Engineered human cardiac tissue

Abstract

The present application relates to methods for generating engineered human pluripotent stem cell-derived cardiac tissue, engineered cardiac tissue produced by such methods, and methods of their use.

Description

[Technical Field]

[0001] The present application relates to methods for generating engineered human pluripotent stem cell-derived cardiac tissue, engineered cardiac tissue produced by such methods, and methods of their use. [Background technology]

[0002] Despite many breakthroughs in cardiac surgery and medicine, heart attacks and heart failure remain the most prominent health problems. Heart disease is also a leading cause of death and disability in children, affecting up to 1 in 100 live births. Studies have shown that adults diagnosed with congestive heart failure (CHF) have an approximately 50% mortality rate within 5 years of diagnosis. An emerging and concerning trend is the sharp increase in the number of children and adults with CHD hospitalized for heart failure. Surgical advances over the past 20 years have dramatically improved survival rates for childhood heart disease patients; now, more than 85% of children with CHD survive into adulthood. As a result, CHD is now considered a lifelong condition.

[0003] Heart failure is characterized by the loss or dysfunction of cardiac muscle cells, either due to ischemic heart disease, hypertensive heart disease, congenital heart disease, or idiopathic cardiomyopathy. Adult myocardium lacks the ability to regenerate, and damaged cardiac tissue is replaced by non-contractile scar tissue. Currently, heart failure can only be resolved through heart transplantation, but the gap between the number of donors and the number of heart failure patients requiring transplants is widening. Current treatment options are inadequate, and new approaches that will fundamentally change patients' outcomes are essential.

[0004] The directed differentiation of human pluripotent stem cells (hPSCs), including both human embryonic stem cells (hES) and human induced pluripotent stem cells (hiPS), toward different cellular endpoints has enabled the generation of organoid models of various human tissues, including cardiac tissue.Tissue engineering techniques have aimed to generate stem cell-derived functional cardiac tissue that can be used for disease modeling and treatment.

[0005] However, existing methods and protocols for producing engineered cardiac tissue have not been able to accurately recapitulate the composition and function (e.g., contractile force) of native cardiac tissue. Various protocols reported in the literature produce cardiac tissue composed of cardiomyocytes and interstitial cells (fibroblasts) (see, e.g., WO 2015 / 040142), but they typically lack endothelial cells, the most abundant non-muscle cell population in the human heart, and other vascular cells (smooth muscle cells). While cardiomyocyte, interstitial cell, and endothelial cell populations can be easily obtained and subsequently combined through different, separate differentiation protocols, combining or mixing cell types from separate differentiation protocols introduces undesirable complexity and variability.

[0006] Furthermore, current protocols and cell culture media compositions attempt to promote the maturation and improved contractile force of tissue-engineered cardiac tissue by enhancing oxidative metabolism (Mills et al., 2017. Proc. Natl. Acad. Sci. USA, 114(40):E8372-E8381). However, oxidative metabolism may not be optimal for survival and engraftment after transplantation into a hypoxic environment in vivo. Oxidative metabolism also inhibits cardiomyocyte proliferation (Mills et al., 2017), which may not be optimal for tissue growth after transplantation in vivo.

[0007] There remains a need in the art for methods for generating pluripotent stem cell-derived cardiac tissue that overcome these drawbacks. Summary of the Invention

[0008] Through detailed studies, the inventors have developed a protocol for generating engineered human stem cell-derived cardiac tissue that contains the appropriate diversity of cell types found in native cardiac tissue and can be derived from a single differentiation protocol.

[0009] According to a first aspect, the present invention provides a method for producing engineered human cardiac tissue comprising endothelial cells, the method comprising culturing a cell suspension comprising a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population in the presence of an FGFR1 agonist and a PDGFR agonist for a time and under conditions sufficient to produce engineered human cardiac tissue comprising endothelial cells.

[0010] According to a second aspect, the present invention provides a method for producing engineered human cardiac tissue, comprising the steps of: i) mixing a cell suspension comprising a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population with a flowable hydrogel composition, wherein the cell suspension is in a serum-free medium; ii) filling the flowable hydrogel composition comprising the cell suspension into a mold and incubating the hydrogel for a time and under conditions sufficient to solidify the hydrogel; and iii) culturing the hydrogel from step ii) in serum-free medium, wherein the human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population is contacted with an FGFR1 agonist and a PDGFR agonist, thereby providing engineered human cardiac tissue comprising cardiomyocytes, stromal cells / fibroblasts, and endothelial cells.

[0011] According to a third aspect, the present invention provides engineered human cardiac tissue comprising at least about 30% hPSC-derived cardiomyocytes, not more than about 35% hPSC-derived stromal cells / fibroblasts, and not more than about 30% hPSC-derived endothelial cells, wherein the listed cells are distributed throughout the hydrogel composition.

[0012] According to a fourth aspect, the present invention provides engineered human cardiac tissue comprising hPSC-derived cardiomyocytes, hPSC-derived stromal cells / fibroblasts, and hPSC-derived endothelial cells, wherein the enumerated cells are derived from a single differentiation process, and the enumerated cells are distributed throughout the hydrogel composition.

[0013] According to a fifth aspect, the present invention provides engineered human cardiac tissue produced according to the method of the first aspect.

[0014] According to a sixth aspect, the present invention provides a method for analyzing the biological effect of at least one test compound or biologically active substance on cardiac cells, the method comprising contacting engineered human cardiac tissue of any one of the second, third or fourth aspects with the test compound or biologically active substance, incubating the tissue in the presence of the test compound or biologically active substance, and analyzing the biological effect.

[0015] According to a seventh aspect, the present invention provides engineered human cardiac tissue of any one of the second, third or fourth aspects for use as a graft in the treatment of diseased or damaged cardiac tissue in a subject in need thereof.

[0016] According to an eighth aspect, the present invention provides a method for treating diseased or damaged cardiac tissue in a subject in need thereof, the method comprising transplanting in the subject engineered human cardiac tissue of any one of the second, third or fourth aspects.

[0017] According to a ninth aspect, the present invention provides the use of engineered human cardiac tissue of any one of the second, third or fourth aspects in the manufacture of a medicament for the treatment of diseased or damaged cardiac tissue in a subject in need thereof. [Brief explanation of the drawings]

[0018] [Figure 1]A) Schematic of the cardiac differentiation and tissue engineering protocol. Human pluripotent stem cells were differentiated into cardiac cells. On day 15, the cells were enzymatically dissociated, mixed with hydrogel, and then placed into a custom-designed PDMS mold. The tissue was cultured on the custom-designed scaffold for approximately 7 days before being removed from the mold for implantation. B) Photographs of the bioengineered cardiac tissue patch (BHTP) are shown. C-E) Immunofluorescence and histological images of fixed, paraffin-embedded, and sectioned BHTPs are shown. The immunofluorescence images show that cells within the fibrin matrix express cardiac-specific markers, including α-actinin, myosin light chain 2 (MLC2v) (panel C), and cardiac troponin T (cTnT) (panel D). Nuclei in C) and D) are labeled with Hoechst. E) Histological images are hematoxylin and eosin staining, showing the overall tissue structure of the BHTP and the cardiac cells distributed within it. [Figure 2] Single-cell RNA-sequencing UMAP plot showing cell clusters present in BHTP (serum-free (sf) "fresh" medium), including cardiomyocytes (purple), endothelial cells (red), fibroblasts / stromal cells (green), smooth muscle cells (blue), and cardiac progenitor cells (gold). [Figure 3] A heatmap showing the expression of cell type-specific marker genes across a wide range of cell types is shown: CM(Prlf), cardiomyocyte (proliferative); CM, cardiomyocyte; Endo, endothelial cell; Fibro / Stromal, fibroblast / stromal cell; and Smc, smooth muscle cell; cardiac progenitor cell. [Figure 4] A UMAP plot showing the expression of cardiomyocyte (proliferative) markers (ANLN, TOP2A and CDK1) across samples is shown. [Figure 5] A UMAP plot showing the expression of cardiomyocyte markers (TNNT2, ACTN2 and MYH7) across samples is shown. [Figure 6] A UMAP plot showing the expression of endothelial cell markers (PECAM1, ENG, EMCN) across samples is shown. [Figure 7]A UMAP plot showing the expression of smooth muscle cell markers (TAGLN and ACTA2) across samples is shown. [Figure 8] A UMAP plot showing the expression of fibroblast / stromal cell markers (LAMB1, COL1A2 and COL1A1) across samples is shown. [Figure 9-1] BHTP (new medium) cardiomyocytes are characterized by decreased expression of genes involved in redox processes and fatty acid oxidation. A) Single-cell RNA sequencing UMAP plot showing cell clusters present in BHTP (new medium) compared to BHTP (old medium) and cardiomyocyte clusters used for downstream gene set enrichment analysis (GSEA). The proliferative cardiomyocyte (CM) subcluster is shown in light blue. B) Heatmap showing expression of genes associated with Gene Ontology (GO) biological processes related to redox, fatty acid oxidation, and muscle contraction. Markers for these processes are upregulated during human development (primary cardiomyocytes) from fetal to adulthood. Markers are also elevated in BHTP (old medium) compared to new medium. C) Dot plot showing exemplary genes for biological processes, demonstrating decreased expression of oxidative stress markers in BHTP (new medium). [Figure 9-2]BHTP (new medium) cardiomyocytes are characterized by decreased expression of genes involved in redox processes and fatty acid oxidation. A) Single-cell RNA sequencing UMAP plot showing cell clusters present in BHTP (new medium) compared to BHTP (old medium) and cardiomyocyte clusters used for downstream gene set enrichment analysis (GSEA). The proliferative cardiomyocyte (CM) subcluster is shown in light blue. B) Heatmap showing expression of genes associated with Gene Ontology (GO) biological processes related to redox, fatty acid oxidation, and muscle contraction. Markers for these processes are upregulated during human development (primary cardiomyocytes) from fetal to adulthood. Markers are also elevated in BHTP (old medium) compared to new medium. C) Dot plot showing exemplary genes for biological processes, demonstrating decreased expression of oxidative stress markers in BHTP (new medium). [Figure 10] Relative gene expression for glycolysis marker (PKM2) and redox markers (ALDH1A1, AGMO, DPYD, GPX3, CYP1B1, PLIN5) after pseudo-bulk analysis of cardiomyocytes in snRNA-seq data obtained from engineered human cardiac tissue prepared in SF medium or MM ("old") medium is shown. Data are presented as log2 relative gene expression normalized to the housekeeping gene TUBA1A. [Figure 11] The new medium, BHTP, retains a higher percentage of proliferative cardiomyocytes. The pie chart shows the percentage (%) of proliferative cardiomyocytes in the total cell population: New medium rep1: 2.66%, New medium rep2: 3.98%, Old medium rep2: 1.12%. The percentage (%) of proliferative cardiomyocytes in the total cardiomyocyte population is as follows: New medium rep1: 7.55%, New medium rep2: 8.93%, Old medium rep2: 2.80%. [Figure 12]Violin plots from single-cell RNA-seq of BHTP (fresh medium) showing FGF receptor expression in different cell clusters. FGFR1 is the predominant FGF receptor expressed in BHTP. FGFR1 is expressed in fibroblasts / stromal cells, cardiomyocytes, endothelial cells, and cardiac progenitor cells. [Figure 13] Violin plots from single-cell RNA-seq of BHTP (fresh medium) showing PDGF receptor expression in different cell clusters. PDGFRB is the predominant PDGF receptor expressed in BHTP. PDGFRB is predominantly expressed in the fibroblast / stromal cell population. [Figure 14-1] Fresh serum-free (SF) human heart tissue is shown to be resistant to hypoxia. A) Schematic of the hypoxia study. B) Staining after hypoxia in control and SF human heart tissue. C) Nuclear staining intensity is higher in SF human heart tissue. D) Cardiomyocyte intensity is unchanged in human heart tissue. E) Lactate dehydrogenase is lower in SF human heart tissue. F) Lactate is elevated in SF human heart tissue. G) Lactate-to-pyruvate ratio is highest in SF human heart tissue after hypoxia. [Figure 14-2] Fresh serum-free (SF) human heart tissue is shown to be resistant to hypoxia. A) Schematic of the hypoxia study. B) Staining after hypoxia in control and SF human heart tissue. C) Nuclear staining intensity is higher in SF human heart tissue. D) Cardiomyocyte intensity is unchanged in human heart tissue. E) Lactate dehydrogenase is lower in SF human heart tissue. F) Lactate is elevated in SF human heart tissue. G) Lactate-to-pyruvate ratio is highest in SF human heart tissue after hypoxia. [Figure 15]Implantation of cardiac tissue patches in vivo. A) A patch was sutured onto the right ventricle of a 6-month-old sheep heart. B) Images showing two BHTPs sutured onto the right ventricle of a 6-month-old sheep heart, demonstrating near-complete coverage of the entire surface of the right ventricle. C) To help protect the patch, the pericardium was closed with three stitches. D)-F) The patch was shown to be intact in situ after a 2.5-hour acute monitoring period (D and E) and after removal (F). [Figure 16] 1 shows functional validation of the patch in vivo. Steady-state pressure-volume (PV) curves are shown before patch implantation and at 15 and 90 minutes after patch implantation. [Figure 17] A highly linear right ventricular (RV) preload recruitable stroke work-end diastolic volume relationship is shown before patch implantation and at 15 and 90 minutes after patch implantation. [Figure 18] Changes in mean aortic blood pressure (BP, Panel A), mean pulmonary artery (PA) blood pressure (Panel B), heart rate (Panel C), right ventricular (RV) stroke volume (Panel D), maximum rate of change in RV blood pressure, an index of RV contractility (RV dP / dtmax, Panel E), and RV preload recruitment stroke work index (PRSWI, Panel F) are shown at 15-minute intervals before (Pre) and after patch implantation. Important: Patch implantation did not result in immediate changes in any of the variables, and with the exception of a slight increase in PA blood pressure (<1 mmHg) and heart rate (10 beats / min), no variables changed over the subsequent observation period up to 120 minutes after patch implantation. DETAILED DESCRIPTION OF THE INVENTION

[0019] definition Definitions of common terms in cell and molecular biology and biochemistry are provided in The Merck Manual of Diagnosis and Therapy, 20 thEdition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 9780911910421, 0911910425); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 2008 (ISBN 3527305424, 9783527305421); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Wemer Luttmann, published by Elsevier, 2006; Janeway’s Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2016 (ISBN 9780815345510, 0815345518); Lewin’s Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4 thed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,USA(2012)(ISBN 1936113414);Davis et al,Basic Methods in Molecular Biology,Elsevier Science Publishing,Inc.,New York,USA(2012)(ISBN 044460149X);Laboratory Methods in Enzymology:DNA,Jon Lorsch(ed.)Elsevier,2013(ISBN 0124199542);Laboratory Methods in Enzymology:RNA,Jon Lorsch(ed.)Elsevier,2013(ISBN:9780124200371,0124200370);Current Protocols in Molecular Biology(CPMB),Frederick M.Ausubel(ed.),John Wiley and Sons,2014(ISBN Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), Immunological Methods, Ivan Lefkovits, Benvenuto Pemis, (eds.) Elsevier Science, 2014 (ISBN: 9781483269993, 148326999X), the contents of all of which are incorporated herein by reference in their entireties.

[0020] As used in this specification and the appended claims, the singular and singular forms "a," "an," and "the" optionally include plural referents unless the content clearly dictates otherwise. For example, "a" cell includes one cell, one or more cells, and a plurality of cells.

[0021] As used herein, the term "about," unless otherwise specified, refers to + / - 10%, more preferably + / - 5%, and more preferably + / - 1% of the specified value. As used herein, "up to about" refers to an amount of the referenced entity that may be zero but does not exceed the specified value. For example, "up to about 10%" refers to an amount between 0% and about 10%.

[0022] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as explicitly supporting both meanings or either meaning.

[0023] Throughout this specification, the term "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0024] Numerical ranges are inclusive of the numbers defining the range. Every maximum numerical limitation given throughout this specification should be understood to include every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification should be understood to include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification should include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0025] The headings provided herein are not intended to limit the disclosure.

[0026] Throughout this specification, references to a particular gene or protein may be used interchangeably, and one of skill in the art will understand in context whether the reference is intended to refer to a particular gene or to the protein encoded by that gene.

[0027] The terms "human pluripotent stem cells" and "hPSCs" refer to cells derived from, obtained from, or arising from human tissue that exhibit pluripotency. hPSCs may be human embryonic stem cells or human induced pluripotent stem cells.

[0028] Human pluripotent stem cells can be derived from the inner cell mass or can be reprogrammed using Yamanaka factors from many fetal or adult somatic cell types. hPSCs can be generated using somatic cell nuclear transfer.

[0029] The terms "human embryonic stem cells," "hES cells," and "hESCs" refer to cells derived from, obtained from, or arising from a human embryo or blastocyst, which are self-renewing, pluripotent, or totipotent, and have the capacity to give rise to all of the cell types present in an adult animal. Human embryonic stem cells (hESCs) can be isolated, for example, from human in vivo preimplantation embryos, in vitro fertilized embryos, or human blastocysts obtained from one-cell human embryos expanded to the blastocyst stage.

[0030] The terms "induced pluripotent stem cells" and "iPSCs" refer to cells derived from, obtained from, or arising from any type of human adult somatic cell that has been reprogrammed to a pluripotent state through the expression of exogenous genes, such as transcription factors, including a preferred combination of OCT4, SOX2, KLF4, and c-MYC. hiPSCs exhibit a level of pluripotency comparable to hESCs, but can be derived from patients and used for autologous therapy, with or without simultaneous gene correction prior to differentiation and cell delivery.

[0031] More generally, the methods disclosed herein can be applied to any pluripotent stem cells derived from any patient, or to hPSCs that are subsequently modified using gene editing to create mutant models, or to mutant hPSCs that are corrected using gene editing, which can be by CRISPR, TALEN, or ZF nuclease technology.

[0032] As used herein, the term "cell culture" refers to any in vitro culture of cells. The term "culturing" refers to the process of growing and / or maintaining and / or manipulating cells. This term includes continuous cell lines (e.g., with an immortalized phenotype), primary cell cultures, finite-life cell lines (e.g., non-transformed cells), and any other cell populations maintained in vitro, such as oocytes and embryos. As used herein, the terms "primary cell culture" and "primary culture" refer to cell cultures obtained directly from cells in vivo, such as from animal or human tissue specimens or biopsies. These cultures can be derived from adult and fetal tissues.

[0033] A "progenitor cell" is a cell that can differentiate along one or more developmental pathways, with or without self-renewal. Typically, progenitor cells are unipotent or oligopotent, capable of at least limited self-renewal.

[0034] The terms "differentiate," "differentiating," and "differentiated" refer to the progression of a cell from an early or earlier stage in a developmental pathway to a later or more mature stage in a developmental pathway. It will be understood that "differentiated" in this context does not mean or imply that the cell has completely differentiated and lost pluripotency or the ability to progress further along a developmental pathway or along another developmental pathway. Differentiation may involve cell division.

[0035] As is well understood in the art, the stage or state of cell differentiation can be characterized by the expression and / or non-expression of one of several markers. In this context, "marker" refers to a nucleic acid or protein encoded by the genome of a cell, cell population, lineage, compartment, or subset, the expression or pattern of which changes throughout development. The expression of nucleic acid markers can be detected or measured by any technique known in the art, including, but not limited to, amplification of nucleic acid sequences (e.g., polymerase chain reaction) and nucleic acid hybridization (e.g., microarray, Northern hybridization, in situ hybridization). The expression of protein markers can be detected or measured by any technique known in the art, including, but not limited to, flow cytometry, immunohistochemistry, immunoblotting, protein arrays, and protein profiling (e.g., 2D gel electrophoresis).

[0036] Such terms are common and well understood by those of skill in the art when characterizing the phenotype of a cell. By way of additional guidance, when a cell is said to be positive for a given marker, e.g., a given gene or gene product, or to express or contain expression of that marker, one of skill in the art would conclude the presence of or evidence of a distinctive signal for that marker upon performing an assay that can detect or quantify the marker in or on a cell. Preferably, the presence of or evidence of a distinctive signal for the marker would be concluded based on a comparison of the results of the assay obtained for the cell with the results of the same assay performed on a negative control (e.g., cells known not to express the marker) and / or a positive control (e.g., cells known to express the marker). For measurement methods that allow for quantitative assessment of a marker, positive cells may generate a signal for the marker that is at least 1.5-fold higher, e.g., at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, or even higher, than the signal for the marker generated by reference cells (e.g., negative control cells) or the average signal for the marker generated by a population of reference or negative control cells. Furthermore, positive cells may generate a signal for the marker that is 3.0 or more standard deviations, e.g., 3.5 or more, 4.0 or more, 4.5 or more, or 5.0 or more standard deviations, above the average signal for the marker generated by a population of reference or negative control cells.

[0037] As used herein, the terms "culture medium" and "cell culture medium" refer to a medium suitable for supporting the growth of cells (i.e., cell cultures, cell lines, etc.) in vitro. The term is not intended to be limited to any particular culture medium. For example, the definition is intended to encompass maintenance media as well as other media for the differentiation or specialization of cells. Indeed, the term is intended to encompass any culture medium suitable for the growth of the cell culture and cells of interest.

[0038] As used herein, "tissue" refers to an aggregate of cells. In some embodiments, the cells in a tissue are coherent or fused.

[0039] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired.

[0040] The terms "treatment," "treating," "treating," and the like are used herein generally to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or adverse effects resulting from the disease are partially or completely stabilized or cured. "Treatment," as used herein, encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease or condition from occurring in a subject who may be susceptible to, but has not yet been diagnosed as having, the disease or condition; (b) suppressing disease symptoms, i.e., arresting their progression; or (c) alleviating disease symptoms, i.e., causing regression of the disease or condition.

[0041] The terms "reduce," "reduced," "reduction," "to a lesser extent," or "inhibit" are all used herein to refer to a statistically significant decrease or lowering of a characteristic, level, or other parameter. In some embodiments, "reduced," "reduction," "reduce," or "inhibit" typically refers to a decrease of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a decrease of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to the reference level. The decrease may preferably be to a level that is accepted as within the normal range for individuals without a given disorder.

[0042] The terms "increased," "increase," "increase," or "enhance," or "activate," or "to a greater extent" are all used herein to generally mean an increase in a characteristic, level, or other parameter by a statistically significant amount. For the avoidance of doubt, the terms "increased," "increase," "to a greater extent," "enhance," or "activate" can refer to an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% compared to a reference level, or any increase between 10-100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 1000-fold, or more compared to a reference level.

[0043] As used herein, a "reference level" refers to the level of a marker or parameter in a normal, otherwise unaffected cell population or tissue (e.g., a cell, tissue, or biological sample obtained from a healthy subject, or a biological sample obtained from a subject at a previous time, e.g., a cell, tissue, or biological sample obtained from a patient before being diagnosed with a disease, or a biological sample that has not been contacted with an agent or composition disclosed herein). Alternatively, a reference level can also refer to the level of a given marker or parameter in a subject, organ, tissue, or cell prior to treatment, e.g., with an agent or by administration of a transplant composition.

[0044] As used herein, "control" or "suitable control" refers to an untreated but otherwise identical cell, subject, organism, or population (e.g., a cell, tissue, or biological sample that has not been contacted with an agent or composition described herein) compared to a cell, tissue, biological sample, or population that has been contacted or treated with a given treatment. For example, a suitable control can be a cell, tissue, organ, or subject that has not been contacted with an agent as described herein or administered a cell as described herein. In one example, the control is a cell, tissue, biological sample, or population cultured in a maturation medium as described in the Examples herein.

[0045] In one or more embodiments described herein, assessing the expression of various genes comprises comparing fold changes. In one embodiment, fold changes are used to measure changes in the expression levels of genes. In one embodiment, gene expression can be expressed as relative expression compared to a housekeeping gene. In one embodiment, fold changes are measured by RPKM. As used herein, the term "RPKM" refers to "Reads Per Kilobase per Million mapped reads." The term RPKM refers to a method of quantifying gene expression from RNA sequencing data by normalizing for total read length and number of sequencing reads. In one embodiment, calculating RPKM provides a normalization for comparing gene coverage values. RPKM values correct for differences in both sequencing depth and gene length of samples. In one example, RPKM can be calculated by the following formula: numReads / (geneLength / 1000 * totalNumReads / 1,000,000) where "numReads" refers to the number of reads mapped to the gene sequence, "geneLength" refers to the length of the gene sequence, and "totalNumReads" refers to the total number of mapped reads of the sample.

[0046] The terms "agonist" or "activator" may be used interchangeably and, as used herein, refer to, for example, an activator of a pathway or signaling molecule. An agonist of a molecule can retain substantially the same or a subset of the biological activities of that molecule (e.g., FGF). For example, an FGF agonist or FGF activator refers to a molecule that selectively activates FGF signaling.

[0047] The term "inhibitor" as used herein refers to a selective inhibitor of, for example, a pathway or signaling molecule. An inhibitor or antagonist of a molecule can inhibit one or more of the activities of the naturally occurring form of that molecule.

[0048] Various embodiments are described hereinafter. Note that a particular embodiment is not intended as an exhaustive description or as limiting the broad aspects discussed herein. An aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Throughout this specification, references to "one embodiment," "an embodiment," or "an exemplary embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment," "in an embodiment," or "an exemplary embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. Furthermore, particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, while some embodiments described herein include some features but not other features included in other embodiments, it is intended that combinations of features from different embodiments are within the scope of the present invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination. Any example or embodiment herein may be applied mutatis mutandis to any other example or embodiment unless otherwise stated.

[0049] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of illustration only. Functionally equivalent methods and systems are clearly within the scope of the present disclosure as described herein.

[0050] Throughout this specification, unless otherwise specified or required by context, reference to a single step, composition of matter, group of steps, or group of compositions of matter is intended to encompass one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0051] The present disclosure will now be described by the following non-limiting examples and with reference to the accompanying drawings. Although the examples herein relate to humans and the language is primarily directed to human matters, the concepts described herein are applicable to other animals. These and other aspects and features of the present disclosure will become more fully apparent from the following description and appended claims, or may be learned by the practice of the disclosure as set forth hereinafter.

[0052] Reference herein to a patent document or other matter offered as prior art should not be construed as an admission that the document or matter was publicly known or that the information it contains was part of the common general knowledge at the priority date of any claim.

[0053] Methods for producing engineered human cardiac tissue The present invention provides a method for producing engineered human cardiac tissue comprising endothelial cells, comprising culturing a cell suspension comprising a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population in the presence of an FGFR1 agonist and a PDGFR agonist for a time and under conditions sufficient to produce engineered human cardiac tissue comprising endothelial cells.

[0054] The present invention also provides a method for producing engineered human cardiac tissue, comprising: i) mixing a cell suspension comprising a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population with a flowable hydrogel composition, wherein the cell suspension is in a serum-free medium; ii) filling the flowable hydrogel composition comprising the cell suspension into a mold and incubating the hydrogel for a time and under conditions sufficient to solidify the hydrogel; and iii) culturing the hydrogel from step ii), wherein the human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population are contacted with an FGFR1 agonist and a PDGFR agonist, thereby providing engineered human cardiac tissue comprising cardiomyocytes, stromal cells / fibroblasts, and endothelial cells.

[0055] In one embodiment, prior to the mixing in step i), an FGFR1 agonist and / or a PDGFR agonist is added to the cell suspension or flowable hydrogel composition in serum-free medium. In another embodiment, prior to the mixing in step i), a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population is cultured in serum-free medium comprising an FGFR1 agonist and a PDGFR agonist. In an alternative embodiment, after the mixing in step i), an FGFR1 agonist and / or a PDGFR agonist is added to the flowable hydrogel composition comprising the cell suspension. In another embodiment, the solidified hydrogel in step ii) is cultured in serum-free medium comprising an FGFR1 agonist and a PDGFR agonist in step iii).

[0056] The present invention provides a method for producing engineered human cardiac tissue, comprising the steps of: i) mixing a cell suspension comprising a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte populations with a flowable hydrogel composition, wherein the cell suspension is in a serum-free medium comprising an FGFR1 agonist and a PDGFR agonist; ii) filling the flowable hydrogel composition comprising the cell suspension into a mold and incubating the hydrogel for a time and under conditions sufficient to solidify the hydrogel; and iii) culturing the hydrogel from step ii) in the serum-free medium defined in step i), thereby providing engineered human cardiac tissue comprising cardiomyocytes, stromal cells / fibroblasts, and endothelial cells. In a further embodiment, an FGFR1 agonist and / or a PDGFR agonist is also added to the flowable hydrogel composition prior to the mixing in step i). In a further embodiment, after the mixing in step ii), an FGFR1 agonist and / or a PDGFR agonist is also added to the flowable hydrogel composition containing the cell suspension. In a further embodiment, the solidified hydrogel of step ii) is cultured in a serum-free medium containing an FGFR1 agonist and a PDGFR agonist in step iii).

[0057] In one embodiment, the cell suspension comprises about 50% to about 80% cardiomyocytes and about 20% to about 50% non-cardiomyocytes. In a preferred embodiment, the suspension comprises about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% cardiomyocytes and about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% non-cardiomyocytes. In a particularly preferred embodiment, the cell suspension comprises about 70% cardiomyocytes and about 30% non-cardiomyocytes. In another embodiment, the cell suspension comprises less than 5% CD31+ endothelial cells. In another embodiment, the cell suspension comprises about 50% to about 80% cardiomyocytes and about 20% to about 50% interstitial cells. In a preferred embodiment, the suspension contains about 50 to about 75%, about 50 to about 70%, about 60 to about 70%, about 50 to about 65%, about 60% to about 80%, about 55% to about 75%, about 55% to about 80%, or about 65 to about 80% cardiomyocytes, and about 25% to about 50%, about 25% to about 45%, about 30% to about 50%, about 25% to about 50%, about 35% to about 50%, about 25% to about 40%, about 25% to about 45%, about 20 to about 45%, or about 20 to about 40% interstitial cells. In preferred embodiments, the suspension comprises about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% cardiomyocytes and about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% interstitial cells. In particularly preferred embodiments, the suspension of cells comprises about 70% cardiomyocytes and about 30% interstitial cells. In preferred embodiments, the cardiomyocytes are actinocytes. + / CTNT + Cardiomyocytes, whereas non-cardiomyocytes or interstitial cells express CD90 + In some embodiments, the suspension of cells consists essentially of a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and stromal cells. In one embodiment, the suspension of cells consisting essentially of a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and stromal cells comprises about 50% to about 80% cardiomyocytes and about 20% to about 50% stromal cells, or any other range of cardiomyocytes and stromal cells described above.

[0058] In a further preferred embodiment, the cell suspension comprising cardiomyocytes and non-cardiomyocytes derived from the hPSC cell culture is obtained from a single differentiation process, i.e., it will be appreciated that the starting population of hPSCs can give rise to both cardiomyocytes and non-cardiomyocytes through induction of differentiation into cardiac lineages, such that it is not necessary to combine, mix, or seed the cell suspension with another cell population, such as endothelial cells, for further culturing the cell suspension and generating cardiac tissue comprising cardiomyocytes, stromal cells / fibroblasts, and endothelial cells.

[0059] It is the inventors' surprising discovery that by culturing a suspension of cells comprising cardiomyocytes and non-cardiomyocytes in the presence of a combination of an FGFR1 agonist and a PDGFR agonist, as described herein, it is possible to generate engineered human cardiac tissue that more appropriately reflects the heterogeneity of cell types present in native human cardiac tissue. That is, the engineered human cardiac tissue comprises cardiomyocytes, stromal cells / fibroblasts, and endothelial cells. In some embodiments, the engineered cardiac tissue may also further comprise vascular smooth muscle cells and / or cardiac progenitor cells. In some embodiments, the engineered human cardiac tissue comprises cardiomyocytes, stromal cells / fibroblasts, endothelial cells, and vascular smooth muscle cells.

[0060] In one aspect, the present invention provides a method for producing a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes, stromal cells / fibroblasts, and endothelial cells, comprising culturing a cell suspension containing a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte population in the presence of an FGFR1 agonist and a PDGFR agonist for a time and under conditions sufficient to produce a population of human pluripotent stem cell (hPSC) culture-derived endothelial cells. In another aspect, the present invention provides the use of an FGFR1 agonist and a PDGFR agonist to produce a population of human pluripotent stem cell (hPSC) culture-derived endothelial cells in a cell suspension containing a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte population. As described above, in some embodiments, the cell suspension contains about 50% to about 80% cardiomyocytes and about 20% to about 50% non-cardiomyocytes, or any other range of cardiomyocytes and non-cardiomyocytes described above. In another embodiment, the cell suspension contains less than 5% CD31+ endothelial cells. As described above, in some embodiments, the cell suspension comprises about 50% to about 80% cardiomyocytes and about 20% to about 50% interstitial cells, or any other range of cardiomyocytes and interstitial cells described above. In preferred embodiments, the cardiomyocytes are Actinin+ / CTNT+ cardiomyocytes, and the non-cardiomyocytes or interstitial cells are CD90+ stromal cells. In some embodiments, the cell suspension consists essentially of a population of cardiomyocytes and interstitial cells derived from human pluripotent stem cell (hPSC) culture. In one embodiment, a cell suspension consisting essentially of a population of cardiomyocytes and interstitial cells derived from human pluripotent stem cell (hPSC) culture comprises about 50% to about 80% cardiomyocytes and about 20% to about 50% interstitial cells, or any other range of cardiomyocytes and interstitial cells described above.

[0061] In one embodiment, the engineered cardiac tissue comprises about 30% to about 70% cardiomyocytes, about 10% to about 35% interstitial cells / fibroblasts, and about 2% to about 30% endothelial cells. In another embodiment, the engineered cardiac tissue comprises about 30% to about 70% cardiomyocytes, about 15% to about 35% interstitial cells / fibroblasts, and about 5% to about 30% endothelial cells. In another embodiment, the engineered cardiac tissue comprises about 30% to about 65% cardiomyocytes, about 15% to about 35% interstitial cells / fibroblasts, and about 5% to about 30% endothelial cells. In another embodiment, the engineered cardiac tissue comprises about 30% to about 60% cardiomyocytes, about 15% to about 35% interstitial cells / fibroblasts, and about 5% to about 30% endothelial cells. In another embodiment, the engineered cardiac tissue comprises about 30% to about 55% cardiomyocytes, about 15% to about 35% stromal cells / fibroblasts, and about 5% to about 30% endothelial cells. In another embodiment, the engineered cardiac tissue comprises about 30% to about 55% cardiomyocytes, about 20% to about 35% stromal cells / fibroblasts, and about 15% to about 30% endothelial cells. In a preferred embodiment, the engineered cardiac tissue comprises at least about 30% cardiomyocytes, no more than about 35% stromal cells / fibroblasts, and no more than about 30% endothelial cells. In some embodiments, the cardiac tissue further comprises up to about 15% vascular smooth muscle cells. In some embodiments, the cardiac tissue further comprises up to about 10% cardiac progenitor cells. In a more preferred embodiment, the engineered cardiac tissue comprises about 30% to about 50% cardiomyocytes, about 20% to about 35% interstitial cells / fibroblasts, and about 15% to about 30% endothelial cells. In a more preferred embodiment, the engineered cardiac tissue comprises about 30% to about 55% cardiomyocytes, about 20% to about 35% interstitial cells / fibroblasts, about 5% to about 30% endothelial cells, up to about 15% vascular smooth muscle cells, and up to about 10% cardiac progenitor cells. In a more preferred embodiment, the engineered cardiac tissue comprises about 30% to about 55% cardiomyocytes, about 20% to about 35% interstitial cells / fibroblasts, about 5% to about 30% endothelial cells, about 0.5% to about 15% vascular smooth muscle cells, and about 0.5% to about 10% cardiac progenitor cells.In more preferred embodiments, the engineered cardiac tissue comprises about 30% to about 55% cardiomyocytes, about 20% to about 35% stromal cells / fibroblasts, about 15% to about 30% endothelial cells, about 0.5% to about 15% vascular smooth muscle cells, and about 0.5% to about 10% cardiac progenitor cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 45% cardiomyocytes, about 30% to about 35% stromal cells / fibroblasts, and about 20% to about 24% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 45% cardiomyocytes, about 20% to about 30% stromal cells / fibroblasts, and about 15% to about 30% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 45% cardiomyocytes, about 30% to about 35% interstitial cells / fibroblasts, and about 26% to about 30% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 45% cardiomyocytes, about 20% to about 30% interstitial cells / fibroblasts, and about 15% to about 30% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 45% cardiomyocytes, about 30% to about 35% interstitial cells / fibroblasts, and about 20% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 45% cardiomyocytes, about 20% to about 30% interstitial cells / fibroblasts, and about 15% to about 25% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 45% cardiomyocytes, about 30% to about 35% interstitial cells / fibroblasts, and about 30% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 45% cardiomyocytes, about 20% to about 30% interstitial cells / fibroblasts, and about 15% to about 25% endothelial cells.

[0062] In some embodiments, the engineered cardiac tissue comprises about 2% to about 5% proliferative cardiomyocytes. In further embodiments, at least about 3% to about 10% of the cardiomyocytes of the engineered cardiac tissue are proliferative as determined by expression of one or more markers associated with cell proliferation. In further embodiments, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cardiomyocytes of the engineered cardiac tissue are proliferative as determined by expression of one or more markers associated with cell proliferation. In one embodiment, the proliferative cardiomyocytes express one or more markers selected from the group consisting of ANLN, TOP2A, CDK1, and E2F1.

[0063] Another characteristic of cardiomyocytes present in engineered cardiac tissue is that they express MMP1 + Therefore, in a preferred embodiment, the cardiomyocytes in the engineered cardiac tissue are MMP1 + is.

[0064] Another characteristic of fibroblasts present in engineered cardiac tissue is that they express MMP1 + , EMP1 + , FOXD1 + , RAB27B + , NR2F1 + , F2RL1 + , SPP1 + , TMEM158 + , PTH LH + , PHLDA2 + , MALL + , MYCT1 + , DUSP4 + , PLAU + , TMEM156 + , CD274 + , MMP10 + , ARRDC4 + , RFX8 + , M.L.P.H. + , T.H.B.D. + , HHEX + , V.G.F. + , OTULINL + , IL33 + , CA12 + , C6orf141+ , MFSD2A + and / or CARD10 + Thus, in a preferred embodiment, the fibroblasts in the engineered cardiac tissue are positive for one or more of these aforementioned markers.

[0065] As described herein, the inventors have identified FGFR1 as the predominant FGF receptor expressed in cardiac tissue engineered according to the methods of the present invention. Thus, in a preferred embodiment, the methods of the present invention comprise culturing cells in a basal medium supplemented with an FGFR1 agonist selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, the methods of the present invention comprise culturing cells in a basal medium supplemented with an FGFR1 agonist selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8, FGF9, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, the methods of the present invention comprise culturing cells in a basal medium supplemented with an FGFR1 agonist that is a member of the FGF1 subfamily, the FGF4 subfamily, the FGF8 subfamily, the FGF9 subfamily, or the FGF19 subfamily. In some embodiments, the methods of the present invention comprise culturing cells in a basal medium supplemented with an FGFR1 agonist selected from the group consisting of FGF1, FGF2, FGF4, FGF5, FGF6, FGF9, FGF16, and FGF20. More preferably, the FGFR1 agonist is FGF2. In one embodiment, the medium contains about 0.5 to 100 ng / ml of FGF2. Preferably, the concentration of FGF2 is about 1 to 50 ng / ml, more preferably about 5 to 20 ng / mL, and most preferably about 10 ng / mL.

[0066] As described herein, the inventors have also identified PDGFRβ as the predominant PDGF receptor expressed in cardiac tissue engineered according to the methods of the present invention. Accordingly, in a preferred embodiment, the methods of the present invention comprise culturing cells in a basal medium supplemented with a PDGFR agonist selected from the group consisting of PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, or PDGF-DD. More preferably, the PDGFR1 agonist is PDGF-BB. In one embodiment, the medium contains about 0.5 to 100 ng / ml of PDGF-BB. Preferably, the concentration of PDGF-BB is about 1 to 50 ng / ml, more preferably about 5 to 20 ng / ml, and most preferably about 10 ng / ml.

[0067] In one embodiment, the serum-free medium further comprises a basal medium containing albumin and transferrin. In an even more preferred embodiment, the basal medium comprises a commercially available supplement containing albumin and transferrin. In a preferred embodiment, such a supplement is B27® supplement or insulin-free B27® supplement. In a preferred embodiment, the B27® supplement or insulin-free B27® supplement is applied in an amount of 0.1-10% B27® or insulin-free B27®, preferably 0.5-8%, more preferably 1-6%, even more preferably 1.5-4%, and most preferably about 4% B27® or insulin-free B27®. In one embodiment, the B27 supplement comprises biotin, DL-α-tocopherol acetate, DL-α-tocopherol, vitamin A (acetate), BSA, fatty acid free fraction V, catalase, human recombinant insulin, human transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine HCl, glutathione (reduced), L-carnitine HCl, linoleic acid, linolenic acid, progesterone, putrescine 2HCl, sodium selenite, and T3 (triiodo-l-thyronine).

[0068] Through detailed studies, the inventors have demonstrated that culturing cell-seeded hydrogels in a culture medium with low levels of calcium is important for preserving the phenotype of engineered cardiac tissue that exhibits glycolytic metabolism and reduced contractile force. Thus, in one embodiment, the basal medium is a low-calcium medium with a calcium concentration below physiological, e.g., less than about 2 mM. In another embodiment, the basal medium is a low-calcium medium with a calcium concentration less than about 1.2 mM. In yet another embodiment, the basal medium is a low-calcium medium with a calcium concentration less than about 1 mM. In even more preferred embodiments, the basal medium is a low-calcium medium with a calcium concentration less than about 0.8 mM, more preferably less than 0.75 mM, even more preferably less than about 0.5 mM, and most preferably about 0.43 mM. In some embodiments, the basal medium is a low-calcium medium with a calcium concentration of about 0.2 mM to about 1 mM. Those skilled in the art will appreciate that any suitable basal medium with the aforementioned preferred calcium concentrations, such as alpha-MEM, DMEM, or RPMI, can be used in the present method. In another preferred embodiment, the basal medium is RPMI medium.

[0069] Typically, the culturing step is carried out for about 5 to about 7 days. Preferably, the culturing step is carried out for about 7 days. In a more preferred embodiment, the method includes replacing the medium in the culturing step with a fresh medium containing an FGFR1 agonist and a PDGFR agonist at least every two days.

[0070] In one aspect, a medium composition suitable for producing a population of human pluripotent stem cell (hPSC)-derived cardiomyocytes, stromal cells / fibroblasts, and endothelial cells is provided, the medium composition comprising a basal medium having a calcium concentration below physiological calcium concentration, an FGFR1 agonist, and a PDGFR agonist. In some embodiments, the medium composition may also comprise commercially available supplements including albumin and transferrin. The amount and concentration of each component of the medium composition are as described herein.

[0071] As shown in the examples below, engineered cardiac tissues produced by the methods of the present invention and those described below have been shown to advantageously exhibit glycolytic metabolism. This may be advantageous because it provides resistance to hypoxia-induced cell death compared to cells that use oxidative phosphorylation. Because in vivo transplantation results in a period of hypoxia, this may result in better survival. Furthermore, glycolysis supports cardiomyocyte proliferation (Mills et al. PNAS 2017), which may be advantageous for promoting an increase in cell number after transplantation.

[0072] In one embodiment, cardiac tissue generated using the methods and engineered cardiac tissue described herein exhibits a log2 relative gene expression level (normalized to TUBA1A) for PKM greater than 2. Another characteristic of cardiac tissue generated using the methods described herein is that after 24 hours in SF culture medium, the culture medium has a lactate concentration of at least 0.5 mM or at least 1 mM. In another embodiment, after 24 hours of culture of cardiac tissue generated using the methods described herein in SF culture medium, the culture medium has a lactate concentration of about 10-20 nmol / million cells / hour.

[0073] As shown in the examples below, engineered cardiac tissue produced by the methods of the present invention and the engineered cardiac tissue of the present invention have been shown to advantageously exhibit resistance to hypoxic conditions. In one embodiment, cardiac tissue produced using the methods and engineered cardiac tissue described herein exhibits a log2 relative gene expression level (normalized to TUBA1A) of less than -4 for ALDH1, AGMO, GPX3, CYP1B1, and PLIN5. Another characteristic of cardiac tissue produced using the methods described herein and the engineered cardiac tissue itself (described below) is that after 20 hours of culture under hypoxic conditions, the cell culture medium from such cultures has a lactate / pyruvate ratio of at least 1:1. Yet another characteristic of cardiac tissue produced using the methods described herein and the engineered cardiac tissue itself (described below) is that after 20 hours of culture under hypoxic conditions, cell death is at least 1-fold lower than control cardiac tissue, and cell death may be at least 2-fold, at least 3-fold, or more lower than control cardiac tissue. Cell death can be assessed using routine methods known to those skilled in the art, including, but not limited to, LDH assays. In preferred embodiments, after 20 hours of culture under hypoxic conditions of cardiac tissue produced using the methods described herein and the engineered cardiac tissue itself (described below), cell death is at least 1-fold, at least 2-fold, at least 3-fold, or more, lower compared to control cardiac tissue, as measured by LDH assay.

[0074] As shown in the examples below, engineered cardiac tissue produced by the methods of the present invention has been shown to advantageously exhibit low contractile force. The contractile force of engineered tissue can be assessed and measured using techniques known to those skilled in the art (see, e.g., Tibucy et al. Circulation. 2017;135(19):1832-1847 or Voges et al. Development 2017;144(6):1118-1127). In preferred embodiments, the engineered cardiac tissue exhibits a contractile activity of less than about 10 mN / mm.

[0075] In one embodiment, the hydrogel is composed of fibrin, collagen I, or Matrigel, or any combination thereof. In a preferred embodiment, the hydrogel is a fibrin hydrogel. In a preferred embodiment, the fibrin is formed by mixing a thrombin solution and a fibrinogen solution. Preferably, in forming the fibrin hydrogel, fibrinogen is present at a concentration of about 10 mg / mL to about 50 mg / mL. More preferably, fibrinogen is present at a concentration of about 20 mg / mL.

[0076] In another embodiment, the hydrogel can be functionalized with one or more bioactive agents. For example, the bioactive agents (e.g., small molecules, polypeptides including cytokines and chemokines, differentiation factors, signaling pathway inhibitors, etc.) can promote, for example, cell viability and further development or differentiation of cells in the resulting engineered tissue. In one embodiment, the one or more bioactive agents can be an agent selected from the group consisting of an antiproliferative agent, an immunosuppressant, a pro-angiogenic compound, an antibody or fragment or portion thereof, an antibiotic or antimicrobial compound, an antigen or epitope, an aptamer, a biopolymer, a carbohydrate, a cell adhesion mediator (such as RGD), a cytokine, a cytotoxic agent, a drug, an enzyme, a growth factor or recombinant growth factor and fragments and variants thereof, a hormone antagonist, a hormone, an immunological agent, a lipid, a metal, a nanoparticle, a nucleic acid analog, a nucleic acid (e.g., DNA, RNA, siRNA, RNAi, and microRNA agents), a nucleotide, a nutraceutical, an oligonucleotide, a peptide nucleic acid (PNA), a peptide, a prodrug, a prophylactic agent, a protein, a small molecule, a therapeutic agent, or any combination thereof.

[0077] In one embodiment, the cell suspension is mixed with the hydrogel in step i) to obtain a cell suspension of about 5×10 6 , about 10×10 6 , about 15×10 6 , about 20×10 6 , about 25×10 6 , about 30×10 6 , about 35×10 6 , about 40×106 , about 45×10 6 , about 50×10 6 , about 55×10 6 , about 60×10 6 , about 65×10 6 , about 70×10 6 , about 75×10 6 , about 80×10 6 , about 85×10 6 , about 90×10 6 , about 95×10 6 , or approximately 100 × 10 6 More preferably, the cell suspension is mixed with the hydrogel in step i) to provide a cell concentration of about 5×10 cells / mL. 6 ~About 50×10 6 Even more preferably, the cell suspension is mixed with the hydrogel in step i) to provide a cell concentration of about 10 x 10 cells / mL. 6 , about 15×10 6 , about 20×10 6 , about 25×10 6 , about 30×10 6 or approximately 35 x 10 6 Provide cell concentration in cells / mL.

[0078] In one embodiment, the engineered human cardiac tissue comprises about 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , about 10×10 6 , about 15×10 6 , about 20×10 6 , about 25×10 6 , about 30×10 6 , about 35×10 6 , about 40×10 6 , about 45×10 6 , about 50×10 6 , about 55×10 6 , about 60×10 6 , about 65×10 6 , about 70×10 6 , about 75×10 6 , about 80×10 6 , about 85×10 6, about 90×10 6 , about 95×10 6 , about 100×10 6 , about 110×10 6 , about 120×10 6 , about 130×10 6 , about 140×10 6 , or approximately 150 × 10 6 cells at the time the hydrogel solidifies. For the method of the first aspect of the invention, that is the cell density of the patch at the end of step (ii) and before the hydrogel is cultured in the serum-free medium defined in step (iii). More preferably, the engineered human cardiac tissue has about 1 x 10 6 ~About 100×10 6 Even more preferably, the engineered human cardiac tissue has about 20×10 cells. 6 cells ~ approx. 150 x 10 6 It has cells.

[0079] In one embodiment, the engineered human cardiac tissue comprises about 1×10 6 , about 2×10 6 , about 3×10 6 , about 4×10 6 , about 5×10 6 , about 10×10 6 , about 15×10 6 , about 20×10 6 , about 25×10 6 , about 30×10 6 , about 35×10 6 , about 40×10 6 , about 45×10 6 , about 50×10 6 , about 55×10 6 , about 60×10 6 , about 65×10 6 , about 70×10 6 , about 75×10 6 , about 80×10 6 , about 85×10 6 , about 90×10 6 , about 95×10 6 , or approximately 100 × 10 6 cells / cm 2When the hydrogel solidifies, it has a cell density of about 1 x 10. In relation to the method of the first aspect of the invention, this is the cell density of the patch at the end of step (ii) and before the hydrogel is cultured in the serum-free medium defined in step (iii). More preferably, the engineered human cardiac tissue has a cell density of about 1 x 10. 6 ~Approx. 50×10 6 cells / cm 2 Even more preferably, the engineered human cardiac tissue has a cell density of about 1 x 10 6 cells / cm 2 ~About 10×10 6 cells / cm 2 The cell density is .

[0080] The engineered human cardiac patch can be fabricated in any shape, size, or configuration using an appropriate mold. In some embodiments, the tissue is shaped to reflect the application site. In one embodiment, the engineered cardiac tissue has a trapezoidal shape.

[0081] Those skilled in the art will be aware of various methods in the literature for inducing hPSCs to differentiate along cardiac lineages to provide a mixed population of hPSC-derived cardiomyocytes and non-cardiomyocytes. In one embodiment, prior to step i), the hPSCs are subjected to one of the following: a) culturing hPSCs in a basal medium containing effective amounts of BMP4, activin A, FGF, and a GSK3 inhibitor for a period of time sufficient to induce mesodermal differentiation of the hPSCs; b) culturing the cells obtained in step a) in a basal medium containing an effective amount of an inhibitor of the Wnt signaling pathway for a period of time sufficient to induce cardiac differentiation of the mesoderm; c) culturing the cells obtained in step b) in a basal medium for a period of time sufficient to provide a mixed population of hPSC-derived cardiomyocytes and non-cardiomyocytes. induces differentiation into cardiac lineages.

[0082] In one aspect, the present invention provides a method for producing a population of cardiomyocytes, stromal cells / fibroblasts, and endothelial cells from a human pluripotent stem cell (hPSC) culture, the method comprising: a) culturing hPSCs in a basal medium containing effective amounts of BMP4, activin A, FGF, and a GSK3 inhibitor for a period of time sufficient to induce mesodermal differentiation of the hPSCs; b) culturing the cells obtained in step a) in a basal medium containing an effective amount of an inhibitor of the Wnt signaling pathway for a period of time sufficient to induce cardiac differentiation of the mesoderm; c) culturing the cells obtained in step b) in a basal medium for a period of time sufficient to provide a mixed population of hPSC-derived cardiomyocytes and non-cardiomyocytes; and d) culturing the cells obtained in step c) in a basal medium containing effective amounts of an FGFR1 agonist and a PDGFR agonist for a period of time sufficient to produce a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes, stromal cells / fibroblasts, and endothelial cells.

[0083] In a preferred embodiment, prior to step i), the hPSCs are subjected to one of the following: a) culturing hPSCs in a basal medium comprising an effective amount of BMP4, activin A, FGF, a GSK3 inhibitor, albumin, and a serum-free supplement containing transferrin, but no insulin, for about 72 hours, wherein the medium is replaced with fresh medium daily; b) culturing the cells obtained in step a) in a basal medium containing an effective amount of an inhibitor of the Wnt signaling pathway and the same serum-free supplements as in a) for about 72 hours, wherein the medium is replaced with fresh medium every day; c) culturing the cells obtained in step b) in a basal medium comprising an effective amount of an inhibitor of the Wnt signaling pathway and a serum-free supplement comprising albumin and transferrin, and including insulin, for about 7 days, wherein the medium is replaced with fresh medium after the second and fourth days of the about 7-day period; d) inducing differentiation into cardiac lineages by culturing the cells obtained in step c) in a basal medium containing albumin, transferrin, and insulin for about 72 hours, wherein the basal medium used in each of steps a) to d) has a calcium concentration of less than about 1.2 mM; This provides a cell suspension containing human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte populations.

[0084] In one embodiment, in step a), hPSCs are cultured in a basal medium containing 1 to 20 ng / ml BMP4, preferably 2 to 15 ng / ml, more preferably 2.5 to 10 ng / ml, more preferably 3 to 8 ng / ml, most preferably 4 to 6 ng / ml, and even more preferably about 5 ng / ml BMP4; 0.1 to 10 ng / ml FGF2, preferably 1 to 9 ng / ml, more preferably 2 to 8 ng / ml, even more preferably 3 to 7 ng / ml, most preferably 4 to 6 ng / ml, and even most preferably about 5 ng / ml FGF2; and 1 to 20 ng / ml activin A, preferably 2.5 to 18 ng / ml, more preferably 5 to 16 ng / ml, even more preferably 7.5 to 14 ng / ml, even more preferably 8 to 12 ng / ml, most preferably 8.5 to 10 ng / ml, and even most preferably about 9 ng / ml activin A.

[0085] The GSK3 inhibitor in the basal medium of step (a) may be selected from the group consisting of, for example, CHIR99021, CHIR98014, SB216763, TWS119, tideglusib, SB415286, and LY2090314. However, any suitable GSK3 inhibitor may be applied in the method of the present invention. In a preferred embodiment, the GSK3 inhibitor in the basal medium of step (a) is CHIR99021.

[0086] Those skilled in the art will appreciate that the concentration of an effective amount of a GSK3 inhibitor will vary depending on the inhibitor's usefulness and inhibition constant. In the context of the present invention, the term "effective amount," as used herein in the context of a GSK3 inhibitor, is intended to mean an enzyme-inactivating concentration. For example, in the case of CHIR99021, the basal medium of step (a) comprises 0.1-10 μM CHIR99021, preferably 0.2-9 μM, more preferably 0.3-8 μM, even more preferably 0.4-7 μM, still more preferably 0.5-6 μM, more preferably 0.6-5 μM, more preferably 0.7-4 μM, more preferably 0.8-3 μM, most preferably 0.9-2 μM, and even most preferably about 1 μM CHIR99021.

[0087] In one embodiment, the serum-free supplement is B27® supplement or insulin-free B27® supplement. In a preferred embodiment, the B27® supplement or insulin-free B27® supplement is applied in an amount of 0.1-10% B27® or insulin-free B27®, preferably 0.5-8%, more preferably 1-6%, even more preferably 1.5-4%, and most preferably about 4% B27® or insulin-free B27®.

[0088] In one embodiment, the Wnt pathway inhibitor is C59 (4-(2-methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide), DKK1, IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide), IWP-4 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]-acetamide), Ant1.4Br, Ant 1.4CI, niclosamide, apicularen, bafilomycin, XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), IWR-1 (4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide), NSC668036 (N-[(1,1-dimethylethoxy)carbonyl]-L-alanyl-(2S)-2-hydroxy-3-methylbutanoyl-L-alanine-(1S)-1-carboxy-2-methylpropyl ester water) The Wnt pathway inhibitor is selected from the group consisting of: benzodiazepine monohydrate), 2,4-diaminoquinazoline, quercetin, ICG-001 ((6S,9aS)-hexahydro-6-[(4-hydroxyphenyl)methyl]-8-(1-naphthalenylmethyl)-4,7-dioxo-N-(phenylmethyl)-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxamide), PKF115-584, BML-284 (2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine), FH-535, iCRT-14, JW-55, JW-67, antibodies against Wnt and Wnt receptors, and Wnt inhibitory nucleic acids. In a preferred embodiment, the Wnt pathway inhibitor is IWP-4, IWR-1, or ICRT14.

[0089] Any pluripotent stem cell population, including human embryonic stem cell populations (hESCs) or human induced pluripotent stem cell populations (iPSCs), can be used as starting material to derive engineered human cardiac tissue using the methods described herein. In one embodiment, the population of pluripotent progenitor cells is a human iPSC population. In one embodiment, the iPSC cells are grown under feeder-free conditions. In another embodiment, cells obtained from a subject can be subjected to a method to generate patient-specific iPSCs, which can then be differentiated using the methods described herein.

[0090] Engineered cardiac tissue The present invention provides engineered human cardiac tissue comprising at least about 30% hPSC-derived cardiomyocytes, up to about 35% hPSC-derived stromal cells / fibroblasts, and up to about 30% hPSC-derived endothelial cells, wherein the cells are distributed throughout the hydrogel composition.

[0091] In another embodiment, the engineered human cardiac tissue comprises hPSC-derived cardiomyocytes, hPSC-derived stromal cells / fibroblasts, and hPSC-derived endothelial cells, wherein the cells are derived from a single differentiation process, and the cells are distributed throughout the hydrogel composition.

[0092] The present invention provides engineered human cardiac tissue produced according to the above methods.

[0093] In some embodiments, the engineered human cardiac tissue comprises cardiomyocytes, stromal cells / fibroblasts, endothelial cells, and vascular smooth muscle cells. In preferred embodiments, the engineered cardiac tissue comprises at least about 30% cardiomyocytes, about 35% or less stromal cells / fibroblasts, and about 30% or less endothelial cells. In one embodiment, the engineered cardiac tissue comprises about 30% to about 70% cardiomyocytes, about 10% to about 35% stromal cells / fibroblasts, and about 2% to about 30% endothelial cells. In another embodiment, the engineered cardiac tissue comprises about 30% to about 65% cardiomyocytes, about 10% to about 35% stromal cells / fibroblasts, and about 2% to about 30% endothelial cells. In another embodiment, the engineered cardiac tissue comprises about 30% to about 60% cardiomyocytes, about 10% to about 35% stromal cells / fibroblasts, and about 2% to about 30% endothelial cells. In another embodiment, the engineered cardiac tissue comprises about 30% to about 55% cardiomyocytes, about 10% to about 35% interstitial cells / fibroblasts, and about 2% to about 30% endothelial cells. In another embodiment, the engineered cardiac tissue comprises about 30% to about 70% cardiomyocytes, about 15% to about 35% interstitial cells / fibroblasts, and about 2% to about 30% endothelial cells. In another embodiment, the engineered cardiac tissue comprises about 30% to about 70% cardiomyocytes, about 20% to about 35% interstitial cells / fibroblasts, and about 2% to about 30% endothelial cells. In another embodiment, the engineered cardiac tissue comprises about 30% to about 70% cardiomyocytes, about 10% to about 35% interstitial cells / fibroblasts, and about 5% to about 30% endothelial cells. In another embodiment, the engineered cardiac tissue comprises about 30% to about 70% cardiomyocytes, about 10% to about 35% stromal cells / fibroblasts, and about 15% to about 30% endothelial cells. In a more preferred embodiment, the engineered cardiac tissue comprises about 30% to about 50% cardiomyocytes, about 20% to about 35% stromal cells / fibroblasts, and about 15% to about 30% endothelial cells. In a more preferred embodiment, the engineered cardiac tissue comprises about 30% to about 55% cardiomyocytes, about 20% to about 35% stromal cells / fibroblasts, and about 15% to about 30% endothelial cells. In some embodiments, the cardiac tissue further comprises up to about 15% vascular smooth muscle cells. In some embodiments, the cardiac tissue further comprises up to about 10% cardiac progenitor cells.In a more preferred embodiment, the engineered cardiac tissue comprises about 30% to about 70% cardiomyocytes, about 10% to about 35% interstitial cells / fibroblasts, about 2% to about 30% endothelial cells, up to about 15% vascular smooth muscle cells, and up to about 10% cardiac progenitor cells. In a more preferred embodiment, the engineered cardiac tissue comprises about 30% to about 60% cardiomyocytes, about 15% to about 35% interstitial cells / fibroblasts, about 5% to about 30% endothelial cells, up to about 15% vascular smooth muscle cells, and up to about 10% cardiac progenitor cells. In a more preferred embodiment, the engineered cardiac tissue comprises about 30% to about 55% cardiomyocytes, about 20% to about 35% interstitial cells / fibroblasts, about 15% to about 30% endothelial cells, up to about 15% vascular smooth muscle cells, and up to about 10% cardiac progenitor cells. In a more preferred embodiment, the engineered cardiac tissue comprises about 30% to about 55% cardiomyocytes, about 20% to about 35% interstitial cells / fibroblasts, about 5% to about 30% endothelial cells, about 0.5% to about 15% vascular smooth muscle cells, and about 0.5% to about 10% cardiac progenitor cells. In more preferred embodiments, the engineered cardiac tissue comprises about 30% to about 55% cardiomyocytes, about 20% to about 35% interstitial cells / fibroblasts, about 15% to about 30% endothelial cells, about 0.5% to about 15% vascular smooth muscle cells, and about 0.5% to about 10% cardiac progenitor cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 60% cardiomyocytes, about 20% to about 35% interstitial cells / fibroblasts, and about 15% to about 30% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 45% cardiomyocytes, about 30% to about 35% interstitial cells / fibroblasts, and about 20% to about 24% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 35% to about 55% cardiomyocytes, about 20% to about 35% interstitial cells / fibroblasts, and about 15% to about 30% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 35% to about 60% cardiomyocytes, about 20% to about 35% interstitial cells / fibroblasts, and about 15% to about 30% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 35% to about 55% cardiomyocytes, about 40% to about 60% interstitial cells / fibroblasts, and about 15% to about 30% endothelial cells.In some embodiments, the engineered cardiac tissue comprises about 30% to about 45% cardiomyocytes, about 30% to about 35% interstitial cells / fibroblasts, and about 26% to about 30% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 45% cardiomyocytes, about 30% to about 35% interstitial cells / fibroblasts, and about 20% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 45% cardiomyocytes, about 30% to about 35% interstitial cells / fibroblasts, and about 30% endothelial cells.

[0094] In some embodiments, the engineered cardiac tissue comprises about 1% to about 10% proliferative cardiomyocytes. In some embodiments, the engineered cardiac tissue comprises about 2% to about 5% proliferative cardiomyocytes. In further embodiments, at least about 3% to about 10% of the cardiomyocytes in the engineered cardiac tissue are proliferative as determined by expression of one or more markers associated with cell proliferation. In further embodiments, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cardiomyocytes in the engineered cardiac tissue are proliferative as determined by expression of one or more markers associated with cell proliferation. In one embodiment, the proliferative cardiomyocytes express one or more markers selected from the group consisting of ANLN, TOP2A, CDK1, and E2F1.

[0095] Another characteristic of cardiomyocytes present in engineered cardiac tissue is that they express MMP1 + Therefore, in a preferred embodiment, the cardiomyocytes in the engineered cardiac tissue are MMP1 + is.

[0096] Another characteristic of fibroblasts present in engineered cardiac tissue is that they express MMP1 + , EMP1 + , FOXD1 + , RAB27B + , NR2F1 + , F2RL1 + , SPP1 + , TMEM158 + , PTH LH + , PHLDA2 +, MALL + , MYCT1 + , DUSP4 + , PLAU + , TMEM156 + , CD274 + , MMP10 + , ARRDC4 + , RFX8 + , M.L.P.H. + , T.H.B.D. + , HHEX + , V.G.F. + , OTULINL + , IL33 + , CA12 + , C6orf141 + , MFSD2A + and / or CARD10 + Thus, in a preferred embodiment, the fibroblasts in the engineered cardiac tissue are positive for one or more of these aforementioned markers.

[0097] In one embodiment, the cardiac tissue described herein exhibits a log2 relative gene expression level (normalized to TUBA1A) for PKM greater than 2. Another characteristic of the cardiac tissue is that after 24 hours in SF culture medium, the SF culture medium has a lactate concentration of at least 0.5 mM or at least 1 mM. In another embodiment, after 24 hours of culture of cardiac tissue produced using the methods described herein in SF culture medium, the culture medium has a lactate concentration of 10-20 nmol / million cells / hour.

[0098] In one embodiment, the engineered cardiac tissue exhibits resistance to hypoxia. In one embodiment, the cardiac tissue described herein exhibits a log2 relative gene expression level (normalized to TUBA1A) of less than -4 for ALDH1, AGMO, GPX3, CYP1B1, and PLIN5. Another characteristic of the engineered cardiac tissue is that after 20 hours of culture under hypoxic conditions, the cell culture medium from such cultures has a lactate / pyruvate ratio of at least 1:1.

[0099] As shown in the Examples below, engineered cardiac tissue produced by the methods of the present invention has been shown to advantageously exhibit low contractile force. In preferred embodiments, the engineered cardiac tissue exhibits contractile activity of less than about 10 mN / mm.

[0100] In one embodiment, the hydrogel composition is composed of fibrin, collagen I, or Matrigel, or any combination thereof. In a preferred embodiment, the hydrogel is a fibrin hydrogel. In another embodiment, the hydrogel can be functionalized with one or more bioactive substances. For example, bioactive substances (e.g., small molecules, polypeptides including cytokines and chemokines, differentiation factors, signaling pathway inhibitors, etc.) can promote, for example, cell viability and further development or differentiation of cells in the resulting engineered tissue. In one embodiment, the one or more bioactive agents can be agents selected from the group consisting of antiproliferative agents, immunosuppressants, pro-angiogenic compounds, antibodies or fragments or portions thereof, antibiotics or antimicrobial compounds, antigens or epitopes, aptamers, biopolymers, carbohydrates, cell adhesion mediators (such as RGD), cytokines, cytotoxic agents, drugs, enzymes, growth factors or recombinant growth factors and fragments and variants thereof, hormone antagonists, hormones, immunological agents, lipids, metals, nanoparticles, nucleic acid analogs, nucleic acids (e.g., DNA, RNA, siRNA, RNAi, and microRNA agents), nucleotides, nutraceuticals, oligonucleotides, peptide nucleic acids (PNAs), peptides, prodrugs, prophylactic agents, proteins, small molecules, therapeutic agents, or any combination thereof.

[0101] In one embodiment, the engineered human cardiac tissue comprises about 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , about 10×10 6 , about 15×10 6 , about 20×10 6 , about 25×10 6 , about 30×10 6 , about 35×106 , about 40×10 6 , about 45×10 6 , about 50×10 6 , about 55×10 6 , about 60×10 6 , about 65×10 6 , about 70×10 6 , about 75×10 6 , about 80×10 6 , about 85×10 6 , about 90×10 6 , about 95×10 6 , about 100×10 6 , about 110×10 6 , about 120×10 6 , about 130×10 6 , about 140×10 6 , or approximately 150 × 10 6 More preferably, the engineered human cardiac tissue has about 1 x 10 cells. 6 ~About 100×10 6 Even more preferably, the engineered human cardiac tissue has about 20×10 cells. 6 cells ~ approx. 150 x 10 6 It has cells.

[0102] In one embodiment, the engineered human cardiac tissue comprises about 1×10 6 , about 2×10 6 , about 3×10 6 , about 4×10 6 , about 5×10 6 , about 10×10 6 , about 15×10 6 , about 20×10 6 , about 25×10 6 , about 30×10 6 , about 35×10 6 , about 40×10 6 , about 45×10 6 , about 50×10 6 , about 55×10 6 , about 60×10 6 , about 65×10 6 , about 70×10 6 , about 75×10 6 , about 80×10 6 , about 85×106 , about 90×10 6 , about 95×10 6 , or approximately 100 × 10 6 cells / cm 2 More preferably, the engineered human cardiac tissue has a cell density of about 1 x 10 6 ~About 50×10 6 cells / cm 2 Even more preferably, the engineered human cardiac tissue has a cell density of about 1 x 10 6 cells / cm 2 ~About 10×10 6 cells / cm 2 The cell density is .

[0103] The engineered human cardiac patch can be fabricated in any shape, size, or configuration using an appropriate mold. In some embodiments, the tissue is shaped to reflect the application site. In one embodiment, the engineered cardiac tissue has a trapezoidal shape.

[0104] The engineered human cardiac tissue may be derived from a human embryonic stem cell population (hESC) or a human induced pluripotent stem cell population (iPSC) and used as a starting material. In one embodiment, the engineered human cardiac tissue is derived from a human iPSC population. In one embodiment, the iPSC cells are grown under feeder-free conditions. In another embodiment, the engineered human cardiac tissue is derived from cells obtained from a patient and subjected to a method to generate patient-specific iPSCs, which can then be differentiated using the methods described herein to generate the engineered human cardiac tissue.

[0105] Treatment method In order to engineer human cardiac tissue for the purpose of transplantation into patients with heart disease and heart failure, there is a need to provide cardiac tissue patches that are durable and adaptable enough for transplantation and have a biocompatible structure that can withstand implantation in a hypoxic environment. There is also a need to provide engineered cardiac tissue with the appropriate variety of cardiomyocyte and non-cardiomyocyte cell types, particularly endothelial cells, so that the transplanted cardiac tissue patch can be vascularized, remain viable, and ultimately functionally integrate into the recipient heart. Thus, there is a need for better tissues for transplantation.

[0106] As described herein, the inventors have also surprisingly determined that certain culture conditions produce hPSC-derived cardiac tissue patches containing cardiomyocytes, stromal cells / fibroblasts, and increased numbers of endothelial cells that are durable enough for transplantation and have improved tolerance to hypoxia. While not wishing to be bound by any particular theory, producing engineered cardiac tissue according to the methods described herein promotes differentiation of progenitor cells toward an endothelial cell fate and into pericytes, which play a supporting role in ECs, providing engineered tissue with reduced oxidation (including reduced fatty acid oxidation), predominantly glycolytic metabolism, and low contractile force. This may result in engineered tissue that is more resistant to hypoxia, making such engineered cardiac tissue more suitable for therapeutic applications such as transplantation.

[0107] Thus, the present invention provides engineered human cardiac tissue as described and defined herein for use as a graft in the treatment of diseased or damaged cardiac tissue in a subject in need thereof.

[0108] The present invention also provides a method for treating diseased or damaged cardiac tissue in a subject in need thereof, comprising transplanting in the subject engineered human cardiac tissue as described and defined herein.

[0109] The present invention also provides the use of engineered human cardiac tissue as described and defined herein in the manufacture of a medicament for the treatment of diseased or damaged cardiac tissue in a subject in need thereof.

[0110] In one embodiment, the subject suffers from cardiomyopathy or cardiac tissue damage. In another embodiment, the cardiomyopathy or cardiac tissue damage is due to acute or chronic stress, atherosclerotic damage of blood vessels, ischemia, myocardial infarction, inflammatory disease, valvular heart disease, or myocarditis. In a further embodiment, the subject suffers from congenital heart disease. In yet a further embodiment, the congenital heart disease is selected from the group consisting of hypoplastic left heart syndrome, tetralogy of Fallot, truncus arteriosus, pulmonary atresia, ventricular septal defect, atrial septal defect, and single ventricle including endocardial cushion defect.

[0111] Screening Method The present invention also provides for the use of engineered human cardiac tissue according to the present invention in an in vitro model for drug toxicity screening or in an in vitro method for testing the modulation of cardiac function by candidate pharmacological agents. In one embodiment, the present invention provides a method for analyzing the biological effect of at least one test compound or bioactive agent on cardiac cells, comprising contacting engineered human cardiac tissue with the test compound or bioactive agent, incubating the tissue in the presence of the test compound or bioactive agent, and analyzing the biological effect.

[0112] kit Also provided herein are kits that include one or more of the following: cells or tissues produced according to the methods described herein; products or compositions comprising the produced cells or tissues (optionally containing an additional therapeutic agent, or optionally the cells containing a reporter system or other modification according to the methods described herein); a combination of at least two selected from agonists, inhibitors, media, devices or other components that can be used in the methods described herein; and instructions for use, e.g., instructions for producing the cells, performing the assays, or administering the cells, tissues, compositions, or products; and a vial or other container for containing one of these aforementioned cells, tissues, compositions, products, agonists, inhibitors, media, etc.

[0113] List of numbered embodiments: 1. A method for producing engineered human cardiac tissue comprising endothelial cells, comprising culturing a cell suspension comprising a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population in the presence of an FGFR1 agonist and a PDGFR agonist for a time and under conditions sufficient to produce engineered human cardiac tissue comprising endothelial cells. 2. A method for producing engineered human cardiac tissue, comprising: i) mixing a cell suspension comprising a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population with a flowable hydrogel composition, wherein the cell suspension is in a serum-free medium; ii) filling the mold with the flowable hydrogel composition containing the cell suspension and incubating the hydrogel for a time and under conditions sufficient to solidify the hydrogel; iii) culturing the hydrogel from step ii) in serum-free medium; A method of contacting a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte populations with an FGFR1 agonist and a PDGFR agonist, thereby providing engineered human cardiac tissue, the engineered human cardiac tissue comprising cardiomyocytes, stromal cells / fibroblasts, and endothelial cells. 3. The method of embodiment 2, wherein the FGFR1 agonist and / or the PDGFR agonist is added to the serum-free medium or the flowable hydrogel composition prior to the mixing in step i). 4. The method of embodiment 2, wherein after the mixing in step i), an FGFR1 agonist and / or a PDGFR agonist is added to the flowable hydrogel composition containing the cell suspension. 5. The method according to any one of embodiments 2 to 4, wherein, prior to the mixing in step i), the human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte populations are cultured in a serum-free medium comprising an FGFR1 agonist and a PDGFR agonist. 6. The method according to any one of embodiments 2 to 5, wherein in step iii), the hydrogel of step ii) is cultured in a serum-free medium containing an FGFR1 agonist and a PDGFR agonist. 7. The method according to any one of embodiments 1 to 6, wherein the cell suspension comprises about 70% cardiomyocytes and about 30% to about 50% non-cardiomyocytes. 8. The method according to any one of embodiments 1 to 7, wherein the cell suspension comprises about 50 to about 75% cardiomyocytes and about 25 to about 50% non-cardiomyocytes. 9. The method according to any one of embodiments 1 to 8, wherein the cell suspension is derived from a single differentiation process. 10. The method of any one of embodiments 1 to 9, wherein the engineered human cardiac tissue further comprises vascular smooth muscle cells and cardiac progenitor cells. 11. Engineered human cardiac tissue - at least about 30% cardiomyocytes; - approximately 35% or less stromal cells / fibroblasts; - approximately 30% or less endothelial cells; 11. The method of any one of embodiments 1 to 10, comprising: 12. Engineered human cardiac tissue -Approximately 30 to 70% of cardiomyocytes, - Approximately 10 to 35% stromal cells / fibroblasts, - Approximately 5 to 30% endothelial cells, -Up to approximately 15% of vascular smooth muscle cells, -Up to approximately 10% cardiac progenitor cells, 12. The method of any one of embodiments 1 to 11, comprising: 13. The method of any one of embodiments 1 to 12, wherein the FGFR1 agonist is selected from FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. 14. The method of embodiment 13, wherein the FGFR1 agonist is FGF2. 15. The method of any one of embodiments 1-14, wherein the PDGFR agonist is selected from PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, or PDGF-DD. 16. The method of embodiment 15, wherein the PDGFR agonist is a PDGFRβ agonist. 17. The method of embodiment 16, wherein the PDGFR agonist is PDGF-BB. 18. The method according to any one of embodiments 1 to 17, wherein the serum-free medium comprises about 0.5 to 100 ng / ml of human PDGF-BB and about 0.5 to 100 ng / ml of human FGF2. 19. The method of any one of embodiments 1-18, wherein the serum-free medium comprises about 10 ng / ml human PDGF-BB and about 10 ng / ml human FGF2. 20. The method of any one of embodiments 1-19, wherein the serum-free medium comprises a basal medium comprising albumin and transferrin. 21. The method of embodiment 20, wherein the serum-free medium comprises a B27 supplement. 22. The method of embodiment 20 or 21, wherein the basal medium is a low-calcium medium having a calcium concentration of less than about 1.2 mM. 23. The method according to any one of embodiments 2 to 22, further comprising replacing the medium in step iii) with fresh cell culture medium at least every 2 days. 24. The method according to any one of embodiments 2 to 23, wherein the culturing in step iii) is carried out for at least 5 days. 25. The method of any one of embodiments 1 to 24, wherein a population of about 3% to about 10% of cardiomyocytes in the engineered human cardiac tissue is proliferative. 26. The method of any one of embodiments 1-25, wherein the engineered human cardiac tissue exhibits glycolytic metabolism. 27. The method of embodiment 26, wherein the cell culture medium obtained after 24 hours of culture with engineered human cardiac tissue has a lactate concentration of 1 mM. 28. The method of any one of embodiments 1 to 27, wherein the engineered human cardiac tissue exhibits a log2 relative gene expression level for PKM of greater than 2 when normalized to TUBA1A expression levels. 29. The method of any one of embodiments 1 to 28, wherein the engineered human cardiac tissue is resistant to hypoxic conditions. 30. The method of any one of embodiments 1-29, wherein the engineered human cardiac tissue results in a lactate / pyruvate ratio of at least 1:1 in the cell culture medium obtained after 20 hours of culture under hypoxic conditions. 31. The method of any one of embodiments 1 to 30, wherein the engineered human cardiac tissue exhibits downregulation of ALDH1, AGMO, DPYD, GPX3, CYP1B1 and PLIN5. 32. Cardiomyocytes produce MMP1 + 32. The method of any one of embodiments 1 to 31, wherein 33. Fibroblasts express MMP1 + , EMP1 + , FOXD1 + , RAB27B + , NR2F1 + , F2RL1 + , SPP1 + , TMEM158 + , PTH LH + , PHLDA2 + , MALL + , MYCT1 + , DUSP4 + , PLAU + , TMEM156 + , CD274 + , MMP10 + , ARRDC4 + , RFX8+ , M.L.P.H. + , T.H.B.D. + , HHEX + , V.G.F. + , OTULINL + , IL33 + , CA12 + , C6orf141 + , MFSD2A + and / or CARD10 + 33. The method of any one of embodiments 1 to 32, wherein 34. The method of any one of embodiments 1-33, wherein the engineered human cardiac tissue exhibits a contractile activity of less than 10 mN / mm2. 35. The method of any one of embodiments 2-34, wherein the hydrogel composition comprises a fibrin hydrogel. 36. The method of embodiment 35, wherein fibrin is formed by mixing a thrombin solution and a fibrinogen solution. 37. The method of embodiment 36, wherein the fibrinogen is present in a concentration of 10 mg / mL to 50 mg / mL. 38. The method of embodiment 36, wherein the fibrinogen is present at a concentration of 20 mg / mL. 39. The cell suspension is mixed with the hydrogel of step i) to obtain approximately 5 × 10 6 ~About 100×10 6 39. The method of any one of embodiments 2 to 38, wherein the cell concentration is provided in cells / mL. 40. The cell suspension is mixed with the hydrogel of step i) to obtain approximately 20 × 10 6 ~About 50×10 6 40. The method of embodiment 39, wherein the cell concentration is provided in cells / mL. 41. The cell suspension is mixed with the hydrogel of step i) to obtain approximately 35 × 10 6 41. The method of embodiment 40, wherein the cell concentration is provided in cells / mL. 42. hPSCs: a) culturing hPSCs in a basal medium comprising an effective amount of BMP4, activin A, FGF, a GSK3 inhibitor, albumin, and a serum-free supplement containing transferrin, but no insulin, for about 72 hours, wherein the medium is replaced with fresh medium daily; b) culturing the cells obtained in step a) in a basal medium containing an effective amount of an inhibitor of the Wnt signaling pathway and the same serum-free supplements as in a) for about 72 hours, wherein the medium is replaced with fresh medium every day; c) culturing the cells obtained in step b) in a basal medium containing an effective amount of an inhibitor of the Wnt signaling pathway and a serum-free supplement containing albumin and transferrin, including insulin, for about 7 days, wherein the medium is replaced with fresh medium after the second and fourth days of the about 7-day period; d) inducing differentiation into cardiac lineages by culturing the cells obtained in step c) in a basal medium containing a serum-free supplement containing albumin, transferrin, and insulin for about 72 hours; wherein the basal medium used in each of steps a) to d) has a calcium concentration of less than about 1.2 mM; 42. The method according to any one of embodiments 1 to 41, thereby providing a cell suspension comprising a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population. 43. - the GSK3 inhibitor is CHIR99021, and / or the inhibitor of the Wnt signaling pathway is IWP-4, and / or - the basal medium is RPMI or DMEM; 43. The method of embodiment 42. 44. The method of any one of embodiments 1 to 43, wherein the engineered human cardiac tissue is in the form of a tissue patch. 45. The method of embodiment 44, wherein the patch is used as an implant in the subject. 46. The method of embodiment 45, wherein the subject is suffering from cardiomyopathy or cardiac tissue damage. 47. The method of embodiment 46, wherein the cardiomyopathy or cardiac tissue damage results from acute or chronic stress, atherosclerotic damage of blood vessels, ischemia, myocardial infarction, inflammatory disease, valvular heart disease, or myocarditis. 48. The method of embodiment 45, wherein the subject has a congenital heart disease. 49. The method of embodiment 48, wherein the congenital heart disease is selected from the group consisting of hypoplastic left heart syndrome, tetralogy of Fallot, truncus arteriosus, pulmonary atresia, ventricular septal defect, atrial septal defect, and single ventricle including endocardial cushion defect. 50. The method of any one of embodiments 1 to 49, wherein the hPSCs are iPSCs. 51. The method of embodiment 50, wherein the iPSCs are derived from a subject with cardiac disease and / or from a subject into which the engineered human cardiac tissue will be transplanted. 52. The method of any one of embodiments 1 to 51, wherein the hPSCs are embryonic stem cells. 53. Engineered human cardiac tissue comprising at least about 30% hPSC-derived cardiomyocytes, not more than about 35% hPSC-derived stromal cells / fibroblasts, and not more than about 30% hPSC-derived endothelial cells, wherein said cells are distributed throughout the hydrogel composition. 54. An engineered human cardiac tissue comprising hPSC-derived cardiomyocytes, hPSC-derived stromal cells / fibroblasts, and hPSC-derived endothelial cells, wherein said cells are derived from a single differentiation process, and said cells are distributed throughout a hydrogel composition. 55. The engineered human cardiac tissue of embodiment 53 or 54, wherein the engineered human cardiac tissue further comprises vascular smooth muscle cells and cardiac progenitor cells. 56. Engineered human cardiac tissue -Approximately 30 to 70% of cardiomyocytes, - Approximately 10 to 35% stromal cells / fibroblasts, - Approximately 5 to 30% endothelial cells, 56. The engineered human cardiac tissue of any one of embodiments 53 to 55, comprising: 57. An engineered human cardiac tissue according to any one of embodiments 53 to 56, wherein up to about 10% of the cardiomyocytes in the engineered human cardiac tissue are proliferative. 58. The engineered human cardiac tissue according to any one of embodiments 53-57, wherein the engineered human cardiac tissue exhibits glycolytic metabolism. 59. The engineered human cardiac tissue of embodiment 53, wherein the cell culture medium obtained after 24 hours of culture with the engineered human cardiac tissue has a lactate concentration of 1 mM. 60. The engineered human cardiac tissue according to any one of embodiments 53 to 59, wherein the engineered human cardiac tissue exhibits a log2 relative gene expression level of PKM greater than 2 when normalized to TUBA1A. 61. An engineered human cardiac tissue according to any one of embodiments 53 to 60, wherein the engineered human cardiac tissue is resistant to hypoxic conditions. 62. The engineered human cardiac tissue according to any one of embodiments 53 to 61, wherein the engineered human cardiac tissue provides a lactate / pyruvate ratio of at least 1:1 in the cell culture medium obtained after 20 hours of culture under hypoxic conditions. 63. An engineered human cardiac tissue according to any one of embodiments 53 to 62, wherein the engineered human cardiac tissue exhibits log2 relative gene expression levels of ALDH1, AGMO, GPX3, CYP1B1 and PLIN5 of less than -4 when normalized to TUBA1A expression levels. 64. Cardiomyocytes produce MMP1 + 64. The engineered human cardiac tissue of any one of embodiments 53 to 63, wherein 65. Fibroblasts produce MMP1 + , EMP1 + , FOXD1 + , RAB27B + , NR2F1 + , F2RL1 + , SPP1 + , TMEM158 + , PTH LH + , PHLDA2 + , MALL + , MYCT1 + , DUSP4 + , PLAU+ , TMEM156 + , CD274 + , MMP10 + , ARRDC4 + , RFX8 + , M.L.P.H. + , T.H.B.D. + , HHEX + , V.G.F. + , OTULINL + , IL33 + , CA12 + , C6orf141 + , MFSD2A + and / or CARD10 + 65. The engineered human cardiac tissue of any one of embodiments 53 to 64, wherein 66. -Approximately 30 to 70% of cardiomyocytes, - Approximately 10 to 35% stromal cells / fibroblasts, - Approximately 5 to 30% endothelial cells, -Up to approximately 15% of vascular smooth muscle cells, -Up to approximately 10% cardiac progenitor cells, 66. The engineered human cardiac tissue of any one of embodiments 53 to 65, comprising: 67. Engineered human cardiac tissue exhibits a resistance of 10 mN / mm 2 67. The engineered human cardiac tissue of any one of embodiments 53 to 66, which exhibits a contractile activity of less than 100 ng / mL. 68. The engineered human cardiac tissue of any one of embodiments 53 to 67, wherein the hydrogel composition comprises fibrin formed by mixing a thrombin solution and a fibrinogen solution. 69. The engineered human cardiac tissue of embodiment 68, wherein fibrinogen is present at a concentration of 10 mg / mL to 50 mg / mL. 70. The engineered human heart tissue of embodiment 69, wherein fibrinogen is present at a concentration of 20 mg / mL. 71. The total number of cells in a tissue is approximately 20 x 10 6 cells ~100×10 6 71. The engineered human cardiac tissue of any one of embodiments 53 to 70, which is a cell. 72. The engineered human cardiac tissue according to any one of embodiments 53 to 71, in the form of a tissue patch. 73. The engineered human cardiac tissue according to any one of embodiments 53 to 72, wherein the hPSCs are iPSCs. 74. The engineered human cardiac tissue according to any one of embodiments 53 to 73, wherein the hPSCs are embryonic stem cells. 75. Engineered human cardiac tissue produced according to the method of any one of embodiments 1 to 52. 76. A method for analyzing the biological effect of at least one test compound or biologically active substance on cardiac cells, comprising contacting engineered human cardiac tissue described in any one of embodiments 53 to 75 with the test compound or biologically active substance, incubating the tissue in the presence of the test compound or biologically active substance, and analyzing the biological effect. 77. The engineered human cardiac tissue of any one of embodiments 53 to 75, for use as a graft in the treatment of diseased or damaged cardiac tissue in a subject in need of treatment. 78. A method for treating diseased or damaged cardiac tissue in a subject in need thereof, comprising transplanting in said subject the engineered human cardiac tissue described in any one of embodiments 53 to 75. 79. Use of engineered human cardiac tissue according to any one of embodiments 53 to 75 in the manufacture of a medicament for the treatment of diseased or damaged cardiac tissue in a subject in need thereof. 80. The engineered human cardiac tissue of embodiment 77, the method of embodiment 78 or the use of embodiment 79, wherein the subject is suffering from cardiomyopathy or cardiac tissue damage. 81. The engineered human cardiac tissue, method or use of embodiment 80, wherein the cardiomyopathy or cardiac tissue damage results from acute or chronic stress, atherosclerotic damage of blood vessels, ischemia, myocardial infarction, inflammatory disease, valvular heart disease, or myocarditis. 82. The engineered human heart tissue according to embodiment 77, the method according to embodiment 78 or the use according to embodiment 79, wherein the subject suffers from a congenital heart disease. 83. The engineered human cardiac tissue, method or use of embodiment 82, wherein the congenital heart disease is selected from the group consisting of hypoplastic left heart syndrome, tetralogy of Fallot, truncus arteriosus, pulmonary atresia, ventricular septal defect, atrial septal defect, and single ventricle including endocardial cushion defect. [Example]

[0114] Materials and Methods human pluripotent stem cells The hESCs utilized were female HES3 (WiCell). The following cell lines were obtained from the CIRM hPSC Repository, funded by the California Institute of Regenerative Medicine: CW30382A (male, designated AA) and CW30318C (female, designated CC) (both obtained from FujiFilm). hPSC lines were maintained in mTeSR-1 (Stem Cell Technologies) / Matrigel (Millipore) and passaged using ReLeSR (Stem Cell Technologies). Quality control was performed using karyotyping and mycoplasma testing. PSCs were plated at 2 × 10 on Matrigel-coated flasks. 4 cells / cm 2 and cultured in mTeSR-1 for 4 days.

[0115] Cardiac differentiation To induce cardiac mesoderm, hPSCs were cultured in RPMI B27 medium (RPMI 1640 GlutaMAX + insulin-free 2% B27 supplement, 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma), and 1% penicillin / streptomycin (ThermoFisher Scientific)) supplemented with 5 ng / ml BMP-4 (RnD Systems), 9 ng / ml activin A (RnD Systems), 5 ng / ml FGF-2 (RnD Systems), and 1 μM CHIR99021 (Stem Cell Technologies). Daily medium changes were required for 3 days for mesoderm induction. Following this, cardiac specification was performed using insulin-free RPMI B27 containing 5 μM IWP-4 (Stem Cell Technologies) for an additional 3 days, followed by 5 μM IWP-4 RPMI B27+ (RPMI 1640 Glutamax+ insulin containing 2% B27 supplement, 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, and 1% penicillin / streptomycin) for an additional 7 days, with medium changes every 2–3 days. For the final 2 days of differentiation, hPSCs were cultured in RPMI B27+insulin.

[0116] The recovery of differentiated cardiac cells involves enzymatic digestion to dissociate the cells, first in PBS (Ca) at 37°C for 1 hour. 2+ and Mg 2+First, in 0.2% type I collagenase (Sigma) containing 20% fetal bovine serum (FBS) in PBS (containing PBS), and second, in 0.25% trypsin-EDTA for 10 min at 37° C. Cells were filtered through a 100-μm mesh cell strainer (BD Biosciences), centrifuged at 300 × g for 3 min, and resuspended in α-MEM Glutamax, 10% FBS, 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, and 1% penicillin / streptomycin. We have also found that recovery is possible using Accutase (Sigma) for approximately 15 minutes, followed by filtration through a 100 pm mesh cell strainer (BD Biosciences), centrifugation at 300 x g for 3 minutes, and resuspension in α-MEM Glutamax, 10% FBS, 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, and 1% penicillin / streptomycin.

[0117] Flow cytometry analysis showed that differentiated cardiac cells express approximately 70% α-actinin + / CTNT + Cardiomyocytes were shown to be approximately 30% CD90 stromal cells.

[0118] Cardiac tissue patch formation and culture Serum-free medium (SF) containing 4% B27 supplement (ThermoFisher Scientific), 10 ng / ml PDGF-BB (RnD Systems), 10 ng / ml bFGF (RnD Systems), 33 μg / ml aprotinin (Sigma) in RPMI 1640 Glutamax (ThermoFisher Scientific) containing 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma) and 1% penicillin / streptomycin (ThermoFisher Scientific) was used for cardiac tissue patch formation and culture.

[0119] A fibrinogen solution of 20-100 mg / mL in sterile SF was prepared as a 2x solution. The SF was preheated to 37°C to facilitate dissolution of the fibrinogen. A wide-bore P1000 pipette tip was used to facilitate mixing of the fibrinogen. Thorough mixing was required and can be facilitated by placing the pipette in a 37°C water bath. The fibrinogen was sterile filtered and kept on ice until needed.

[0120] Flowable fibrin hydrogel was prepared by mixing the following reagents in a polypropylene tube in the order listed below: Reagents were thawed on ice and kept on ice throughout the entire procedure. i) Add fibrinogen (Sigma) solution to a final concentration of 10-50 mg / ml. ii) Add an equal volume of the fibrinogen solution to the cell suspension in SF to give approximately 35 million cells per ml and mix thoroughly. iii) Add thrombin (Sigma) to a final concentration of 2 U / ml and mix well. iv) The solution is pipetted into the PDMS mold. v) Incubate the hydrogel at 37°C for 30-60 minutes to induce gelation of the cell-matrix mixture.

[0121] In this example, human cardiac tissue is cultured after gelation of the cell matrix material. SF medium is added to cover the human cardiac tissue, and the medium is changed every 2-3 days. At the end of the culture period, the cardiac tissue is removed from the PDMS mold.

[0122] For control human cardiac tissue, instead of SF, human cardiac tissue was cultured in α-MEM GlutaMAX (ThermoFisher Scientific), 10% fetal bovine serum (FBS) (ThermoFisher Scientific), 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma), 1% penicillin / streptomycin (ThermoFisher Scientific), and 33 μg / mL aprotinin (Sigma) for 2 days, and then cultured in 4% B27-(insulin-free) (ThermoFisher Scientific), 1% GlutaMAX (ThermoFisher Scientific), 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, and 1% penicillin / streptomycin (ThermoFisher Scientific). The medium was replaced every 2–3 days with maturation medium (MM) containing glucose-, glutamine-, and phenol red-free DMEM (ThermoFisher Scientific) supplemented with 1 mM glucose, 100 μM palmitic acid (bound to bovine serum albumin in B27 by incubation at 37°C for 2 hours; Sigma), and 33 μg / mL aprotinin (Sigma).

[0123] Preparation of PDMS mold for cardiac tissue patch A polydimethylsiloxane (PDMS) mold was prepared and seeded with a suspension of cells in a flowable fibrin hydrogel as described above. To create a mold capable of fabricating a tissue patch measuring approximately 3 cm x 5 cm, 7.0 g of PDMS base agent was mixed with 0.7 g of PDMS curing agent (Sylgard® 184 Silicone Elastomer PDMS Kit). The polymer was placed in a vacuum desiccator chamber and degassed. Pressure was released and degassed every 5–10 min for a total of approximately 20 min to remove all air bubbles. After degassing, approximately 7 g of polymer was poured into a custom patch mold. The custom patch mold was prepared with an array of holes into which PDMS could flow and settle, creating an array of protrusions or "pillars" on the PDMS mold. The "pillars" facilitate the formation of a mesh-like structure in the cardiac tissue patch when the cell-seeded hydrogel was filled into the mold.

[0124] The custom patch mold containing PDMS was placed in a vacuum desiccator and degassed for an additional 30 minutes, releasing the pressure every 5-10 minutes to ensure all air bubbles were removed from the "pillar" holes. The custom patch mold containing PDMS was then baked at 65 °C for 8 minutes, then rotated 180° and baked for an additional 27 minutes (35 minutes total), then removed from the oven and allowed to cool to room temperature. The baked PDMS mold was then removed from the custom patch mold and sterilized with 70% ethanol followed by UV irradiation.

[0125] Isolation of cardiac nuclei Cardiac nuclei were isolated as previously described (O. Bergmann and S. Jovinge, J. Vis. Exp. (65) (2012)) with minor modifications. Briefly, BHTPs were electrically homogenized in 15 mL of lysis buffer (0.32 M sucrose, 10 mM Tris-HCl (pH = 8), 5 mM CaCl2, 5 mM magnesium acetate, 2 mM EDTA, 0.5 mM EGTA, 1 mM DTT, and 1x Complete protease inhibitors) (IKA). The lysate was then homogenized using 25 strokes in a 40 mL Dounce tissue homogenizer (Wheaton). The cell lysate was then filtered through a 100 μM cell strainer, followed by 70 μM and 40 μM cell strainers (BD Falcon), and then centrifuged at 1000 × g (Beckman Coulter Allegra X-15R) for 5 minutes to pellet the nuclei. The nuclear pellet was then resuspended in 3 mL of 1 M sucrose buffer (1 M sucrose, 10 mM Tris-HCl (pH = 8), 5 mM magnesium acetate, 1 mM DTT, and 1 × Complete protease inhibitor). The isolated cardiac nuclei pellet was washed once with PBS (Thermo Fisher Scientific) and centrifuged at 1000 × g to pellet the nuclei before FACS sorting.

[0126] Single-nucleus RNA-seq library preparation and sequencing Isolated nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific) and then sorted using an Influx cell sorter (BD) with a 70 μM nozzle and 60 psi pressure setting. Sorted nuclei were counted using a hemocytometer to calculate nuclear density and then loaded into a Chromium Controller 4 (10X Genomics) for gel bead emulsion (GEM) formation. Approximately 5,000 nuclei were loaded per sample for library preparation. After GEM formation, library preparation was performed using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 according to the manufacturer's recommended protocol. Libraries were sequenced on a NovaSeq 6000 (Illumina) at a resolution of approximately 100,000 reads per nucleus. Bioinformatics analysis for single-nucleus RNA sequencing: Raw fastq reads from each sample were mapped, processed, and counted using Cell Ranger (v3.0.2). The counts were then aggregated to generate a table of unique molecular identifier (UMI) counts for 33,939 genes for each sample. All preprocessing and filtering steps for the dataset were then performed using the R statistical programming language (v3.6.0). Cell quality was assessed independently for each sample by examining the total number of cells, the distribution of total UMI counts, the number of unique genes detected per sample, and the percentage of ribosomal and mitochondrial content per cell. Briefly, after removing mitochondrial genes, ribosomal genes, and unannotated genes, genes with at least one count in at least 20 cells were selected for downstream analysis, assuming a minimum cluster size of 20 cells. All genes on the X and Y chromosomes were removed before clustering.For each sample, we used the R package Seurat (v3.0.2) to separately perform SC Transform normalization (C Hafemeister and R Satija, Genome Biol 20(1), 296 (2019), data integration of replicates (A Butler et al., Nat Biotechnol 36(5), 411 (2018); T Stuart et al., Cell 177(7), 1888 (2019); T Stuart and R Satija, Nat Rev Genet 20(5), 257 (2019)), data scaling, and graph-based clustering. Data integration of replicates for each group was performed using CCA from the Seurat package with 30 dimensions and 3000 integration anchors (T Stuart et al., Cell 177(7), 1888(2019)), followed by data scaling. Cell clustering was performed using 20 principal components (PCs) and an initial resolution of 0.3. Marker genes for annotating clusters were identified as significantly upregulated genes for each cluster. This was performed using the limma R package (v3.40.2) with a modified t-test that applied robust empirical Bayes variance reduction, taking into account mean-variance trends, followed by a TREAT test with a log fold change threshold of 0.5 and a false discovery rate (FDR) cutoff of <0.05. Furthermore, to aid in the interpretation of the clusters, we used heat maps showing the expression of previously published marker genes. Visualization of the dataset was primarily performed using nonlinear dimensionality reduction (UMAP) plots (Becht et al., Nat Biotechnol (2018)).

[0127] Hypoxia Media: MM, SF, MM+HEPES, SF+HEPES The medium was prepared 24 hours before the experiment (MM or SF + 10 mM HEPES (Sigma-Aldrich)) and placed in a modular incubator chamber (MIC-101, Billups-Rothenberg Inc.). A degassing process was performed for 24 hours using a N2 gas flush at 50 L / min for 10 minutes.

[0128] During this time, the culture medium of the human cardiac tissue patches prepared according to the method described above was replaced with MM or SF (without HEPES). Just before the hypoxia experiment, control samples of culture medium (for LCMS) were collected from four different positions around and on top of the tissue at 24 h.

[0129] Once the samples were collected, the medium was replaced with degassed HEPES-containing medium, and the patch was placed in a modular incubator chamber equipped with a humidified water reservoir and an anaerobic indicator (Thermos Scientific, BR0055B). The chamber was then flushed with pure N gas at 50 L / min for 10 min and placed in a cell incubator at 37°C for 20 h.

[0130] After 20 hours of hypoxia, the tissues were removed from the chamber, media samples were taken for LCMS analysis (as above), and the tissues were then processed for immunostaining.

[0131] immunostaining Human cardiac tissue patches were fixed with 1% paraformaldehyde (Sigma) for 60 minutes at room temperature and washed three times with PBS. They were then stained with a primary antibody against α-cardiac actinin (Sigma, A-7811, 1:1000) in blocking buffer (5% FBS and 0.2% Triton X-100 (Sigma) in PBS) overnight at 4°C on a shaker. They were then washed twice for 2 hours in blocking buffer, followed by staining with goat anti-mouse IgG conjugated to Alexa Fluor 555 (Thermofisher, A-21422, 1:400) and Hoescht (1:1,000) overnight at 4°C. The human cardiac tissue patches were washed twice for 2 hours in blocking buffer and imaged in situ using a Leica Thunder microscope with a 5x objective. The entire patch was imaged and stitched together by computer.

[0132] To determine the intensity of nuclear and cardiomyocyte (α-cardiac actinin) staining, a custom batch processing file was created in Matlab R2013a (Mathworks) to subtract background, calculate image intensities, and export batch data to an Excel (Microsoft) spreadsheet.

[0133] mass spectrometry Metabolites were extracted from conditioned medium using a method described previously (PMID 31690627) with minor modifications. Specifically, medium samples were combined with four volumes of extraction buffer containing a 1:1 (v / v) mixture of methanol and acetonitrile, and 2.5 μM D-camphor-10-sulfonic acid (Wako) and 2.5 μM 4-morpholineethanesulfonic acid (Sigma-Aldrich) were added as internal standards. The mixture was vortexed briefly and centrifuged at 16,000 × g and 4°C for 20 min. The supernatant was lyophilized using an EZ-2 centrifugal evaporator (GeneVac), resuspended in a 1:1 (v / v) mixture of water and acetonitrile, recentrifuged, and the supernatant transferred to HPLC vials. Calibration standards were diluted with untreated medium and then extracted in parallel with the samples.

[0134] Metabolites were separated by LC using a 1290 Infinity II pump (Agilent) equipped with an InfinityLab Poroshell 120 HILIC-Z column (Agilent, 2.7 μm particle size, 2.1 mm i.d. × 100 mm length, PEEK lining). Buffer A was 90:10 (v / v) acetonitrile / water containing 10 mM ammonium acetate and 5 μM medronate (Sigma-Aldrich), and buffer B was water containing 10 mM ammonium acetate and 5 μM medronate. Medronate was included as an additive to improve sensitivity and reduce peak tailing (PMID 29976062). The column was deactivated with 0.5% (v / v) phosphoric acid before use according to the manufacturer's protocol. The LC gradient was as follows: 0 min, 10% B, 250 μL / min; 2 min, 10% B, 250 μL / min; 12 min, 40% B, 250 μL / min; 14 min, 40% B, 250 μL / min; 14.5 min, 10% B, 500 μL / min; 18.4 min, 10% B, 500 μL / min; 18.5 min, 10% B, 250 μL / min. The autosampler temperature was 4°C, the column temperature was 25°C, and the injection volume was 3 μl. MS analysis was performed using an Agilent 6470 QQQ with the following parameters: gas temperature 200°C, gas flow rate 11 L / min, nebulizer pressure 40 psi, sheath gas temperature 400°C, sheath gas flow rate 12 L / min, capillary voltage 3000 V in both negative and positive modes, and nozzle voltage 0 V in negative mode and 500 V in positive mode. Multiple-reaction monitoring transitions were calibrated and optimized using metabolite standards. Acquisition was performed with a dwell time of 10 ms.

[0135] LCMS data were extracted using Skyline (version 20.2.0.343-a7a9e8c4f) (PMID 31984744). Metabolite peak areas were normalized to an internal standard and converted to absolute amounts using calibration standards.

[0136] LDH assay Supernatants from human cardiac tissue were collected after hypoxia, and a lactate dehydrogenase (LDH) assay (Pierce) was performed on these samples according to the manufacturer's instructions to determine the extent of cell death.

[0137] Relative gene expression analysis For relative gene expression analysis between old and new media in major cardiac cell types, pseudobulk samples were created by summing the counts of each technical replicate for a given cell type. To account for differences in sequencing depth for comparison of gene expression levels between samples, counts per million (CPM) values of several housekeeping genes were calculated. TUBA1A was selected as a housekeeping gene because its CPM value did not vary between experimental conditions. To account for differences in gene length, transcripts per million (TPM) values of several genes of interest were then calculated and normalized to the TPM value of TUBA1A in each condition to calculate relative fold change (FC) values, which were then scaled using log2 transformation.

[0138] Animal testing Large animal model of right heart failure The study conformed to National Health and Medical Council of Australia guidelines and was approved by the Murdoch Children's Research Institute Animal Ethics Committee.

[0139] Anesthesia and surgical preparation were performed in a large animal facility. Border-Leicester crossbred castrated sheep, 6-9 months old and weighing 27.3 ± 1.3 kg (n = 6, mean ± SD), were premedicated with ketamine 5 mg / kg and xylazine 0.1 mg / kg intramuscularly and then anesthetized with 4% isoflurane administered by mask. After transporting the animals to the laboratory on a trolley, they were transferred to the operating table, placed supine, and their necks and chests were shaved and washed with disinfectant. After insertion of a cuffed endotracheal tube, anesthesia was maintained with isoflurane (2-3%) and nitrous oxide (10-20%) in O2-enriched air via an electronically controlled ventilator (WATO EX-20Vet, Mindray, Shenzhen, China), combined with intravenous infusion of ketamine (1-1.5 mg / kg / h), midazolam (0.1-0.15 mg / kg / h), and fentanyl (2-2.5 μg / kg / h). Percutaneous arterial O2 saturation was continuously monitored with a pulse-oximetry sensor attached to the ear. A midline neck incision allowed insertion of a 7-Fr arterial sheath into the right common carotid artery for blood pressure monitoring and periodic blood gas analysis (ABL800, Radiometer, Copenhagen, Denmark). During this time, ventilation was adjusted to maintain an arterial blood O2 partial pressure (PaO2) of 100-120 mmHg and a CO2 partial pressure (PaCO2) of 35-40 mmHg. A triple-lumen venous cannula was inserted into the superior vena cava via the right external jugular vein, and fluid infusion (combined sodium lactate at a rate of 15 ml / kg / h) and anesthetics (ketamine, midazolam, and fentanyl at the doses listed above) were administered. Body temperature was maintained at 39-40°C using a heating pad and towels.

[0140] After a supplemental intramuscular injection of buprenorphine (150 mg) for pain relief, the skin on the front of the chest was incised from the base of the neck to the xiphoid process, and a midline sternotomy was performed to expose the lungs and heart. After insertion and expansion of a thoracic retractor, an adjustable polyvinyl snare was placed around the inferior vena cava just below the heart. The pericardium was then incised between the apex and the ascending aorta to create a pericardial cradle to support the heart. Fluid-filled catheters were then inserted into the left and right atrial appendages to measure left and right cardiac filling pressures. A fluid-filled catheter and a 3.5-Fr micromanometer catheter (SPR-877, Millar Instruments, Houston, TX, USA) were inserted into the pulmonary trunk through separate purse-string sutures to measure mean pulmonary artery pressure and high-fidelity pulmonary artery pressure, respectively. A 6-Fr arterial sheath was also inserted into this artery through another purse-string suture. A sequential flow probe (16 or 18 mm PAU, Transonic Systems, Ithaca, NY, USA) was then placed around the pulmonary trunk to measure right ventricular stroke volume. The animals were then anticoagulated with an intravenous bolus of heparin sodium (40 IU / kg), followed by additional boluses of heparin (20 IU / kg) at hourly intervals. A 5-Fr combined micromanometer-conductance catheter (Ventri-Cath 507, Millar Instruments, Houston, TX, USA) was then introduced into the right ventricle via a sheath in the pulmonary trunk to obtain high-fidelity pressure and volume signals.

[0141] After instrumentation, a model of right ventricular (RV) dysfunction under RV volume overload was created by combining two interventions. First, similar to the approach described by Agger et al. (Agger et al., Interact Cardiovasc Thorac Surg 10(6), 962(2010)), a stable degree of pulmonary regurgitation was created by catching two of the tricuspid valves that make up the pulmonary valve with six or seven interrupted 4 / 0 silk sutures on a curved needle inserted at the base of the pulmonary trunk. Severe pulmonary regurgitation was created by ligating the sutures on both the input and output sides, thereby fixing the pulmonary valve leaflets to the inner surface of the pulmonary trunk. RV dysfunction was then induced by injecting 65,000 polystyrene microbeads (90 μm diameter, Polysciences, Warrington, PA, USA, catalog number 07315) via a catheter inserted into the ascending aorta through a small aortotomy and positioned within the origin of the right coronary artery, according to the method of Huang et al. (Y. Huang et al., Asaio j 43(5), M408 (1997)). The microbead suspension was briefly ultrasonically mixed immediately before injection, followed by manual shaking. After this, the microbeads were delivered in 2.5 ml of isotonic saline. To prevent abnormal cardiac rhythms, a bolus of lignocaine (25 mg) was administered intravenously before microbead administration.

[0142] Surgical implantation of BHTP The trapezoidal BHTP was carefully secured to the outer surface of the right ventricle in the area previously affected by the microbead injection. Fixation of the patch to the right ventricle was achieved using six interrupted sutures with 5 / 0 prolene, with four sutures placed at the corners of the patch and the remaining sutures placed at the midpoint and opposite sides of the trapezoid's base. The pericardium was then closed over the patch using a combination of interrupted and running sutures with 4 / 0 silk. After closure, the pericardium was covered with surgical gauze soaked in isotonic saline, and the gauze was kept moist with additional isotonic saline applied at 15-minute intervals during the 2-hour monitoring period.

[0143] Functional evaluation of a large animal model of right heart failure with implanted BHTP Experimental protocol General hemodynamics (heart rate, aortic and pulmonary artery blood pressure, right ventricular stroke volume) and right ventricular pressure-volume measurements were performed immediately before RV patch application and repeated at 15-minute intervals for 2 hours after RV patch implantation. Two data sets were recorded on the computer at each time point. The first was steady-state data from a 20- to 30-second block. The second, used to perform the gold standard assessment of RV function using pressure-volume analysis, involved brief (approximately 10-second) clamping of an adjustable snare around the inferior vena cava to temporarily reduce venous blood return to the heart and, therefore, RV preload.

[0144] At the end of the study, the animals were humanely sacrificed with an overdose of sodium pentobarbitone (100 mg / kg) injected into the left atrial catheter.

[0145] Physiological Data Processing and Analysis Fluid-filled catheter pressures in the aorta, pulmonary artery, left atrium, and right atrium were measured with atmospheric pressure-referenced transducers at the left atrial level and were calibrated with a water manometer before each test. Trunk pulmonary artery blood flow (i.e., right ventricular output) was measured with a flowmeter (Model T206, Transonic Systems, Ithaca, NY, USA). Instantaneous RV pressure and volume were obtained from a micromanometer-conductance catheter via an interfaced pressure-volume signal processing system (MV Ultra, Millar Instruments, Houston, TX, USA).

[0146] Analog catheter, micromanometer, and flow probe signals were digitized (iNET-100B, GW Instruments, Somerville, MA, USA) at a sampling rate of 1 kHz and recorded using programmable acquisition and analysis software (Spike2, Cambridge Electronic Design, Cambridge, UK). During analysis of physiological data, no filtering was used except for a 48 Hz low-pass filter to remove electrical interference from the signal, and steady-state analysis was typically performed on ensemble-averaged signals generated from >30 beats.

[0147] Right ventricular pressure and volume data RV volume was calculated from the conductance catheter signal using standard methodology incorporating estimates of blood resistivity, parallel conductance, and gain constants. (4-9) Blood resistivity was measured in small samples (<0.5 ml) of aortic blood (Rho-calibrated cuvette, Millar Instruments, Houston, TX, USA) before and after RV patch implantation. Parallel conductance, i.e., the offset of the conductance catheter signal related to the conductivity arising from structures surrounding the blood pool in the RV cavity, was obtained by recording changes in RV volume data during injection of 4 ml of hypertonic (10%) saline into the right atrium at 30-minute intervals after RV patch implantation (Baan et al., Circulation 70(5), 812 (1984); P Steendijk et al., Am J Physiol Heart Circ Physiol 281(2), H755 (2001); White and A.N. Redington, Physiol Meas 21(3), R23 (2000)). The gain constant was calculated as the ratio of the RV stroke volume obtained from the conductance catheter to the RV stroke volume obtained from the flow probe under steady-state conditions immediately before each transient occlusion of the inferior vena cava.

[0148] The rate of change of RV pressure was calculated using a high-fidelity three-point differential algorithm, and the maximum rate of rise of RV pressure (RV dP / dtmax) was used as a measure of RV contractility. RV end-diastole was defined at the origin of the rising edge of the RV pressure waveform using an automated curvature-based feature extraction algorithm (JP Mynard et al., Annu Int Conf IEEE Eng Med Biol Soc 2007, 1691 (2007)). The corresponding RV end-diastolic volume was then measured at this point. RV stroke work was calculated as the area of the RV pressure-volume loop. The RV preload recruitment stroke work index (PRSWI), which constitutes the slope of the highly linear relationship between RV stroke work on the Y-axis and RV end-diastolic volume on the X-axis, was obtained from a series of pressure-volume loops generated during the transient reduction in RV preload caused by tightening the snare around the inferior vena cava. PRSWI represents a robust and sensitive measure of ventricular pumping function (D Burkhoff et al., Am J Physiol Heart Circ Physiol 289(2), H501(2005); DD Glower et al., Circulation 71(5), 994(1985)).

[0149] statistical analysis Results were analyzed using GraphPad Prism version 9 (GraphPad Software Inc., La Jolla, CA, USA). Time courses of hemodynamic and RV pressure-volume data were analyzed using one-way repeated measures analysis of variance (ANOVA), and specific comparisons were assessed by partitioning within-animal sums of squares into individual degrees of freedom. Data are presented as mean ± SD, and significance was determined at P < 0.05.

[0150] Example 1. Characterization of bioengineered cardiac tissue patches Human and control cardiac tissue patches were prepared according to the protocol described in the Materials and Methods section above and compared using single-cell analysis, also as described above. Briefly, after 7 days of culture in SF medium (also referred to as "new medium") or maturation medium MM (also referred to as "old medium"), the tissue patches were removed from their molds and frozen. The frozen patches were then detached, homogenized, and nuclei were isolated from the cells in each patch. The nuclei were then sequenced and analyzed.

[0151] Single-nuclear RNA sequencing (snRNA-seq) of 5,889 single cardiac nuclei from human cardiac tissue patches cultured in SF medium (also called "new medium") or maturation medium MM (also called "old medium") revealed five major cellular clusters (Figure 2). Analysis of marker genes in these five clusters allowed us to identify the major cardiac cell types in the human cardiac tissue patches, including cardiomyocytes (TNNT2, ACTN2, MYH7), fibroblasts / stromal cells (LAMB1, COL1A2, COL1A1), endothelial cells (PECAM1, ENG, EMCN), smooth muscle cells (TAGLN, ACTA2), and progenitor / proliferative cells (ANLN, TOP2A, CDK1) (Figures 2-8). Culturing human cardiac tissue patches in "new medium" was accompanied by a major shift in cellular composition, characterized by a significant expansion of the relative proportions of endothelial and smooth muscle cells (Figure 2). Human cardiac tissue patches cultured in the "old medium" consisted solely of cardiomyocytes (approximately 40%), interstitial cells / fibroblasts (approximately 55%), and cardiac progenitor cells (approximately 5%), whereas the "new medium" retained cardiomyocytes (approximately 40%), interstitial cells / fibroblasts (approximately 25%), cardiac progenitor cells (approximately 5%), endothelial cells (approximately 20%), and smooth muscle cells (approximately 10%).

[0152] Table 1 Proportion of cell types present in human cardiac tissue patches cultured in new medium (New Medium rep1 and New Medium rep2) compared to human cardiac tissue patches cultured in old medium (Old Medium rep2), where proportions are expressed as percentages of the total population.

[0153] [Table 1]

[0154] The "new medium" cardiomyocytes were characterized by the suppression of biological processes related to redox processes, fatty acid oxidation, and muscle contraction, as well as marker genes related to these biological processes (Figure 9). In addition, a subpopulation of cardiomyocytes expressing proliferation markers was identified, and this population was more prevalent in the new medium (approximately 3%) compared to the old medium (approximately 1%) (Figure 4).

[0155] The fibroblast growth factor receptor FGFR1 was the predominant FGF receptor expressed in human cardiac tissue patches (Figure 12), and the platelet-derived growth factor (PDGF) receptor PDGFRB was the predominant PDGF receptor expressed in human cardiac tissue patches (Figure 13). FGFR1 was expressed in cardiomyocytes, stromal cells / fibroblasts, endothelial cells, and smooth muscle cells, whereas PDGFRB was highly enriched in stromal cells / fibroblasts in human cardiac tissue patches.

[0156] Example 2. Hypoxia-resistant cardiac tissue patch Human cardiac tissue patches and control cardiac tissue patches were prepared according to the protocol described above in the section entitled Materials and Methods and were subjected to hypoxia also as described above.

[0157] Briefly, after 5 days of culture in SF medium (also called "fresh medium") or maturation medium MM, tissue patches were subjected to hypoxia for 20 hours, and these patches and their respective culture media were then evaluated (Figure 14A).

[0158] After 20 hours of hypoxia, SF human cardiac tissue patches showed higher nuclear intensity, indicating more viable cells (Figure 14B, C), with no change in cardiomyocyte content, and α-actinin staining revealed both viable and nonviable cells (Figure 14B, D). There was less cell death in SF human cardiac tissue patches, marked by lactate dehydrogenase in the culture medium (Figure 14E).

[0159] SF human cardiac tissue patches also demonstrated enhanced glycolytic metabolism as evidenced by increased lactate production both before and after hypoxia (Figure 14F), and the ability to adapt to hypoxia as evidenced by an increased lactate-to-pyruvate ratio in response to hypoxia (Figure 14G).

[0160] These data demonstrate that the cardiac tissue patches of the present invention have improved resistance to hypoxic conditions compared to stem cell-derived cardiac tissue prepared according to existing methods, indicating improved survival and engraftment potential after transplantation.

[0161] Example 3. Functional validation of engineered cardiac tissue in vivo Human cardiac tissue patches were sutured onto the right ventricle as described above, and cardiac function was monitored for up to 2 hours after implantation (Figures 15-18). The human cardiac tissue patches were robust and durable, maintaining their integrity after implantation (Figure 15). There was no change in the shape of the pressure-volume loops after patch implantation, and there was a slight rightward shift at 90 minutes after patch implantation, consistent with a slight increase in right ventricular size after patch implantation (Figure 16). After patch implantation, a highly linear relationship was obtained between right ventricular preload recruitment stroke work and end-diastolic volume (Figure 17). There was only a slight change in the slope of these relationships after patch implantation, indicating that right ventricular pump function was maintained after patch implantation (Figure 18). No changes were observed in mean aortic blood pressure (P = 0.71), RV stroke volume (P = 0.62), RV dP / dtmax (P = 0.52), or RV PRSWI (P = 0.61) (Figure 18). Although there was a small linear increase in heart rate (P = 0.006) and mean pulmonary artery blood pressure (P = 0.0002) over the 2 hours following patch implantation (Figure 18), these changes were likely due to the long-term effects of anesthesia rather than the patch. These data demonstrate that human cardiac tissue patches are well tolerated after implantation in vivo and are not associated with adverse acute events.

Claims

1. 1. A method for producing engineered human cardiac tissue comprising endothelial cells, comprising culturing a cell suspension comprising a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population in the presence of an FGFR1 agonist and a PDGFR agonist for a time and under conditions sufficient to produce engineered human cardiac tissue comprising endothelial cells.

2. 1. A method for producing engineered human cardiac tissue, comprising: i) mixing a cell suspension comprising a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population with a flowable hydrogel composition, wherein the cell suspension is in a serum-free medium; ii) filling the flowable hydrogel composition containing the cell suspension into a mold and incubating the hydrogel for a time and under conditions sufficient to solidify the hydrogel; iii) culturing the hydrogel from step ii) in serum-free medium; contacting the human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte populations with an FGFR1 agonist and a PDGFR agonist, thereby providing engineered human cardiac tissue, the tissue comprising cardiomyocytes, stromal cells / fibroblasts, and endothelial cells.

3. 3. The method of claim 2, wherein the FGFR1 agonist and / or the PDGFR agonist is added to the serum-free medium or the flowable hydrogel composition prior to the mixing in step i).

4. 3. The method of claim 2, wherein after the mixing in step i), the FGFR1 agonist and / or the PDGFR agonist is added to the flowable hydrogel composition containing the cell suspension.

5. 5. The method of claim 2, wherein, prior to the mixing in step i), the human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte populations are cultured in a serum-free medium comprising an FGFR1 agonist and a PDGFR agonist.

6. The method according to any one of claims 2 to 5, wherein in step iii), the hydrogel of step ii) is cultured in a serum-free medium containing the FGFR1 agonist and the PDGFR agonist.

7. The method of any one of claims 1 to 6, wherein the cell suspension comprises about 70% cardiomyocytes and about 30% to about 50% non-cardiomyocytes.

8. The method of any one of claims 1 to 7, wherein the cell suspension comprises about 50 to about 75% cardiomyocytes and about 25 to about 50% non-cardiomyocytes.

9. The method according to any one of claims 1 to 8, wherein the cell suspension is derived from a single differentiation process.

10. 10. The method of any one of claims 1 to 9, wherein the engineered human cardiac tissue further comprises vascular smooth muscle cells and cardiac progenitor cells.

11. the engineered human cardiac tissue comprises: - at least about 30% cardiomyocytes, - about 35% or less stromal cells / fibroblasts, - about 30% or less endothelial cells, The method according to any one of claims 1 to 10, comprising:

12. the engineered human cardiac tissue comprises: - about 30 to about 70% cardiomyocytes, - about 10 to about 35% stromal cells / fibroblasts, - about 5 to about 30% endothelial cells, - up to about 15% of vascular smooth muscle cells, - up to about 10% cardiac progenitor cells, The method according to any one of claims 1 to 11, comprising:

13. 13. The method of any one of claims 1 to 12, wherein the FGFR1 agonist is selected from FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

14. The method of claim 13, wherein the FGFR1 agonist is FGF2.

15. The method of any one of claims 1 to 14, wherein the PDGFR agonist is selected from PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, or PDGF-DD.

16. 16. The method of claim 15, wherein the PDGFR agonist is a PDGFRβ agonist.

17. 17. The method of claim 16, wherein the PDGFR agonist is PDGF-BB.

18. The method according to any one of claims 1 to 17, wherein the serum-free medium comprises about 0.5 to 100 ng / ml of human PDGF-BB and about 0.5 to 100 ng / ml of human FGF2.

19. The method according to any one of claims 1 to 18, wherein the serum-free medium comprises about 10 ng / ml human PDGF-BB and about 10 ng / ml human FGF2.

20. The method of any one of claims 1 to 19, wherein the serum-free medium comprises a basal medium containing albumin and transferrin.

21. 21. The method of claim 20, wherein the serum-free medium comprises a B27 supplement.

22. 22. The method of claim 20 or 21, wherein the basal medium is a low calcium medium having a calcium concentration of less than about 1.2 mM.

23. 23. The method of any one of claims 2 to 22, further comprising replacing the medium in step iii) with fresh cell culture medium at least every two days.

24. The method according to any one of claims 2 to 23, wherein the culturing in step iii) is carried out for at least 5 days.

25. 25. The method of any one of claims 1 to 24, wherein about 3% to about 10% of the population of cardiomyocytes in the engineered human cardiac tissue are proliferative.

26. 26. The method of any one of claims 1 to 25, wherein the engineered human cardiac tissue exhibits glycolytic metabolism.

27. 27. The method of claim 26, wherein the cell culture medium obtained after 24 hours of culture with the engineered human cardiac tissue has a lactate concentration of 1 mM.

28. 28. The method of any one of claims 1 to 27, wherein the engineered human cardiac tissue exhibits a log2 relative gene expression level for PKM of greater than 2 when normalized to TUBA1A expression levels.

29. 29. The method of any one of claims 1 to 28, wherein the engineered human cardiac tissue is resistant to hypoxia.

30. 30. The method of any one of claims 1-29, wherein the engineered human cardiac tissue produces a lactate / pyruvate ratio of at least 1:1 in the resulting cell culture medium after 20 hours of culture under hypoxic conditions.

31. 31. The method of any one of claims 1 to 30, wherein the engineered human cardiac tissue exhibits downregulation of ALDH1, AGMO, DPYD, GPX3, CYP1B1 and PLIN5.

32. The cardiomyocytes are ＋ The method according to any one of claims 1 to 31, wherein

33. The fibroblasts are ＋ , EMP1 ＋ , FOXD1 ＋ , RAB27B ＋ , NR2F1 ＋ , F2RL1 ＋ , SPP1 ＋ , TMEM158 ＋ , PTHLH ＋ , PHLDA2 ＋ , MALL ＋ , MYCT1 ＋ , DUSP4 ＋ , PLAU ＋ , TMEM156 ＋ , CD274 ＋ , MMP10 ＋ , ARRDC4 ＋ , RFX8 ＋ , M.L.P.H. ＋ , THBD ＋ , HHEX ＋ , VGF ＋ , OTULINL ＋ , IL33 ＋ , CA12 ＋ , C6orf141 ＋ , MFSD2A ＋ and / or CARD10 ＋ The method according to any one of claims 1 to 32, wherein

34. 34. The method of any one of claims 1 to 33, wherein the engineered human cardiac tissue exhibits a contractile activity of less than 10 mN / mm2.

35. The method of any one of claims 2 to 34, wherein the hydrogel composition comprises a fibrin hydrogel.

36. 36. The method of claim 35, wherein the fibrin is formed by mixing a thrombin solution and a fibrinogen solution.

37. 37. The method of claim 36, wherein the fibrinogen is present at a concentration of 10 mg / mL to 50 mg / mL.

38. 37. The method of claim 36, wherein the fibrinogen is present at a concentration of 20 mg / mL.

39. The cell suspension is mixed with the hydrogel of step i) to obtain approximately 5×10 ６ ~Approx. 100×10 ６ 39. The method of any one of claims 2 to 38, providing a cell concentration in cells / mL.

40. The cell suspension is mixed with the hydrogel of step i) to obtain approximately 20×10 ６ ~Approx. 50×10 ６ 40. The method of claim 39, wherein the cell concentration is provided in cells / mL.

41. The cell suspension is mixed with the hydrogel of step i) to obtain approximately 35×10 ６ 41. The method of claim 40, providing a cell concentration in cells / mL.

42. hPSCs comprising: a) culturing hPSCs for about 72 hours in a basal medium comprising an effective amount of BMP4, activin A, FGF, a GSK3 inhibitor, a serum-free supplement comprising albumin and transferrin, but no insulin, wherein the medium is replaced with fresh medium daily; b) culturing the cells obtained in step a) in a basal medium containing an effective amount of an inhibitor of the Wnt signaling pathway and the same serum-free supplement as in a) for about 72 hours, wherein the medium is replaced with fresh medium every day; c) culturing the cells obtained in step b) in a basal medium containing an effective amount of an inhibitor of the Wnt signaling pathway and a serum-free supplement containing albumin and transferrin, and including insulin, for about 7 days, wherein the medium is replaced with fresh medium after the second and fourth days of the about 7-day period; d) inducing differentiation into cardiac lineages by culturing the cells obtained in step c) in a basal medium containing a serum-free supplement containing albumin, transferrin, and insulin for about 72 hours; wherein the basal medium used in each of steps a) to d) has a calcium concentration of less than about 1.2 mM; The method of any one of claims 1 to 41, thereby providing a cell suspension comprising a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte and non-cardiomyocyte cell population.

43. - the GSK3 inhibitor is CHIR99021, and / or - the inhibitor of the Wnt signaling pathway is IWP-4, and / or - The method of claim 42, wherein said basal medium is RPMI or DMEM.

44. 44. The method of any one of claims 1 to 43, wherein the engineered human cardiac tissue is in the form of a tissue patch.

45. 45. The method of claim 44, wherein the patch is used as an implant in a subject.

46. 46. The method of claim 45, wherein the subject is suffering from cardiomyopathy or cardiac tissue damage.

47. 47. The method of claim 46, wherein the cardiomyopathy or cardiac tissue damage results from acute or chronic stress, atherosclerotic damage of blood vessels, ischemia, myocardial infarction, inflammatory disease, valvular heart disease, or myocarditis.

48. 46. The method of claim 45, wherein the subject has a congenital heart disease.

49. 49. The method of claim 48, wherein the congenital heart disease is selected from the group consisting of hypoplastic left heart syndrome, tetralogy of Fallot, truncus arteriosus, pulmonary atresia, ventricular septal defect, atrial septal defect, and single ventricle including endocardial cushion defect.

50. 50. The method of any one of claims 1 to 49, wherein the hPSCs are iPSCs.

51. 51. The method of claim 50, wherein the iPSCs are derived from a subject with a cardiac disease and / or from a subject into which the engineered human cardiac tissue will be transplanted.

52. 52. The method of any one of claims 1 to 51, wherein the hPSCs are embryonic stem cells.

53. An engineered human cardiac tissue comprising at least about 30% hPSC-derived cardiomyocytes, not more than about 35% hPSC-derived stromal cells / fibroblasts, and not more than about 30% hPSC-derived endothelial cells, wherein said cells are distributed throughout the hydrogel composition.

54. An engineered human cardiac tissue comprising hPSC-derived cardiomyocytes, hPSC-derived stromal cells / fibroblasts, and hPSC-derived endothelial cells, wherein the cells are derived from a single differentiation process and the cells are distributed throughout a hydrogel composition.

55. 55. The engineered human cardiac tissue of claim 53 or 54, wherein the engineered human cardiac tissue further comprises vascular smooth muscle cells and cardiac progenitor cells.

56. the engineered human cardiac tissue comprises: - about 30 to about 70% cardiomyocytes, - about 10 to about 35% stromal cells / fibroblasts, - about 5 to about 30% endothelial cells, 56. The engineered human cardiac tissue of any one of claims 53 to 55, comprising:

57. 57. The engineered human cardiac tissue of any one of claims 53 to 56, wherein up to about 10% of the cardiomyocytes in the engineered human cardiac tissue are proliferative.

58. 58. The engineered human cardiac tissue of any one of claims 53 to 57, wherein said engineered human cardiac tissue exhibits glycolytic metabolism.

59. 54. The engineered human cardiac tissue of claim 53, wherein the cell culture medium obtained after 24 hours of culture with the engineered human cardiac tissue has a lactate concentration of 1 mM.

60. 60. The engineered human cardiac tissue of any one of claims 53 to 59, wherein the engineered human cardiac tissue exhibits a log2 relative gene expression level of PKM greater than 2 when normalized to TUBA1A.

61. 61. The engineered human cardiac tissue of any one of claims 53 to 60, wherein the engineered human cardiac tissue is resistant to hypoxic conditions.

62. 62. The engineered human cardiac tissue of any one of claims 53-61, wherein the engineered human cardiac tissue produces a lactate / pyruvate ratio of at least 1:1 in the resulting cell culture medium after 20 hours of culture under hypoxic conditions.

63. 63. The engineered human cardiac tissue of any one of claims 53 to 62, wherein the engineered human cardiac tissue exhibits log2 relative gene expression levels of ALDH1, AGMO, GPX3, CYP1B1 and PLIN5 of less than -4 when normalized to TUBA1A expression levels.

64. The cardiomyocytes are ＋ 64. The engineered human cardiac tissue of any one of claims 53 to 63, wherein:

65. The fibroblasts are ＋ , EMP1 ＋ , F0XD1 ＋ , RAB27B ＋ , NR2F1 ＋ , F2RL1 ＋ , SPP1 ＋ , TMEM158 ＋ , PTHLH ＋ , PHLDA2 ＋ , MALL ＋ , MYCT1 ＋ , DUSP4 ＋ , PLAU ＋ , TMEM156 ＋ , CD274 ＋ , MMP10 ＋ , ARRDC4 ＋ , RFX8 ＋ , M.L.P.H. ＋ , THBD ＋ , HHEX ＋ , VGF ＋ , OTULINL ＋ , IL33 ＋ , CA12 ＋ , C6orf141 ＋ , MFSD2A ＋ and / or CARD10 ＋ 65. The engineered human cardiac tissue of any one of claims 53 to 64, wherein:

66. - about 30 to about 70% cardiomyocytes, - about 10 to about 35% stromal cells / fibroblasts, - about 5 to about 30% endothelial cells, - up to about 15% of vascular smooth muscle cells, - up to approximately 10% cardiac progenitor cells, 66. The engineered human cardiac tissue of any one of claims 53 to 65, comprising:

67. The engineered human cardiac tissue has a resistance of 10 mN / mm ２ 67. The engineered human cardiac tissue of any one of claims 53 to 66, exhibiting a contractile activity of less than 100 ng / ml.

68. 68. The engineered human cardiac tissue of any one of claims 53 to 67, wherein the hydrogel composition comprises fibrin formed by mixing a thrombin solution and a fibrinogen solution.

69. 69. The engineered human cardiac tissue of claim 68, wherein the fibrinogen is present at a concentration of 10 mg / mL to 50 mg / mL.

70. 70. The engineered human cardiac tissue of claim 69, wherein the fibrinogen is present at a concentration of 20 mg / mL.

71. The total number of cells in the tissue is about 20 x 10 ６ Cell ~100×10 ６ 71. The engineered human cardiac tissue of any one of claims 53 to 70, which is a cell.

72. 72. The engineered human cardiac tissue of any one of claims 53 to 71, wherein the engineered human cardiac tissue is in the form of a tissue patch.

73. 73. The engineered human cardiac tissue of any one of claims 53-72, wherein the hPSCs are iPSCs.

74. 74. The engineered human cardiac tissue of any one of claims 53 to 73, wherein the hPSCs are embryonic stem cells.

75. 53. Engineered human cardiac tissue produced according to the method of any one of claims 1 to 52.

76. 76. A method for analyzing the biological effect of at least one test compound or biologically active substance on cardiac cells, the method comprising contacting engineered human cardiac tissue according to any one of claims 53 to 75 with said test compound or said biologically active substance, incubating said tissue in the presence of said test compound or said biologically active substance, and analyzing said biological effect.

77. 76. The engineered human cardiac tissue of any one of claims 53 to 75, for use as a graft in the treatment of diseased or damaged cardiac tissue in a subject in need thereof.

78. 100. A method for treating diseased or damaged cardiac tissue in a subject in need thereof, the method comprising transplanting in said subject the engineered human cardiac tissue of any one of claims 53-75.

79. 80. Use of the engineered human cardiac tissue of any one of claims 53 to 75 in the manufacture of a medicament for the treatment of diseased or damaged cardiac tissue in a subject in need thereof.

80. 80. The engineered human cardiac tissue of claim 77, the method of claim 78 or the use of claim 79, wherein the subject is suffering from cardiomyopathy or cardiac tissue damage.

81. 81. The engineered human cardiac tissue, method or use of claim 80, wherein said cardiomyopathy or said cardiac tissue damage results from acute or chronic stress, atherosclerotic damage of blood vessels, ischemia, myocardial infarction, inflammatory disease, valvular heart disease, or myocarditis.

82. 80. The engineered human cardiac tissue of claim 77, the method of claim 78 or the use of claim 79, wherein the subject is suffering from congenital heart disease.

83. 83. The engineered human heart tissue, method or use of claim 82, wherein said congenital heart disease is selected from the group consisting of hypoplastic left heart syndrome, tetralogy of Fallot, truncus arteriosus, pulmonary atresia, ventricular septal defect, atrial septal defect, and single ventricle including endocardial cushion defect.

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