeres in day 26 hHOs (left) and hHMAs (right), n > 11. Scale = 5pm.

[0051] FIG. 38 is a bar graph representing the quantification of sarcomere length, measured from z-line to z-line between TNNT2+ signal, from high-magnification confocal IF images of sarcomeres in hHOs and hHMAs. nOrganoids / sarcomeres > 11 / 33. Value = mean ± s.e.m., Student’s t- test.

[0052] FIG. 39 is a heatmap depicting normalized log2 fold change for genes related sarcomere organization, cardiac ventricle morphogenesis, and ventricular muscle tissue development in day 15 hHOs, day 26 hHOs, day 15 hHMAs, and day 26 hHMAs. Red correlates to maximum relative expression and blue correlates to minimum relative expression.

[0053] FIG. 40 includes gene set enrichment plots of differential gene expression between hHMAs and hHOs for Phagocytosis, Recognition, GO Biological Process 2023.

[0054] FIG. 41 includes representative confocal IF images of Day 26 hHMAs for DAPI (blue),CD45 (green), and CALR (red), n = 18 organoids. Scale Bar = 200pm.

[0055] FIG. 42 includes representative confocal IF images of Day 26 hHMAs for DAPI (blue),CD45 (green), MERTK (red), and CALR (cyan), n = 14. Scale Bar = 10pm.

[0056] FIG. 43A is a bar graph representing the quantification of CD45+ signal in CALR+ / - regions of interest in Day 26 hHMAs. n = 18 organoids. For graph: Value = mean ± s.e.m., Student’s t-test.

[0057] FIG. 43B is a bar graph representing the quantification of MERTK+ punctae in CD45 cells and all other cells in Day 26 hHMAs. n = 14 organoids. For graph: Value = mean ± s.e.m., Student’s t-test.

[0058] FIG. 44 includes gene set enrichment plots of differential gene expression between hHMAs and hHOs for Collagen Fibril Organization, GO Biological Process 2023.

[0059] FIG. 45 includes representative confocal IF image for DAPI (blue) and COL1A1 (red), displaying the exterior of day 26 hHMAs at low magnification, n > 7. Scale Bar = 200pm.

[0060] FIG. 46A and FIG. 46B include representative high magnification confocal IF images of MPs for DAPI (blue), CD45 (green), TNNT2 (red), and Cx43 (white) in day 20 hHMAs. N = 10. Scale = 10pm.

[0061] FIG. 47 is a bar graph representing RT-qPCR analyses on day 20 hHMAs for AREG, a protein expressed by cardiac embryonic MPs when Cx43 GAP junctions are formed, n > 13. Value = mean ± s.e.m., Student’s t-test.

[0062] FIG. 48A includes live-cell confocal IF imaging for NucBlue™ (blue) and mCherry (red) positive MPs. n > 39. Scale = 20pm.

[0063] FIG. 48B includes representative live-cell confocal IF imaging for Fluo4 dye (green) and mCherry (red) positive MPs. n > 8. Scale = 20pm.

[0064] FIG. 48C includes representative live-cell confocal IF image for FluoVolt™ dye (green) and mCherry (red) positive MPs. n > 8. Scale = 10 pm.

[0065] FIG. 49A includes graphs showing Ca2+ transients measured using Fluo-4 live-cell confocal imaging of representative hHMAs in an mCherry-MP (bottom) and an adjacent cardiomyocyte (top), n > 8

[0066] FIG. 49B shows action potentials measured using FluoVolt™ live-cell confocal imaging of representative hHMAs in an mCherry-MP (bottom) and an adjacent CM (top), n > 8.

[0067] FIG. 50 is a dot plot displaying differential gene expression of ion channels in at least 0.01% of MPs with an average normalized expression of 0.1. Color is indicative of the normalized average gene expression between each cluster and the size of the circle is indicative of the percentage of cells within the cluster that express the respective gene.

[0068] FIG. 51 is a line graph showing average action potential waveforms of CMs and mCherry-MPs captured by FluoVolt™ live-cell imaging, n > 7.

[0069] FIG. 52A, FIG. 52B, and FIG. 52C are bar graphs representing quantified APD90 (FIG. 52A), APD50 (FIG. 52B), and APD30 (FIG. 52C) of CMs and mCherry-MPs captured by Fluovolt™ live-cell imaging, n > 7. Value = mean ± s.e.m., Student’s t-test.

[0070] FIG. 53 is a heatmap depicting normalized log2 fold change for genes involved in Phase 0, 1, 2, 3, and 4 of the cardiac action potential in day 15 hHOs, day 26 hHOs, day 15hHMAs, and day 26 hHMAs. Red correlates to maximum relative expression and blue correlates to minimum relative expression.DETAILED DESCRIPTIONA. Introduction

[0071] Tissue-resident MPs play an important role in both heart development and maintenance in mouse heart development. They have been shown to contribute to electrical conductance, valvular remodeling, lymphatic channel development, and endothelial network patterning, however their role in human heart development remains poorly understood. There is reason to believe they play an important role in human heart development. For one, MPs are present in human embryonic hearts based on single cell RNA sequencing data. Different subpopulations of MPs can be identified, suggesting that MPs may play multiple roles in heart development. Congenital heart defects (CHDs) are common in Downs Syndrome patients, and the underlying cause of the CHDs in Downs Syndrome patients is not completely understood. However, there was a study that demonstrated macrophage inhibitory factor is highly expressed in patients with Downs syndrome. Furthermore, patients with bicuspid stenosis, the most common CHD, display increased MP presence versus non-diseased tricuspid valves in the same patients, suggesting a potential link between immune dysregulation and heart development.

[0072] In one embodiment, the present disclosure provides an hPSC differentiation protocol that generates human heart organoids (hHOs) with integrated MPs (i.e., human heart-macrophage assembloid (hHMA)), which allows for the investigation of tissue-resident MPs and their impact on human heart development and disease. Although there are known hHOs, hHOs with integrated MPs described herein is the first of its kind and is completely derived from hPSCs.

[0073] Without being bound by theory, the inventors have discovered an embryonic MP integration method by differentiating monocytes from hPSCs and adding them to developing hHOs. They discovered that the differentiated monocytes migrate into the hHO, and the monocytes differentiate into MPs over time and can persist in the hHO. The method provides hHOs with integrated MPs that are advanced, three-dimensional hHO models derived from hPSCs. The hHOs accurately recapitulate human cardiac development, including significant aspects of function (electrophysiology, metabolism, cardiac chambers, cardiac-relevant cell types), which allows for investigation of tissue-resident MPs and their impact on human heart development and disease.B. Definitions

[0074] The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0075] The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, elements, compositions, steps, integers, operations, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Although the open-ended term “comprising,” is to be understood as a non-restrictive term used to describe and claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of’ or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and / or process steps, 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,” the alternative embodiment excludes 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 characteristics 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 characteristics can be included in the embodiment.

[0076] Any method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed, unless otherwise indicated.

[0077] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, threeor more compounds, four or more compounds, or greater numbers of compounds. The term "plurality" refers to "two or more."

[0078] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0079] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0080] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0081] Throughout this disclosure, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art. Particularly in reference to a given quantity, number or percentage, “about” is meant to encompass deviations of plus or minus ten percent (± 10). For example, about 5% encompassesany value between 4.5% to 5.5%, such as 4.5, 4.6, 4.7, 4.8, 4.9, 5, 4.1, 5.2, 5.3, 5.4, or 5.5. Accordingly, unless otherwise indicated, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0082] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0083] As will be understood by one skilled in the art, for any and all purpose, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member.

[0084] As used herein, any reference to “days of hHO differentiation” starts counting from day zero of hHO differentiation.

[0085] The term “integrate” or “integrated” as used herein refers to a component that may exist within the exterior or the interior of another component. For example, “integrated MPs” are MPs that may exist and / or persist within the exterior and / or the interior of an hHO. For example, some integrated MPs may stick to the outside of the hHO, while others may migrate inside the hHO. Integrated MPs may adopt an M0 (unpolarized), Ml (pro-inflammatory), and / or M2 (antiinflammatory) phenotype.

[0086] The term “tissue-resident” as used herein refers to MPs that are seeded during embryonic development, persist throughout a subject’s life (or an hHO’s life), and adopt an M2 phenotype that is tailored to the tissue in which they reside (e.g., cardiac tissue). The number of tissue resident MPs in a subject may decrease due to aging, disease, or dysfunction. The tissue resident MPs with an M2 phenotype may be replaced with recruited Ml MPs. Ml MPs may be recruited from bone marrow, have short half-lives (about 30 days), and / or only reside in diseased dysfunctional tissue.

[0087] The term “differentiate” or “differentiated” as used herein refers to the process in which a cell, such as an hPSC, changes from one type to a different type. A cell that underwentdifferentiation may be described herein as differentiated. For example, a cell may change to a more specialized type.

[0088] Human pluripotent stems cells (hPSCs) will be understood to include human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs).

[0089] The term “monocyte” or “monocytes” as used herein refers to viable, cluster of differentiation 14 (CD 14) positive (i.e., CD14+) cells. Monocytes may exist in suspension and / or are non-tissue bound. Monocytes may express CD 163, CD45, or a combination thereof. A viable cell may be a healthy cell that has the capacity to sustain growth, development, or reproduction.

[0090] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. In particular, this disclosure utilizes routine techniques in the field of immunology and organoid production.C. Method of generating an hHO with integrated MPs

[0091] In one embodiment, the present disclosure provides a method for generating a human heart organoid (hHO) with integrated macrophages (MPs). The hHO with integrated MPS may be referred to herein as a human heart-macrophage assembloid (hHMA). Assembloids are generated by putting in contact with two different organ tissue types followed by developmental self- organization-driven integration.

[0092] The method may comprise adding monocytes to a medium comprising an hHO, where the hHO has differentiated for a number of days. The method may also comprise agitating the hHO, along with the monocytes and the medium, to generate an hHO with integrated MPs. In a certain embodiment, the method of generating an hHO with integrated MPs may include adding monocytes to a medium comprising an hHO, where the hHO has differentiated for at least 5 days counting from day zero, and agitating the hHO, monocytes, and medium to generate an hHO with integrated MPs (i.e., an hHMA). In some embodiments, the method of generating an hHO with integrated MPs may occur in vitro.

[0093] Monocytes, a type of white blood cell (i.e., leukocyte), may differentiate into MPs or monocyte-derived dendritic cells. In certain embodiments described herein, monocytes may differentiate into MPs. In some embodiments, monocytes can express high levels of the CD14 cell surface receptor (CD 14++CD 16" monocytes). In some embodiments, monocytes express low levels of CD14 and additional co-expression of the CD16 receptor (CD14+C16++monocytes). In some embodiments, the monocytes express high levels of CD14 and low levels of CD16 (CD14++CD16+monocytes). In some embodiments, the monocytes are viable. In a particular embodiment, the monocytes are viable, CD14+monocytes.

[0094] Viable monocytes, in other words, may be healthy cells that have the capacity to sustain growth, development, or reproduction. Examples of viable monocytes are monocytes that can stay alive or can divide and multiply. In certain embodiments, viable monocytes may be monocytes that are able to differentiate into MPs. In certain embodiments, viable monocytes may be monocytes that are able to integrate into an hHO. In some embodiments, cell (e.g., monocyte) viability may be measured by the proportion of live, healthy cells within a population.

[0095] In some embodiments, monocytes may be derived from human pluripotent stem cells (hPSCs). In some embodiments, monocytes may be generated by following a step-wise hPSC embryoid body differentiation protocol, such as that described in Monkley, S. et al. (2020) Optimised generation of iPSC-derived macrophages and dendritic cells that are functionally and transcriptionally similar to their primary counterparts. PLoS One, 15: 1-17, which is incorporated by reference in its entirety. In some embodiments, the monocytes may be cultured in a media, such as Roswell Park Memorial Institute (RPMI) media.

[0096] As described herein, differentiation (also known as cellular differentiation) may refer to the process in which a cell, such as a stem cell (e.g., an hPSC or bone marrow-derived stem cell), changes from one type to a different type of cell. A cell that underwent differentiation may be described herein as a differentiated cell. For example, a cell may differentiate into a more specialized type. For example, a less specialized cell may develop or mature to possess a distinct form and / or function (e.g., the cell may turn into a different type of cell). Differentiation may happen multiple times during development of multicellular organisms to form complex systems of tissues and cell types. Differentiation may change a cell’s shape, size, polarity, membrane potential, metabolic activity, responsiveness to signals, etc. Differentiation may occur in a cell without changing the cell’s DNA sequence itself. A cell that can differentiate into all cell types of an adult organism is known as pluripotent (also known as embryonic stems cells in animals). In some embodiments, cells may change their gene expression profiles during differentiation. A cell may also differentiate due to modifications in gene expression.

[0097] In some embodiments, after cells have differentiated into monocytes (e.g., stem cell- derived monocytes), the monocytes may be purified. In some embodiments, the monocytes may be purified using magnetic-activated cell sorting (MACS). In some embodiments, monocytes may be purified using MACS for CD 14 expression. In some embodiments, the monocytes may express CD45, CD14, CD68, CD163, or a combination thereof.

[0098] hHOs may be formed via known methods. For example, an early embryonic hHO can be formed from differentiation of hiPSCs as described by International Patent Publication No. WO 2021 / 257812, which is hereby incorporated by reference in its entirety. For example, hHOs may be generated by forming a cellular aggregate of pluripotent stem cells, activating Wnt signaling in the cellular aggregate to cause the cellular aggregate to differentiate into a three- dimensional cardiac mesoderm, and inhibiting Wnt signaling in the cardiac mesoderm to form the hHO. In some embodiments, hHOs may be generated via self-organization. In some embodiments, hHOs may be three-dimensional. The same hPSCs used to generate monocytes may also be used to generate hHOs. For example, hHOs may be generated by hiPSCs or hESCs. Non-limiting examples of hPSCs include the hiPSC lines iPSC-Ll, AICS-0037-172, and iPSCORE_16_3, hESC line H9, and a combination thereof. In some embodiments, hHOs may be differentiated from hiPSC embryoid bodies to the cardiac lineage. The hHOs may be differentiated from hiPSCs to the cardiac lineage from / between zero and 7 days through a timewise 3-step (activation / inhibition / activation) Wnt pathway modulation strategy. hHOs may include myocardial tissue, endocardial tissue defining at least one chamber, and epicardial tissue disposed on at least an outer surface of the myocardial tissue. In specific embodiments, hHOs are cultured in RPMI media. In some embodiments, the hHOs may beat.

[0099] The developing / differentiating hHO may express factors / proteins known to facilitate / promote monocyte adhesion, monocyte-macrophage differentiation, macrophage migration, macrophage viability, or a combination thereof. In some embodiments, no substances are added to the medium, monocytes, or hHO to promote MP survival and / or MP integration into the hHO. In some embodiments, the hHO may express vascular cell adhesion protein 1 (VCAM1), colony stimulating factor 1 (CSF1), also known as macrophage colony-stimulating factor (M- CSF), or both VCAM1 and CSF1. VCAM1 may be an adhesion molecule that monocytes bind to in cardiovascular endothelial cells. CSF1 may be a mediator for monocyte-MP differentiation and / or MP chemotaxis. In some embodiments, hHOs may express VCAM1 and / or CSF1 at day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 of hHO differentiation, counting from day zero. In certain embodiments, expression of VCAM1 and / or CSF1 occurs on day 5 of hHO differentiation. In certain embodiments, expression of VCAM1 and / or CSF1 occurs on day 8 of hHO differentiation. In certain embodiments, an increased expression of VCAM1 and / or CSF1 occurs on day 8 of differentiation. In some embodiments, VCAM1 and / or CSF1 may be expressed in any of the cell types in the hHO cell population.

[0100] In some embodiments, monocytes are added to a medium comprising an hHO. In some embodiments, monocytes are added to a medium comprising an hHO to generate an hHO with integrated (and in some instances, tissue-resident) MPs.

[0101] In some embodiments, the monocytes being added to a medium comprising an hHO may be viable, CD 14+ monocytes. In some embodiments, the monocytes may be added on day zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 of hHO differentiation, counting from day zero. In some embodiments, monocytes are added on day 5 of hHO differentiation, counting from day zero. In some embodiments, monocytes are added on day 8 of hHO differentiation, counting from day zero. In certain embodiments, monocytes may be added to a medium comprising an hHO more than once. For example, monocytes may be added to a medium comprising an hHO 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, monocytes may be added on or after day zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of hHO differentiation, counting from day zero. For example, monocytes may be added on or after day 5 of hHO differentiation, counting from day zero. In some embodiments, monocytes may be added on days 8, 12, and 16 of hHO differentiation, counting from day zero. In some embodiments, monocytes may be added every three to five days of hHO differentiation, counting from day zero. In some embodiments, monocytes may be added at certain intervals and certain levels to maintain an appropriate level of monocytes in the culture medium comprising the hHO. In some embodiments, the appropriate level of monocytes may be maintained through day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 of hHO differentiation, counting from day zero. In certain embodiments, the appropriate level of monocytes may be maintained though day 20 of hHO differentiation. In a certain embodiment, monocytes may be added to the medium comprising an hHO one or more times, such as once, twice, three, four, five, or six times, on or after day 5 up to and including day 20 of hHO differentiation.

[0102] In some embodiments, about 1-100,000 monocytes may be included in each addition to a medium comprising an hHO. In some embodiments, about 10,000-40,000 monocytes may be added in each addition. For example, about 10,000, about 15,000, about 20,000, about 25,000, about 30,000, about 35,000, or about 40,000 monocytes may be added in each addition. In a certain embodiment, about 20,000 monocytes are added in each addition. In a specific embodiment, about 20,000 monocytes are added to a medium comprising an hHO three times. Thus, the total number of monocytes added to the medium comprising an hHO over time may be about 60,000 monocytes. In some embodiments, if monocytes are added more than once, a different amount of monocytes may be added with each addition. For example, about 10,000 monocytes may be added in the first addition, about 20,000 in the second addition, and about 30,000 in the third addition or vice versa.

[0103] In some embodiments, the monocytes may be added to a medium comprising more than one hHO. In some embodiments, the medium may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hHOs.

[0104] In some embodiments, the monocytes being added to the medium comprising an hHO may be freshly generated. “Freshly generated” monocytes refers to monocytes that are taken from a fresh culture of monocytes, such as from monocytes proliferating in a fresh, liquid culture. In other words, fresh monocytes have not been preserved in any way. For example, fresh monocytes have not been frozen and / or cryopreserved. In other embodiments, the monocytes being added to the medium comprising an hHO may be preserved. For example, the monocytes may have been frozen and / or cryopreserved before being added to the medium comprising the hHO.

[0105] As referred to herein, the media / medium that hPSCs are differentiated to monocytes in may be any medium that enables monocyte / macrophage production (i.e., hPSC to monocyte differentiation medium). For example, the medium may a cell growth medium, and may include a medium supplement. As referred to herein, the media / medium that organoids are differentiated in (i.e., medium to which the monocytes will be added) may be any medium that can be used for cardiac organoid differentiation and / or maintenance (i.e., organoid differentiation medium). In certain embodiments, the organoid differentiation medium may be RPMI (such as RPMI / B27), Dulbecco's Modified Eagle Medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), etc.). In some embodiments, the medium may be a liquid medium. In some embodiments, the medium may be a non-gel liquid suspension culture. A “non-gel” may refer to a media that does not contain hydrogel, agarose, etc. In some embodiments, a hydrogel may contain crosslinked 3D networks of hydrophilic polymer chains. In some embodiments, a hydrogel may be a biphasic material, a mixture of porous, permeable solids, and at least 10% by weight or volume of interstitial fluid composed completely or mainly by water. In some embodiments, the medium may not contain a natural hydrogel. In some embodiments, the medium may not contain a synthetic hydrogel. A suspension culture, as referred to herein, may include cells and / or cell aggregates dispersed and / or growing in a moving, liquid medium. In other words, the medium may not be an adherent medium. In some embodiments, the media / medium may be referred to as a cell media / medium and / or a culture media / medium. In some embodiments, the medium may include, or be supplemented with, B27, insulin, penicillin-streptomycin, or a combination thereof.

[0106] In some embodiments, the medium containing an hHO and monocytes may be agitated. Agitation may comprise any form of movement that disturbs a component. For example, agitationmay disturb a liquid cell culture medium, including the components inside, such as monocytes and / or hHOs.

[0107] Agitating a medium, including agitating the monocytes and hHO in the medium, may include resuspending the medium, monocytes, and hHO. Resuspension, as referred to herein, may refer to the process of taking a component out of suspension, followed by replacing the component back in suspension. Resuspension may be achieved via pipette resuspension. Pipette resuspension, as used herein, may refer to the process of taking a component out of suspension using a pipette, followed by placing the component back in suspension using a pipette. For example, monocytes and / or hHOs may be taken up via pipette from a liquid media, and then placed back into the medium. In some embodiments, all the contents of a container, such as an Eppendorf® tube, may be taken up into a pipette, followed by placing all the contents back into the same or different / new container. In a certain embodiment, all the medium, monocytes, and hHO are taken up into the pipette, and are replaced back into a container. The pipette resuspension process may include multiple rounds of taking contents up and replacing them. For example, one act of pipette resuspension may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 rounds of taking contents of a container up via pipette and placing them back in a container. The act of pipette resuspension may occur one or more times, such as once, twice, three, four, five, or six times during hHO differentiation. For example, pipette resuspension may occur three times during hHO differentiation. In one embodiment, pipette resuspension occurs on the same day as monocytes are added to the medium (the first medium or fresh medium). Pipette resuspension may occur on or after day 5 up to day 20 of hHO differentiation, counting from day zero. For example, pipette resuspension may occur on days 8, 12, and 16 of hHO differentiation, counting from day zero. Pipette resuspension may be performed using any type of pipette, such as a transfer pipette, micropipette, multichannel pipette, serological pipette, etc.

[0108] Agitation may additionally or alternatively include shaking the medium, monocytes, and hHO. For example, the medium, monocytes, and hHO may be shaken at certain time intervals (e.g., every 3-5 days), perhaps by using a vortex. Additionally or alternatively, the medium, monocytes, and hHO could be shaken constantly, for example on a shaker table. Additionally or alternatively, agitation may include centrifuging the media, monocytes, and hHO. For example, centrifugation may occur before shaking the media, monocytes, and hHO.

[0109] In some embodiments, the medium containing an hHO may be replaced. For instance, the medium containing the hHO may be in a tube. To replace the medium, a certain amount of medium may be taken out of the tube (and may be discarded), followed by adding a certain amountof medium to the tube. In certain embodiments, the hHO is not replaced and may remain in the tube while the medium is replaced. In some embodiments, the medium may be replaced with the same type of medium (e.g., RPMI medium). In some embodiments, the medium may be replaced with a different type of medium. In some embodiments, the medium may be replaced 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the medium may be replaced on day 8 of hHO differentiation, counting from day zero. In some embodiments, the medium may be replaced three times during hHO differentiation. In some embodiments, the medium may be replaced on days 10, 14, and 18 of hHO differentiation, counting from day zero. In some embodiments, the medium may be replaced by using a pipette.

[0110] The medium, as described herein, may be referred to as a first or a fresh medium. A first medium may be the initial (i.e., original) medium including the hHO. In other words, the first medium may be the medium including the hHO, where the first addition of monocytes is added. A fresh medium, as referred to herein, may be a subsequent (i.e., new, second, third, fourth) medium that replaces the first or subsequent medium. When medium is replaced, all of the medium or a portion of the medium may be removed and replaced with fresh medium. Monocytes may be added to the first medium or to a fresh medium, and the first or fresh medium may be agitated.

[0111] In some embodiments, replacing the medium that the hHO is in may occur on the same day monocytes are added. If the medium is replaced on the same day monocytes are added, the medium may be replaced before or after the addition of monocytes. In a certain embodiment, the medium may be replaced before the addition of monocytes. Alternatively, the medium may be replaced on a day that monocytes are not added.

[0112] Replacing the medium may additionally or alternatively occur on the same day as agitating the medium. If the medium is replaced on the same day as agitating the medium, agitation may occur before or after the media is replaced. In a certain embodiment, the agitation may occur after the medium is replaced. In other embodiments, replacing the medium may occur on a day that the medium is not agitated.

[0113] Additionally or alternatively, monocytes may be added on the same day the medium is agitated. If the monocytes are added on the same day the medium is agitated, the agitation may occur before or after the addition of monocytes. In a specific embodiment, agitation may occur after the addition of monocytes. In other embodiments, monocytes may be added on a day that the medium containing an hHO is not agitated.

[0114] In some embodiments, monocytes added to the media containing an hHO may differentiate into MPs. In some embodiments, monocytes may differentiate into MPs uponintegration into the hHO. In some embodiments, MPs may integrate into an hHO by means of the hHO’s expression of factors / proteins known to facilitate / promote monocyte adhesion, monocytemacrophage differentiation, macrophage migration, macrophage viability, or a combination thereof, such as VCAM1 and / or CSF1. Additionally or alternatively agitating the medium, monocytes, and hHO may help facilitate MP integration into the hHO.

[0115] In some embodiments, hHOs may not contain MPs before the addition of monocytes. In other words, there may not be an endogenous population of MPs in the developing hHO prior to the addition of monocytes. In some embodiments, there is not a significant endocardial derived MP population in the hHO prior to adding monocytes.

[0116] In some embodiments, monocytes are added to a medium comprising an hHO in an amount and frequency to achieve an amount of integrated MPs of about 0.1% to about 10%, about 1% to about 10%, about 1% to about 9%, about 2% to about 8%, about 3% to about 7%, about 4% to about 6%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6% about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 5% to about 10%, about 6% to about 10%, about 7% to about 10%, about 8% to about 10%, or about 9% to about 10% of the total cell population in the hHO. In a certain embodiment, the amount of monocytes added to a medium comprising an hHO results in an amount of integrated MPs of about 1% to about 5% of the total cell population in the hHO. In some embodiments, the amount of monocytes added to a medium comprising an hHO results in an amount of integrated MPs of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10.0% of the total cell population in the hHO.

[0117] In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the integrated MPs have migrated inside of the hHO. In a certain embodiment, a majority (over 50%) of the integrated MPs have migrated inside of the hHO. In an example embodiment, 70% of the integrated MPs may have migrated inside of the hHO.

[0118] In some embodiments, integrated MPs may last / persist in an hHO for at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days, counting from day zero of hHO differentiation. In certain embodiments, integrated MPs may persist in an hHO for at least 60 days. For example, the integrated MPs may not dedifferentiate back into monocytes or hPSCs. In some embodiments, the integrated MPs that persist for at least 60 daysmay be integrated into the exterior of the hHO. In some embodiments, the integrated MPs that persist for at least 60 days may be integrated into the interior of the hHO. Additionally or alternatively, the integrated MPs that persist for at least 60 days may express be tissue-resident MPs. In some embodiments, the tissue resident M2 may adopt an M2 phenotype. Additionally or alternatively, the integrated MPs may adopt an MO phenotype and / or an Ml phenotype.

[0119] In some embodiments, integrated MPs function similar to MPs found in a human heart. For example, integrated MPs may function similar to MPs in a human heart at the same stage of development (i.e., embryonic heart development). In some embodiments, integrated MPs may express CD45, CD163, CD14, CD163, CD68, CSFR1, AIF1, CD74, CD206, or a combination thereof. Integrated MPs may express proteins found in typical cardiac embryonic MPs. Integrated MPs may express genes highly relevant to positive regulation of cytokine production, regulation of IL- 16 production, regulation of IL-8 production, regulation of TNF production, phagocytosis, or a combination thereof, all of which are known functions of tissue-resident MPs.

[0120] In some embodiments, the integrated MPs may contribute to electrical conductance, valvular remodeling, lymphatic channel development, endothelial network patterning, or a combination thereof. For example, integrated MPs may form gap junctions with cardiomyocytes. For example, integrated MPs may form connexin 43 (Cx43) gap junctions with CMs in the hHO interstitium and / or on the outside of the hHO. hHOs with integrated MPs may also demonstrate increased expression of amphiregulin (AREG), a protein secreted by MPs when Cx43 gap junctions form between MPs and CMs. Calcium (Ca2+) transients and action potentials in integrated MPs may also be in sync with adjacent CMs. In some embodiments, MPs may integrate into the hHO’s conductance system.

[0121] As discussed, the method disclosed herein generates an hHO with integrated MPs. In some embodiments, the hHO may possess similar characteristics to a human heart on a tissue, cellular, and / or molecular level during human heart development and disease.

[0122] In some embodiments, integrated MPs may enhance cell-cell communication within the hHOs with integrated MPs. In some embodiments, cells within the hHOs with integrated MPs may communicate with unique proteins compared to control hHOs. In some embodiments, MPs may remodel hHOs, for example, by promoting efferocytosis, extracellular matrix organization, sarcomere growth, and / or ventricular morphogenesis. In some embodiments, MPs may enhance catabolism in hHOs with integrated MPs. In some embodiments, MPs may express unique ionchannels compared to other cell types in the hHOs with integrated MPs. In some embodiments,MPs expressing unique ion-channels may affect the local electrophysiology of hHOs with integrated MPs.

[0123] In some embodiments, hHOs with integrated MPs may include similar cell populations compared to a human heart at the same stage of development. In some embodiments, integrated MPs may elicit cell population changes in a developing / differentiating hHO. For example, compared to an hHO without integrated MPs, an hHO with integrated MPs may have expansion of ventricular cardiomyocytes (VCMs) and valvular cells (VCs), and decreases in stromal cells (SCs), conductance cells (CCs), and epicardial cells (EPCs). In some embodiments, day 26 EPCs in hHOs with integrated MPs may show positive regulation of vascular endothelial growth factor (VEGF) as compared to hHOs without integrated MPs. In some embodiments, cardiac fibroblasts (CFs) may show increased expression of genes related to extracellular matrix organization as compared to control hHOs with no integrated MPs.

[0124] In some embodiments, the hHO with integrated MPs can physically beat. In some embodiments, the hHO with integrated MPs can physically beat similar to a human heart at the same stage of development. In some embodiments, hHOs may maintain beating function (i.e., maintain a beat) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days. In some embodiments, hHOs may maintain beating function for at least 60 days. Maintaining a beat, as referred to herein, may include maintaining hHO structure and function. In other words, the hHO may be self- sustained. In some embodiments, the hHO may have detectable calcium signaling. In some embodiments, the hHO may have measurable electrophysiology. In some embodiments, the hHO may contain chambers. For example, the hHO may contain 4 chambers. In some embodiments, the hHO may be composed of cell types typically found in a human heart. For example, an hHO may be composed of cardiomyocytes, epicardial cells, endocardial cells, endothelial cells, cardiac fibroblasts, or a combination thereof. In some embodiments, the hHO contains cardiomyocytes, epicardial cells, endocardial cells, endothelial cells, cardiac fibroblasts, and MPs. In some embodiments, the hHO contains tissue-resident MPs. In some embodiments, the tissue-resident MPs adopt an M2 phenotype. Additionally or alternatively, in some embodiments, the hHO contains Ml and / or M2 MPs.

[0125] In some embodiments, hHOs may be used for research purposes, such as disease / condition modeling, drug testing, and identification of therapeutic targets. In some embodiments, hHOs may be used for investigating tissue-resident MPs and their impact on human heart development and disease. In some embodiments, the hHO may be used for disease modelingcongenital heart defects (CHDs), such as those from maternal diabetes or those present in patients with Downs Syndrome.EXAMPLES

[0126] The following examples are merely illustrative, and do not limit this disclosure in any way.

[0127] Statistics and reproducibility. All analyses were performed using GraphPad 9 software. All data presented a normal distribution. Statistical significance was evaluated with a standard unpaired Student t-test (2-tailed; P < 0.05) when appropriate. For multiple-comparison analysis, 1-way ANOVA was applied when appropriate (P < 0.05). The specific number of independent organoids used and the number of independent experiments (batches) for every experiment are indicated in the “BRIEF DESCRIPTION OF THE DRAWINGS” section herein. If not explicitly stated, images and data presented were from organoids derived from the hESC H9 cell line.

[0128] Data availability. Bulk RNA-seq and single-cell RNA sequencing (scRNA-seq) data sets have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus repository under accession numbers GSE153185 and GSE280807, respectively. Proteomics data sets have been deposited in the Metabolomics Workbench repository under accession MSV000096319.

[0129] EXAMPLE 1 : Generation of hHOs

[0130] Stem cell culture. The following human pluripotent stem cell lines were used for this study: ESC-H9, ESC-H9-mCherry, ESC-H1, hiPSC-Ll. Pluripotency and genomic stability were tested for all hPSC lines used. hPSCs were cultured in Essential 8® Flex medium with 1% penicillin / streptomycin (Gibco®) in 6-well plates on growth factor reduced Matrigel® (Coming®) inside an incubator at 37°C and 5% CO2. hPSCs were passaged using ReLeSR passaging reagent upon reaching 60-80% confluency. ESC-H9 derived hHOs and hHMAs were used for most studies in this report unless otherwise noted.

[0131] Self-assembling hHO differentiation. A detailed protocol which describes the generation and differentiation of human heart organoids is provided. Human embryonic stem cell (hESC) line H9 (WiCell®, WA09) was grown to 60-80% confluency on a 6-well plate and dissociated using Accutase® (Innovative Cell Technologies) to obtain a single-cell solution. H9s were collected and centrifuged at 300 g for 5 minutes (min) and resuspended in Essential 8® Flex medium (Gibco®) containing 2pM ROCK inhibitor (Thiazovivin) (Sigma- Aldrich®). H9s werecounted using a MOXI Z® cell counter (Orflo®) and 10,000 cells were seeded in 100|jL per well in a round bottom 96 well ultra-low attachment plate (Costar®). The plate was then centrifuged at 100 g for 3min and subsequently placed inside a 37°C and 5% CO2 incubator (these same incubation conditions were used for the entire protocol). After 24 hours (h), 50 pL of medium was removed from each well and 200 pF of fresh Essential 8® Flex Medium with 1% penicillin / streptomycin was added to obtain a final volume of 250 pL per well. The plate was incubated for 24-h. For every media change onward, 166pE of medium was removed from each well and 166pE of the listed medium was added to each well. After incubation, medium was removed from each well and RPMI with B27 supplement without insulin (Gibco®) supplemented with 1% penicillin / streptomycin (Gibco®) (hereafter termed “RPMVB27 minus insulin”) containing CHIR99021 (CHIR99201 is a commercially available chemical compound with the CAS #252917-06-9. It is a GSK3 inhibitor / WNT activator), Bone Morphogenetic Protein 4 (BMP4, a commercially available growth factor), and Activin A (a commercially available growth factor) was added to each well to obtain final concentrations of 4 pM CHIR99021, 36 pM (1.25 ng / mE) BMP4, and 8 pM (1.00 ng / mE) Activin A. The plate was subsequently incubated, and, after exactly 24-h, the medium was removed from each well and replaced with fresh RPMI / B27 minus insulin. 24-h later, spent medium was removed from each well and RPMI / B27 minus insulin with Wnt-C59 (Selleck®) was added to obtain a final concentration of 2 pM Wnt-C59 inside each well. The plate was then incubated for 48-h. Afterwards, the medium was removed and replaced with fresh RPMI / B27 minus insulin and incubated for another 48-h. Following the incubation, spent medium was removed and replaced with RPMI with B27 supplement (with insulin) and 1% penicillin streptomycin (hereafter termed RPMI / B27). The plate was incubated for 24-h. Afterwards, medium was removed from each well and RPMI / B27 containing CHIR99021 was added to obtain a final concentration of 2 pM CHIR99021 per well. The plate was incubated for 1 hour. After 1-h, the medium was removed from each well and fresh RPMI / B27 was added to each well. The plate was incubated for 48-h. From days 9 to 20, every 48-h, media changes were performed by removing media from each well and adding fresh RPMI / B27.

[0132] EXAMPLE 2: A developmentally inspired strategy to efficiently integrate autologous hPSC-derived embryonic monoc tes in hHOs.

[0133] The following protocol was developed to incorporate MPs in hHOs. To ensure hHOs did not contain endogenous MPs, scRNA-seq data Uniform Manifold Approximation and Projections (UMAPs) of combined 26-day (d) and 15-d hHOs was analyzed, which showed no gene expression of any classic MP markers, such as PTPRC (CD45), CD68, CD163, or CD14. Also, no endocardial-derived MPs were detected, although numerous NFATC1+I CDH5+cellstypically associated with the endocardium in the hHOs were found. After verifying that there were no pre-existing MPs in hHOs, tests were run to see if autologous hPSC-derived monocytes would differentiate into cardiac tissue-resident MPs if added to hHOs with the right timing. To estimate this timing, MP integration events observed in the developing mouse and human heart were relied upon. In the mouse, the first heartbeats occur at E8.5, and yolk sac-derived MPs do not appear until E10.5. The epicardium seems to be required for cardiac seeding by the yolk sac-derived MPs, thus, the addition of monocytes to the hHOs must occur after the day 7 CHIR99201 exposure, which induces the formation of the pro-epicardial organ in the hHO model. Coronary vasculature in the mouse heart forms at E13.5, in hHOs early vasculature is detected as early as day 12 of hHO differentiation. Thus, it was determined that the first monocyte addition should occur between day 7 and day 12 of hHO differentiation. hHOs also exhibited increased expression of genes related to leukocyte adhesion, monocyte differentiation, and positive regulation of MP chemotaxis around day 7 of differentiation (FIG. 1), supporting this time window for monocyte integration. It was also noted that VCAM1 expression, an adhesion molecule necessary for cardiac-macrophage interaction and recruitment, was high at day 11 and colocalized with TNNT2 (FIG. 2), further suggesting cardiac cells were primed, a finding previously reported in embryonic mouse hearts. Several studies have demonstrated it takes approximately four days for monocytes to differentiate into MPs in vitro. Considering all these observations, it was reasoned that monocyte additions should start on day 8 of differentiation.

[0134] Human monocyte differentiation. The monocytic lineage differentiation protocol is based on a 5 sequential step protocol with some minor adjustments. The hPSC lines used were ESC-H9 or PSC-L1. To generate a more robust protocol with a higher yield of CD14+ cells, the protocol was modified by using 60 embryoid bodies (EBs) of known cell number (3xl04) per 6- well plate with 10 EBs per well instead of using 30 embryoid bodies (EBs) of known cell number (30xl03) per 6-well plate with 5 EBs per well. With these adjustments, at day 0, the differentiation and generation of EBs was started simultaneously by seeding undifferentiated single cell hPSCs at a concentration of 3xl04cells per well in a 96 Ultralow attachment plate with round-bottom wells (Coming®) in 100 pl media (Essential 8® Flex complete with 2pM ROCK inhibitor (Thiazovivin) and 80 ng / ml BMP4). The plate was centrifuged at 200xg, for 3 min. at room temperature. On day 2, the EBs were transferred to growth factor-reduced Matrigel coated wells (Coming®), five EBs per well in 6-well plates (Coming®) in the same media described above. For the rest of the differentiation, the Yanagimachi et al., 2013 protocol was followed, which is hereby incorporated by reference in its entirety: Yanagimachi, M.D., et al. (2013). Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent StemCells under Serum- and Feeder Cell-Free Conditions. PLoS One 8, 1-9. Every four days, from day 18 to day 42, the suspension cells were positively sorted by MACS using CD 14 + MicroBeads (Miltenyi Biotech®) generating 7 batches of CD 14+ monocytic lineage-directed cells. The Cao et al, 2019 protocol was adapted for magnetic-antibody cell sorting (MACS) purification, which is hereby incorporated by reference in its entirety: Cao, X., et al. (2019). Differentiation and Functional Comparison of Monocytes and Macrophages from hiPSCs with Peripheral Blood Derivatives. Stem Cell Reports 12, 1282-1297. Yields from day 26 to day 42 were significantly larger than those from day 18 and day 22 collections. Thus, suspension cells between day 26 and day 42 were positively sorted by MACS using CD 14 + MicroBeads and used for additions to human heart organoids.

[0135] Monocytes were purified using MACS for CD 14, since >94% of human cardiac tissueresident MPs express CD 14. CD14+isolated monocytes also expressed PTPRC (CD45), CD 14, CD68, and CD163, classical monocyte markers. Monocyte additions started on day 8, with followup additions on days 12 and 16 to mimic sustained MP seeding during development (FIG. 3).

[0136] Generation of hHMAs. On day 8 of hHO differentiation, CD14-MACS purified monocytes were added to human heart organoids. In more detail, before collecting the cells from Day 26 through Day 42 of monocyte factory culture, Fluorescent Activated Cell Sorting (FACS) buffer (0.5% BSA and 2mM EDTA in phosphate-buffered saline (PBS), pH=7.4) was prewarmed to room temperature. Enough Step 4 Medium from the Yanagimachi, M.D., et al. (2013) protocol was prepared so that 3mL of Step 4 Medium can replenish each monocyte factory well. To dislodge any unattached monocytes, spent monocyte factory medium was pipetted very gently in each well up and down no more than two times using a 1-ml pipet and the whole cell suspension was collected in a 50-ml tube, making sure that the monocyte factories did not become damaged. Step 4 Medium was quickly added to the monocyte factories. After collecting the monocytes, any lingering CD 14 Microbeads were washed off from the monocytes and the monocytes to organoid medium was equilibrated by gently resuspending pelleted monocytes in lOmL of RPMI +ins +B27 medium, centrifuging at 300xg for 3 min at room temperature and discarding the supernatant. This step was repeated two more times. After the last centrifuge spin, CD 14+ cells were resuspended in 1ml of RPMI +ins +B27 +P / S medium and the volume of monocytes needed to add 20,000 monocytes per organoid was calculated using RPMI +ins +B27 as the diluent. On day 10, day 14, and day 18, an RPMI +ins +B27 media change was performed. On days 12 and 16, the monocyte addition was repeated. By day 20, MP integrated organoids were ready for analysis.

[0137] Monocytes were delivered into the standard hHO-containing medium in organoid plates to promote contact and resuspended twice with each addition. No significant differences in hHO morphology were noted early on after monocyte additions of 10,000, 20,000, and 30,000 cells per day (FIG. 4), but a dose-dependent incorporation of CD45+ cells was noted (FIG. 5). Z- stack IF confocal microscopy images showed a preference for CD45+cells to populate the interior compartments of the organoids versus the hHO exterior (FIG. 5 and FIG. 6). By day 20 of hHO differentiation, adding 20,000-30,000 monocytes on days 8, 12, and 16 achieved physiologically relevant CD45+populations (about 1-3% of total cells) in hHOs, consistent with scRNA-seq data from human embryonic heart scRNA-seq datasets (FIG. 7 and FIG. 8). Monocytes and hHOs were differentiated from three different hPSC lines and similar results for monocyte integration were observed, as determined by CD45+positive content in hHOs (FIG. 9). Thus, it was concluded that hHOs can be successfully seeded and integrated with autologous MPs. This new organoid model may be referred to herein as human heart-macrophage assembloids (hHMAs), or hHOs with integrated MPs.

[0138] Organoid, and assembloid dissociation. Culture medium was carefully removed and discarded from the hHO. The hHO was washed once with PBS to remove any loosely attached cells. RPMI+B27 with 2% BSA (w / v) was prepared and filtered to make sterile. Cardiomyocyte Dissociation Medium was warmed to 37°C prior to dissociation. 500pL of the Cardiomyocyte Dissociation Medium was applied to the organoid and shaken at 37 °C at 250rpm for 5min. The dissociation medium was then collected from the organoid containing RPMI+B27 with 2% BSA. The dissociation medium addition, incubation, and collection was repeated until 20min passed. The collection was filtered with a 40pm filter and then centrifuged at 300g for 5min. The supernatant was decanted, and the pelleted cells were resuspended in a smaller volume of FACS buffer for flow cytometry or RPMI+B27 + 2% BSA for scRNA-seq. After the cells were resuspended, the cells were filtered again with a 40pm filter.

[0139] Flow cytometry. Following CD 14 MACS purification, monocytes were washed once with FACS Buffer and blocked with Human BD Fc Block™ (BD Biosciences) for lOmins at room temperature. Cells were then stained with CD45-FITC (HI30; BD Biosciences) and CD14-AF647 (63D3.rMAb; BD Biosciences) or CD163 (MOPC-21; BD Biosciences) at 4°C for 30min in the dark. Stained single-cell suspensions were washed twice with FACS buffer, resuspended in 300pL, and filtered through a 100pm cell strainer. Cell debris was excluded from the analysis with gating from the FSC-SSC plot and singlets were selected from FSC-A vs FSC-H and SSC- A and SSC-H plots. For dissociated organoids, the same protocol was followed except singlets were not ruled out during the analysis. Live / cell staining was accomplished with 7-amino-actinomycin a (7-AAD), demonstrating >90% viability in all dissociated organoid samples. MPs were gated for CD45-FITC cells vs SSC-A.

[0140] EXAMPLE 2: Integrated monocytes adopt embryonic cardiac tissue-resident MP fates and persist in hHMAs over time.

[0141] To verify that the added monocytes did become cardiac tissue-resident MPs, immunofluorescence (IF) microscopy was used to validate colocalization of CD45, a general hematopoietic cell marker, with CD163, a monocyte-MP marker (FIG. 10).

[0142] Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Following CD 14- MACS purification, monocytes were collected, pelleted, and preserved in RNAprotect® (Qiagen®) at -20°C. Organoids were harvested on day 20 and similarly stored in RNAprotect® at -20°C. RNA extraction was conducted using the Qiagen® RNEasy® Mini Kit, mostly following the manufacturer's protocol. Organoid samples were lysed with the Fisherbrand™ Bead Mill 4 Homogenizer (Fisher Scientific™) at speed setting 2 for 30 seconds. RNA concentration was determined using a NanoDrop™ One (Thermo Scientific™), and only samples with a concentration of at least 10 ng / pL proceeded to reverse transcription. cDNA synthesis was performed using the Quantitect® Reverse Transcription Kit (Qiagen®) and stored at -20°C. Primers for real-time qPCR were designed with the PrimerQuest® tool (Integrated DNA Technologies®). SYBR Green (Thermo Scientific™) served as the DNA-intercalating dye for the reaction vessel. Real-time qPCR was conducted on the QuantStudio™ 5 Real-Time PCR system (Applied Biosystems®) with a total reaction volume of 20 pL. Gene expression was normalized to Hypoxanthine phosphoribosyltransferase 1 (HPRT1) for each sample, and fold change was calculated using the double delta CT method. mRNA expression data are presented as fold change (FC) and / or log2FC relative to the control.

[0143] Gene expression analysis by qRT-PCR showed a significant level of genes expressed by embryonic cardiac tissue-resident MPs in day 20 hHMAs vs non-integrated hHOs (FIG. 11). scRNA-seq UMAPs of hHMAs showed specific expression of embryonic tissue-resident MP markers in the expected assigned MP cluster (FIG. 12). The lack of CCR2 expression, a chemokine receptor not expressed by embryonic-derived tissue-resident MPs in the heart, was also noted. Tissue-resident MPs seed the heart early in embryonic development and persist in the heart for long periods into adulthood, significantly supported by endogenously -produced factors produced by cardiac cells. To test the longevity of the MPs in hHMAs, hHMAs were cultured until day 60. It was noticed that hHMAs morphologically changed over time compared to hHOs (FIG. 13-15). While the area of the organoids did not significantly change, hHMAs had significantly highercircularity indexes than hHOs. hHMAs also presented less organoid-associated debris. IF microscopy studies confirmed the presence of MPs through day 60 of hHMA culture (FIG. 16- 18). MP numbers peaked at day 28 and then gradually decreased about 25% by day 36, remaining stable up to day 60 (FIG. 17), but always stayed within physiologically relevant levels (1-3% of the total cardiac cell population). MPs did not affect beating in the hHMAs. Moreover, from day 15 to day 26, MPs had decreased expression of genes associated with Ml or M2 MPs, CCL2. and MIF50, confirming their tissue-resident MP fate. Gene ontology of differentially expressed genes between MPs at day 26 hHMAs vs. day 15 hHMAs showed significantly higher levels of genes encoding enzymes for lipid and cholesterol metabolism, suggesting metabolic maturation, a process associated with tissue-resident MPs.

[0144] Optical coherence tomography. To study the structure of day 26 hHMAs, optical coherence tomography (OCT) was employed on day 26 hHOs and hHMAs. To perform OCT imaging, a Spectral-Domain Optical Coherence Tomography (SD-OCT) system was used. A superluminescent diode (EXALOS®, EXC250023-00) was used as the light source, with a center wavelength of about 1300 nm and a 3 dB spectrum range of about 180 nm. The spectrometer (Wasatch Photonics, Cobra 1300) featured a 2048-pixel InGaAs line-scan camera (Sensors Unlimited, Inc.®, GL2048) and achieved a maximum A-scan rate of 147 kHz. Imaging involved a 5X objective lens with transverse and axial resolutions of about 2.83 pm and about 3.04 pm, respectively. Sixteen fixed Day 26 organoids were imaged and analyzed from each group. Customized MATLAB® code was used to re-scale OCT images for isotropic pixel sizes in the x, y, and z dimensions. Amira software (Thermo Scientific™) and ImageJ was employed for segmentation, 3D rendering and other visualization. Total volume and internal cavities of the organoids were quantified from the manual segmentation using Amira.

[0145] OCT imaging revealed significantly larger interconnected hypodense regions within hHMAs not present in hHOs, suggesting MPs contributed to chamber morphogenesis and maturation. OCT imaging also showed that hHMAs were considerably more compact than hHOs, without altering the number of chambers within the organoids, suggesting improved cardiac muscle compaction. This data confirms that integrated monocytes acquire embryonic cardiac tissue-resident MPs fates, persist in hHMAs without exogenous cytokine additions, and alter the structure of the native organoids, suggesting developmental impact.

[0146] EXAMPLE 3: Single-cell transeriptomics of hHMAs reveals the effect of tissueresident MPs on cardiac development.

[0147] scRNA-seq. Libraries were prepared using the lOx Chromium Next GEM Single Cell 3' Kit, v3.1 (lOx Genomics™) and associated components. Completed libraries were quality controlled and quantified using a combination of Agilent® 4200 TapeStation® HS DNA1000 and Invitrogen™ Collibri™ Library Quantification qPCR assays. Libraries from each pool were pooled in equimolar proportions, and the pool was quantified again using the Invitrogen™ Collibri™ qPCR assay. These two project pools were then pooled in equal proportions and this final pool again quantified by qPCR. The pool was loaded onto one lane of an Illumina® NovaSeq™ 6000 S4 flow cell (vl.5) and sequencing was performed in a 2xl50bp paired end format using 300 NovaSeq™ vl.5 reagent cartridge. Base calling was done by Illumina® Real Time Analysis (RTA) v3.4.4 and output of RTA was demultiplexed and converted to LastQ format with Illumina® Bcl2fastq v2.20.0. Lor Bcl2fastq only the first 28 bases of read #1 and 100 bases of read #2 were output. The first 28bp of read 1 includes the lOx cell barcodes and unique molecular identifiers (UMIs), read 2 is the cDNA read. Demultiplexed LastQ files, were processed using lOx Genomics™ Cellranger count (v7.1.1) software using standard protocol including introns and exons data using Ensemble Homo_sapiens.GRCh38.110 genome. lOx count matrices were processed using standard Seurat (v5.0.2) pipeline and scCustomize helper functions. Cells were slightly filtered (nEeature_RNA > 2000, nEeature_RNA < 7500, nCount_RNA > 1500, nCount_RNA < 40000, percent_mito < 15). Samples were separately estimated to Cell Cycling and scaled by 'S. Score' and 'G2M. Score'. To avoid over-integration and keep heterogeneity samples were integrated using fast integration using reciprocal PCA (RPCA) by Satijalab guidelines, available at Hao, Y., et al. Dictionary learning for integrative, multimodal, and scalable single-cell analysis. Nat Biotechnol 42, 293-304 (2024)., which is hereby incorporated by reference in its entirety. UMAP dimensional reduction was generated with Louvain algorithm using the built-in Seurat implementation. Integration with published data was performed using Harmony (bioinformatics tool) (vO.l Broad) on SCT transformed data, with individual dataset counts regressed using (percent_mito, percent_ribo, S. Score, G2M. Score, nCount_RNA, nEeature_RNA). Enrichr and Hallmark (bioinformatics tools) were used to assess gene ontologies. When Eeature plots are presented, the color intensity represents the relative gene expression value per gene. Liana(bioinformatics tool) was used to elucidate ligand-receptor relationships and scDiffCom (bioinformatics tool) was used to identify differential ligand-receptor relationships between hHMAs and hHOs. Phantasus (bioinformatics tool) was used for hierarchal clustering and pseudobulk heatmap analyses.

[0148] scRNA-seq was performed on day 15 hHOs, day 15 hHMAs, day 26 hHOs, and day 26 hHMAs to determine how MP integration altered organoid cell populations and geneexpression (FIG. 19-24B). Cluster assignments were made for each scRNA-seq sample based on the top 5 differentially expressed genes per cluster. MPs constituted 1.7% of all cells on day 15 and 2.1% of all cells on day 26 hHMAs (FIG. 19-20), comparable to embryonic human hearts. Furthermore, significant cluster population changes were noted with MP integration (FIG. 20-21). In day 26 hHMAs, cardiac fibroblasts (CFs), ventricular cardiomyocytes (VCMs), and valvular cells (VCs) had significantly larger populations, while conductance cells (CCs), cardiac progenitor cells (CPCs), endothelial cells (ENCs), epicardial cells (EPCs), stromal cells (SCs), and proliferating pro-epicardial derived cells (PEDCs) had significantly smaller populations as compared to day 26 hHOs. Integrated scRNA-seq datasets from previously published human embryonic hearts revealed that hHMAs were remarkably similar and that all the key cell populations found in age-matched embryonic human hearts were present in the hHMAs (FIG. 22). Dot plot analyses further demonstrated that cluster assignments appropriately expressed genes commonly associated with each cluster (FIG. 23), MPs specifically expressed PTPRC, CD14, CD163, CSF1R, and CD68, all of which are cardiac tissue-resident MP markers. Gene set enrichment analyses (GSEA) were also performed to show that hHMAs significantly expressed gene sets associated with tissue-resident MPs function, such as the innate immune response, leukocyte migration, and IL-10 signaling (FIG. 24A-B).

[0149] EXAMPLE 4: Tissue-resident MPs program cell-cell communication in hHMAs.

[0150] One of the advantages of this model is the effective control of the organoid environment, allowing performance of in vitro cell communication studies in organoids and through the collection of cell culture media without interference from other organs or other difficult-to-control exogenous factors. scRNA-seq ligand-receptor (L-R) and differential L-R (Diff L-R) analysis were used to identify the production and interaction of macrophage-associated cytokines with cardiac cells. It was noted that MPs had a disproportionately large number of interactions with every other cardiac cell type in hHMAs (FIG. 25). Interestingly, of the top 20 most prevalent L-R interactions from MPs in hHMAs, 7 involved SPP1 (osteocalcin) (FIG. 26), which is also expressed by MPs in human embryonic hearts, suggesting a potentially unexplored critical role for SPP1 in heart development. Diff L-R interactions significantly upregulated in non- MP clusters were genes associated with regulating the integrin-mediated signaling pathway, cellmatrix adhesion, and molecular processes related to proteoglycan binding (FIG. 27). Diff L-R analyses between day 26 hHMAs and hHOs found that tissue-resident MPs upregulate interactions from CFs, VCs, proliferating PEDCs, and SCs while decreasing cell signaling from VCMs. Overall, MP inclusion resulted in the global upregulation of L-R interactions compared to control hHOs. Gene ontology analyses on the most represented ligands produced by MPs showedcontributions to epithelial-to-mesenchymal transition, the complement cascade, angiogenesis, the inflammatory response, and myogenesis (FIG. 28). Furthermore, it was confirmed that the top 15 represented MP ligands were expressed more in day 15 and 26 hHMAs compared to hHOs.

[0151] Extracellular vesicle (EV) collection and. isolation. EVs are a newly recognized cellular communication mechanism, and macrophages are known for EV release and cardiovascular physiology. Culture medium was collected from day 26 hHOs and hHMAs across one 96-well plate, totaling approximately 15 mL of medium containing EVs per sample. The medium was centrifuged at 400 x g for lOmin to remove large debris, then at 2,000 x g for 30min to eliminate large particles, such as apoptotic bodies. To isolate the EVs, the media was placed into ultracentrifuge tubes, balanced with PBS, and centrifuged at 100,000g for 90min at 4°C. After removing the supernatant, the pellet was resuspended in 1 mL of PBS. The sample volume was adjusted to two-thirds of the ultracentrifuge tube, and the sample was spun again at 100,000g for 90min at 4°C. Finally, the supernatant was discarded, and the pellet was resuspended in 1 mL of PBS.

[0152] EC-MS proteomics. LC-MS proteomic analyses of the EVs present in the cell culture medium of day 20-26 hHMAs was performed and compared to media from hHOs at the same timepoints (FIG. 29). For proteolytic digestion, SDS was added to the EV samples to 4% (w / v) and samples were digested overnight using S-trap™ (Protifi™) according to manufacturers’ instructions, using trypsin added to 500ng. After digestion, peptides were eluted from the S-trap™, dried in a vacuum centrifuge and frozen at -20°C. Digested samples were re-suspended in 2% acetonitrile (ACN) / 0.1% trifluoroacetic acid (TFA) to 20pL. An injection of lOpL was automatically made using an EASYnLC 1000 (Thermo Scientific™) onto an Acclaim™ PepMap™ RSLC 0.1mm x 20mm C18 trapping column (Thermo Scientific™) and washed for about 5 minutes with buffer A. Bound peptides were then eluted over 35min onto a Acclaim™ PepMap™ RSLC 0.075mm x 150mm resolving column (Thermo Scientific™) with a gradient of 5%B to 19%B from Omin to 19min, 19%B to 40%B from 19min to 24min and 40%B to 90B% from 24min to 25min. After the gradient, the column was washed with 90%B for lOmin (Buffer A = 99.9% Water / 0.1% Formic Acid, Buffer B = 80% Acetonitrile / 0.1% Formic Acid / 19.9% Water) at a constant flow rate of 300nl / min. Eluted peptides were sprayed into a Q-Exactive™ (Thermo Scientific™) mass spectrometer using a Nanospray Flex™ ion source (Thermo Scientific™). Survey scans were taken in the Orbi trap and the top ten ions in each survey scan are then subjected to automatic higher energy collision induced dissociation (HCD). The resulting MS / MS spectra are converted to peak lists using Mascot Distiller, v2.8.5 (Matrix Sciences®) and searched against a reference protein database containing all human sequences available fromUniprot appended with common laboratory contaminants (downloaded from www.thegpm.org, cRAP project) using the Mascot2 searching algorithm, v 2.8.3. The Mascot output was then analyzed using Scaffold, v5.3.3 (www.proteomesoftware.com) to validate protein identifications probabilistically. Assignments validated using the Scaffold 1% FDR confidence filter are considered valid. Proteomics results for each group were entered into the STRING database to create a STRING network utilizing evidence and medium confidence of 0.4. The desired biological and molecular processes determined on the protein network from gene ontology were presented. Mascot parameters for all databases were as follows: allow up to two missed tryptic sites, fixed modification of carbamidomethyl cysteine, variable modification of oxidation of methionine, peptide tolerance of + / - lOppm, MS / MS tolerance of 0.02 Da, and FDR calculated using randomized database search.

[0153] hHMA EVs showed 150 unique proteins, compared to 55 unique proteins in hHOs (FIG. 30). Many unique proteins were expressed predominantly by MPs, such as LYZ, LPL, CD44, FERMT3, FTL, HLA-DRA, ITGB2, ITGAX, CAPG, and SOD2. In contrast, other proteins were expressed by different types of cells but exclusively in the presence of MPs, such as CALR, LDHA, TRAP1, MFGE8, PRDX6, VCP, RAN, and VCL (FIG. 31), suggesting MP- directed programming of cardiac cells is necessary for their production. Proteins found in the EVs of both hHMAs and hHOs were predominantly housekeeping cytoskeletal proteins (e.g., keratins). Gene ontology and STRING analyses of EV-proteins in hHMAs showed increased activation of endosomal transport, cellular response to LDL, integrin binding, ATP synthase activity, cell- matrix-adhesion, regulation of macroautophagy, actin-filament organization, and positive regulation of cell migration (FIG. 32-33). Whereas gene ontology and STRING analyses of the 20 most represented unique EV-proteins in hHOs were related to system development, regulation of cell growth, and regulation of cell population proliferation. In summary, these data show EVs from MPs significantly contribute to cell-to-cell signaling and communication within the developing human heart and identify new proteins and genes of interest for future developmental studies.

[0154] EXAMPLE 5: MPs functionally remodel hHMAs by promoting efferocytosis, ECM organization, sarcomere growth, and ventricular morphogenesis.

[0155] To determine if MPs exhibit preferential localization in certain parts of the hHMAs, IF staining and quantification of day 20 hHMAs in low-magnification confocal microscopy images was used, which showed MP predominantly localized to TNNT2+ areas, WT1+ areas, and chamber regions of the organoids.

[0156] IF staining. The IF staining protocol has been described previously. hHOs were transferred from the round bottom ultra-low attachment 96 well plate to 1.5 mL microcentrifuge tubes (Eppendorf®) using a cut 200 pL pipette tip (to increase tip bore diameter as to not disturb the organoid). Organoids were washed one time with PBS to remove any loosely attached cells. Organoids were fixed in 4% paraformaldehyde (VWR) in PBS for 30min. Following, organoids were washed using PBS- Glycine (1.5 g / L) three times for 5min each. Organoids were then blocked and permeabilized using a solution containing 10% Donkey Normal Serum (Sigma- Aldrich®), 0.5% Triton™ X- 100 (Sigma- Aldrich®), and 0.5% BSA (Thermo Scientific™) in PBS on a thermal mixer at 300rpm at 4°C overnight. Organoids were then washed 3 times using PBS and incubated with primary antibodies within a solution containing 1% Donkey Normal Serum, 0.5% Triton X-100, and 0.5% BSA in PBS (hereafter termed “Antibody Solution”) on a thermal mixer at 300rpm at 4°C for 24-h. Following, organoids were washed 3 times for 5min each using PBS. Organoids were then incubated with secondary in Antibody Solution on a thermal mixer at 300rpm at 4°C for 24-h in the dark. Subsequently, organoids were washed 3 times for 5min each using PBS and mounted on glass microscope slides (Fisher Scientific™). 90 pm Polybead Microspheres (Polyscience, Inc.®) were placed between the slide and a No. 1.5 coverslip (VWR) to provide support pillars such that the organoids could retain three dimensionality. Organoids were transferred to the glass microscope slides using a cut 200pL pipette tip and mounted using a clearing solution described previously.

[0157] Confocal microscopy and image analyses. IF images were acquired using a Nikon® Instruments Al Confocal Laser Microscope. Images were analyzed using Fiji. For cell quantification of TNNT2, MYL3, and CD45 area was determined by quantifying the total positive pixel signal for each antigen and dividing by the total pixel shadow of the organoid for a given z- stack. This was done for a minimum of 3 z-stack images per organoid and averaged amongst each other to generate a percentage of area that is positive for a given protein. The EE ratio (FIG. 6) was determined by quantifying CD45+ area in z-stacks inside of the organoids (interior) and dividing the CD45+ area in in z-stacks that are on the surface (exterior) of the organoids. Sarcomere length was determined by measuring the distance between z-lines in high- magnification IF images using Fiji (FIG. 38). The region of interest (ROI) tool was used to trace the CALR+ areas in hHMAs (FIG. 43A). CD45+ signal that was within the CALR+ region of interest was considered (CALR+ area) and CD45+ signal that was outside the CALR ROI was considered (CALR- area). MERTK puncta quantifications were done by counting the number MERTK+ puncta within CD45+ cells as compared to other cells in frame of the high- magnification image (FIG. 43B). Nuclei (DAPI+ areas) were quantified and assigned to CD45+Cells or Other Cells for the analyses. Furthermore, the Fiji ROI tool was used to outline TNNT2+, WT1+, or hollow regions (cavities) within a minimum of 3 z-stacks per organoid. CD45+ signal was categorized into TNNT2+ area, WT1+ area, or cavities if it fell within these respective ROIs. The CD45+ signal was then summed and used as the denominator for each categorization, providing a CD45+ Cells % for each ROI.

[0158] WT1 is expressed by VCs, CFs, SCs, and EPCs in hHMAs, indicating MPs accumulate more with cardiac interstitial cells than cardiomyocytes. A recent study showed MPs enhanced the contractility and morphogenesis of VCMs from hPSC-derived engineered heart tissues. Combined with the findings of a higher composition of VCMs in day 26 hHMAs (FIG. 20-21), evidence that MPs enhance hHMA contractility and ventricular cardiomyocyte morphogenesis through transcriptomics and IF microscopy was next discovered. Differential gene expression showed gene set enrichment for cardiac muscle contraction in hHMAs vs. hHOs (FIG. 34). IF microscopy of day 26 hHMAs showed significantly higher contractile protein expression (TNNT2+, MYL3+) than hHOs (FIG. 35-36). High-magnification confocal images of sarcomeres in day 26 hHMAs revealed longer sarcomere structures compared to hHOs (FIG. 37-38), a sign of cardiomyocyte maturity. Genes involved in sarcomere organization, cardiac ventricle morphogenesis, and ventricular muscle tissue development were elevated in hHMAs at day 15 and day 26 compared to hHOs (FIG. 39). Furthermore, MPs are known to participate in homeostatic functions like efferocytosis and cardiomyocyte exopher removal in the heart. GSEA for differentially expressed genes between hHMAs and hHOs indicated significant enrichment in phagocytosis recognition pathways (FIG. 40), highlighting the involvement of MPs in clearing apoptotic cells and cellular debris within hHMAs. Phase-contrast microscopy of hHMAs and hHOs over a time course from day 20 to day 60 showed significantly less cellular debris, which was defined as the shadow surrounding the organoids from phase-contrast images. This is supported by pseudo-bulk transcriptomic data that indicated upregulated gene expression for processes associated with phagocytosis, apoptotic cell clearance, and ECM remodeling in hHMAs, particularly at day 26, compared to hHOs. CALR is an “eat-me” signal expressed by both live and apoptotic cells that is recognized by MPs. Confocal imaging of day 26 hHMAs displayed localization of CD45+ MPs with CALR+ cells (FIG. 41, FIG. 42, and FIG. 43A-B), indicating physiological efferocytotic function. Quantitative analyses revealed that a significant proportion of CD45+ MPs were associated with CALR+ regions. IF staining for MERTK, a receptor involved in cardiac MP efferocytosis and disposal of cardiac exophers, showed significantly increased MERTK+puncta in CD45+ MPs compared to other cell types within day26 hHMAs. These findings underscore the role of MPs in facilitating efferocytosis within hHMA culture, a function traditionally associated with MPs in cardiac tissue.

[0159] MPs are also known to interact with, and remodel, the ECM. GSEA identified significant upregulation of genes involved in collagen fibril organization from hHMAs compared to hHOs (FIG. 44), suggesting MP-driven ECM remodeling. Confocal images of day 26 hHMAs stained for COL1A1 (alpha-1 type collagen) display altered collagen deposition in the MP- containing regions (FIG. 45). Pseudo-bulk transcriptomic data also shows increased relative expression in genes related to ECM assembly, cardiac epithelial-to-mesenchymal transition, and collagen fibril organization in hHMAs versus hHOs. RT-qPCR analyses further corroborated these findings by showing increased expression of COL1 Al in day 20 hHMAs compared to hHOs. Together, these findings suggest that autologous cardiac MPs alter the ECM within hHMAs.

[0160] EXAMPLE 6: MPs enhance catabolic processes in hHMAs.

[0161] GSEA of differentially expressed genes between hHMAs and hHOs identified significant upregulation of glycolytic processes in hHMAs. A further breakdown of the top 10 gene ontology processes from the 250 most differentially expressed genes in day 26 hHMAs compared to hHOs showed significant enrichment in various catabolic processes, including glycolysis, mitochondrial function, and fatty acid metabolism. These findings indicate that MPs are needed for modulating metabolic activity in hHMAs. Feature plots of VCMs on day 26 hHMAs and hHOs showed increased expression of IGFBP2, IGF2, PGK1, ENO1, and GPI. A heatmap illustrating the normalized log2 fold change (log2FC) of crucial metabolic genes revealed upregulation of glycolysis, insulin-like growth factor receptor (IGFR) signaling, mitochondrial electron transport (cytochrome C to oxygen), fatty acid beta-oxidation, lactate metabolism, and intracellular oxygen homeostasis in hHMAs. These findings demonstrated that MPs in hHMAs enhance catabolic processes, including glycolysis and mitochondrial function, contributing to the overall metabolic regulation and energy homeostasis in hHMAs.

[0162] EXAMPLE 7: MPs form GAP junctions with cardiomyocytes and contribute to the electrophysiology profile of hHMAs.

[0163] Studies in the last decade have highlighted the contribution of MPs to cardiac electrophysiology. hHMA staining was to determine if MPs participated in the electrophysiology of hHMAs. High-magnification confocal IF images of day 20 hHMAs stained for CD45, TNNT2, and connexin 43 (CX43) showed the formation of GAP junctions between MPs and cardiomyocytes (FIG. 46A-B). CX43 expression in the regions of MP-cardiomyocyte contact suggested functional coupling between the two cell types. Analysis of bulk RNA-seq from day 20hHMAs and hHOs revealed a significant upregulation of AREG (FIG. 47), a protein associated with the formation of CX43-mediated GAP junctions between MPs and cardiomyocytes in mice hearts, indicating that MPs in hHMAs form GAP junctions with cardiomyocytes. Live-cell confocal imaging of genetically labeled mCherry MPs (FIG. 48A-C and FIG. 49A-B) showed live MPs integrated in hHMAs, and subsequent functional imaging with Fluo-4 dye (for Ca2+ activity) and FluoVolt™ dye (Invitrogen™) (for membrane potential) illustrated MPs with synchronized calcium signaling and action potentials alongside adjacent cardiomyocytes. To elucidate which MP- specific ion channels may be contributing to signaling within hHMAs, a dot plot of differential ion channel gene expression in hHMAs showed MPs express predominantly calcium and potassium ion channels, with high specificity for CACNA1F, KCNA3, KCNJ10, KCNMA1, KCNN4. TRPA1, TRPC2. TRPM2, TRPV2, and TRPV4 (FIG. 50).

[0164] Live-cell imaging for calcium and voltage recordings. Intracellular Ca2+and ion signaling was visualized using Fluo4 and FluoVolt™ dyes, respectively, imaged and analyzed as previously described: Volmert, B., et al. (2023). A patterned human primitive heart organoid model generated by pluripotent stem cell self-organization. Nat. Commun. 14( 1 ) : 8245. Both dyes were prepared via the manufacturer’s instructions. Additionally, NucBlue™ (Thermo Scientific™) was used to visualize cell nuclei. NucBlue™ was prepared by adding two drops per milliliter to basal media (RPMI +ins). hHOs were washed twice using 166 pL of RPMI +ins, then 166 pL of the NucBlue™ solution was added to achieve a final concentration of 100 nM. Organoids were incubated for 30 min at 37°C and 5% CO2. Organoids were then washed twice using basal medium and transferred to a chambered coverglass slide (Cellvis®) using a truncated 200 pL pipette tip. Images were acquired using a Cellvivo microscope (Olympus 1X83) under normoxic culture conditions at 37°C. Samples were excited with 360 nm (for NucBlue™), 488nm (for Fluo4 / FluoVolt™), and 594nm (for mCherry-MPs) light. Live-cell imaging videos were collected for at least 20 seconds at a frame rate of 100 frames / second. Data was processed using the Fiji and analyzed using the multi-measure tool. Samples were excited at 494 nm excitation and emissions were collected at 506nm. Data was processed using Fiji. Baseline Fo of fluorescence intensity was calculated using the lowest 50 intensity values in the acquired dataset. Fluorescence change AF / Fo was calculated using the equation:AF > (F - Fp) Fo Fo

[0165] EADs, DADs, and SCaEs were quantified by identifying the presence of at least one of these phenomena in one hHMA video file.

[0166] Quantified values for action potential duration (APD) at 90%, 50%, and 30% repolarization (APD90, APD50, APD30) demonstrated significant differences between cardiomyocytes and MPs, with MPs displaying prolonged APD90, APD50, and APD30 as compared to cardiomyocytes (FIG. 51 and 52A-C). Moreover, heatmap analysis of normalized log2FC pseudo-bulk gene expression data (FIG. 53) revealed upregulation of genes associated with Phase 2, 3, and 4 of the cardiac action potential in day 26 hHMAs when compared to hHOs. These findings suggest that MPs actively contribute to the electrophysiological activity and maturation of hHMAs.

Claims

WHAT IS CLAIMED IS:

1. A method of generating a human heart organoid (hHO) with integrated macrophages, the method comprising: adding monocytes to a first medium comprising an hHO, wherein the hHO has differentiated for at least 5 days counting from day zero; agitating the hHO, the monocytes and the first or a fresh medium to generate an hHO with integrated macrophages.

2. The method of claim 1, wherein the monocytes are added to the first or fresh medium comprising the hHO in an amount and frequency to achieve an amount of integrated macrophages of about 1% to about 5% of the total cell population in the hHO.

3. The method of claims 1 or 2, wherein the monocytes are added to the first or fresh medium comprising the hHO one or more times, such as once, twice, three, four, five or six times, on or after day 5 up to and including day 20 of hHO differentiation counting from day zero.

4. The method of claim 3, wherein the monocytes are added to the first or fresh medium comprising the hHO every three to five days of hHO differentiation counting from day zero.

5. The method of any one of the previous claims, wherein about 10,000 to about 40,000 monocytes are included in each addition.

6. The method of claim 5, wherein about 20,000 monocytes are included in each addition.

7. The method of any one of the previous claims, wherein the monocytes are freshly generated.

8. The method of any one of the previous claims, wherein at least about 50%, about 60%, about 70%, about 80%, or about 90% of the integrated macrophages have migrated inside of the hHO.

9. The method of any one of the previous claims, wherein the agitating step comprises: resuspending the hHO, the monocytes and the first or fresh medium; shaking the hHO, the monocytes and the first or fresh medium; orcentrifuging the hHO, the monocytes and the first or fresh medium followed by shaking the hHO, the monocytes and the first or fresh medium.

10. The method of claim 9, wherein the agitating step comprises resuspending the hHO, the monocytes and the first or fresh medium by pipette resuspension.

11. The method of claim 10, wherein the pipette resuspension occurs one or more times, such as once, twice, three, four, five or six times, on or after day 5 up to and including day 20 of hHO differentiation counting from day zero.

12. The method of claim 11, wherein the pipette resuspension occurs on the same day as the monocytes are added to the first or fresh medium comprising the hHO.

13. The method of any one of claims 9-12, wherein resuspending the hHO, the monocytes and the first or fresh medium occurs after the addition of monocytes.

14. The method of any one of the previous claims, wherein the first and fresh medium is a nongel liquid suspension culture.

15. The method of any one of the previous claims, wherein the first medium is replaced at least once with fresh medium.

16. The method of any one of the previous claims, wherein the hHO beats.

17. The method of any one of the previous claims, wherein the macrophages integrated into the hHO express CD45, CD163, CD14, CD163, CD68, CSFR1, AIF1, CD74, CD206, or a combination thereof.

18. The method of any one of the previous claims, wherein the integrated macrophages form gap junctions with cardiomyocytes.

19. The method of any one of the previous claims, wherein the hHO expresses one or more protein that promotes monocyte adhesion, monocyte-macrophage differentiation, macrophage migration, macrophage viability, or a combination thereof.

20. The method of claim 19, wherein the protein is VCAM1, CSF1, or a combination thereof.

21. An hHO with integrated macrophages prepared according to the method of any one of the previous claims.

22. A method of generating an hHO with integrated macrophages, the method comprising: adding monocytes to a first medium comprising an hHO, wherein the hHO has differentiated for at least 5 days counting from day zero; and wherein the monocytes are added to the first or fresh medium at least three times; and wherein about 10,000 to about 40,000 monocytes are included in each addition; and agitating the hHO, the monocytes and the first or fresh medium by pipette resuspension to generate an hHO with integrated macrophages; wherein the first and fresh medium is a non-gel liquid suspension culture; and wherein the hHO beats.

23. An hHO with integrated macrophages prepared according to the method of claim 22.

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