Engineering human heart tissue

Abstract

The present application relates to methods for producing engineered human pluripotent stem cell-derived cardiac tissues, engineered cardiac tissues produced by such methods, and methods of use thereof.

Description

[0001] field

[0002] 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 use thereof.

[0003] background

[0004] Despite many breakthroughs in cardiovascular surgery and medicine, heart attacks and heart failure remain the most prominent health challenges. Heart disease is also the leading cause of death and disability in children, affecting one percent of live births. Among adults diagnosed with congestive heart failure (CHF), studies have found that the mortality rate is approximately 50% within 5 years of diagnosis. An emerging and worrying trend is the sharp increase in the number of children and adults with CHD who are hospitalized due to heart failure. Over the past 20 years, advances in surgical procedures have greatly improved the survival rates of children with heart disease, and now more than 85% of children with CHD survive into adulthood. As a result, CHD is now considered a lifelong disease.

[0005] Heart failure is characterized by the loss or dysfunction of cardiomyocytes, whether due to ischemic heart disease, hypertensive heart disease, congenital heart disease, or idiopathic cardiomyopathy. The adult myocardium cannot regenerate, and damaged myocardial tissue is replaced by non-contractile scar tissue. Currently, heart failure can only be addressed with heart transplantation, but there is a growing gap between the number of donors and the number of heart failure patients who require a transplant. Current treatment options are inadequate, and new approaches that fundamentally change the trajectory of patients are necessary.

[0006] The directed differentiation of human pluripotent stem cells (hPSC), including human embryonic stem cells (hES) and human induced pluripotent stem cells (hiPS), to different cell endpoints has been able to produce organoid models of various human tissues (including cardiac tissue). Tissue engineering technology seeks to produce stem cell-derived functional cardiac tissue, which can be used for disease modeling and treatment. However, the existing methods and schemes for producing engineered cardiac tissues cannot accurately reproduce the composition and function (e.g., contractility) of natural cardiac tissue. Various schemes reported in the literature have generated cardiac tissues composed of cardiomyocytes and stromal cells (fibroblasts) (see, e.g., WO2015 / 040142), but they generally lack endothelial cells (which represent the most abundant non-cardiomyocyte population in the human heart) and other vascular cells (smooth muscle cells). Although cardiomyocyte populations, stromal cell populations, and endothelial cell populations can be easily obtained by different individual differentiation schemes, and then combined, the combination or mixing of cell types from different differentiation schemes brings undesirable complexity and variability.

[0007] In addition, current protocols and cell culture medium formulations attempt to promote high maturation and contractility 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 implantation after implantation into the hypoxic environment in vivo. Oxidative metabolism also inhibits cardiomyocyte proliferation (Mills et al., 2017), which may not be optimal for tissue growth after implantation in vivo.

[0008] There remains a need in the art for methods of generating pluripotent stem cell-derived cardiac tissue that overcome these shortcomings. SUMMARY OF THE INVENTION

[0010] 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 that could be derived from a single differentiation protocol.

[0011] 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 population of cardiomyocytes and non-cardiomyocytes derived from a culture of human pluripotent stem cells (hPSCs) in the presence of a FGFR1 agonist and a PDGFR agonist for a period of time under conditions sufficient to produce engineered human cardiac tissue comprising endothelial cells.

[0012] According to a second aspect, the present invention provides a method for producing engineered human cardiac tissue, the method comprising the following steps: i) mixing a cell suspension comprising a population of cardiomyocytes and non-cardiomyocytes derived from a human pluripotent stem cell (hPSC) culture with a flowable hydrogel composition, wherein the cell suspension is in a serum-free culture medium; ii) loading the flowable hydrogel composition comprising the cell suspension into a mold, and incubating the hydrogel for a period of time under conditions sufficient to solidify the hydrogel; and iii) culturing the hydrogel from step ii) in a serum-free culture medium; wherein the population of cardiomyocytes and non-cardiomyocytes derived from a human pluripotent stem cell (hPSC) culture is contacted with a FGFR1 agonist and a PDGFR agonist, thereby providing engineered human cardiac tissue, wherein the tissue comprises cardiomyocytes, stromal cells / fibroblasts and endothelial cells.

[0013] According to a third aspect, the present invention provides engineered human cardiac tissue comprising at least about 30% hPSC-derived cardiomyocytes, about 35% or less hPSC-derived stromal cells / fibroblasts, and about 30% or less hPSC-derived endothelial cells, wherein the above cells are located throughout the hydrogel composition.

[0014] 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 above cells are derived from a single differentiation process, wherein the above cells are located throughout a hydrogel composition.

[0015] According to a fifth aspect, the present invention provides engineered human cardiac tissue produced according to the method of the first aspect.

[0016] According to the sixth aspect, the present invention provides a method for analyzing the biological effect of at least one test compound or bioactive agent on cardiomyocytes, which comprises contacting the engineered human cardiac tissue of any one of the second aspect, the third aspect or the fourth aspect 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.

[0017] According to a seventh aspect, the present invention provides use of the engineered human cardiac tissue of any one of the second, third or fourth aspects as an implant for treating diseased or damaged cardiac tissue in a subject in need thereof.

[0018] According to an eighth aspect, the present invention provides a method for treating diseased or damaged cardiac tissue in a subject in need thereof, comprising implanting the engineered human cardiac tissue of any one of the second, third or fourth aspects into the subject.

[0019] According to a ninth aspect, the present invention provides use of the engineered human cardiac tissue of any one of the second, third or fourth aspects in the manufacture of a medicament for treating diseased or damaged cardiac tissue in a subject in need thereof.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown: A) Schematic overview of the cardiac differentiation and tissue engineering protocol. Human pluripotent stem cells were differentiated into cardiomyocytes, and on day 15, the cells were enzymatically dissociated and mixed with hydrogels, which were then placed in a custom-designed PDMS mold. The tissue was cultured on a custom-designed scaffold for approximately 7 days and then removed from the mold for implantation. B) Photograph of a bioengineered cardiac tissue patch (BHTP). CE) Immunofluorescence and histological images of fixed, paraffin-embedded, and sectioned BHTPs. Immunofluorescence images show that cells within the fibrin matrix express cardiac-specific markers, including α-actin, myosin light chain 2 (MLC2v) (Figure C), and cardiac troponin T (cTnT) (Figure D). The nuclei in C) and D) were labeled with Hoechst. E) Histological images are hematoxylin and eosin staining, showing the overall tissue structure of the BHTP and the cardiomyocytes distributed therein.

[0022] Figure 2 Single cell RNA sequencing UMAP plots are shown, showing cell clusters present in BHTP (serum-free (sf) "new" medium). Cell clusters present in BHTP include cardiomyocytes (purple), endothelial cells (red), fibroblasts / stromal cells (green), smooth muscle cells (blue), and cardiomyocyte progenitor cells (gold).

[0023] Figure 3 A heat map showing the expression of cell type specific marker genes in a wide range of cell types is shown. CM(Prlf), cardiomyocyte (proliferative); CM, cardiomyocyte; Endo, endothelial; Fibro / Stromal, fibroblast / stromal cell; and Smc, smooth muscle cell; cardiac progenitor cell.

[0024] Figure 4 UMAP plots are shown, showing the expression of cardiomyocyte (proliferative) markers (ANLN, TOP2A and CDK1) in the samples.

[0025] Figure 5 UMAP plots showing the expression of cardiomyocyte markers (TNNT2, ACTN2, and MYH7) in the samples are shown.

[0026] Figure 6 UMAP plots showing the expression of endothelial markers (PECAM1, ENG, EMCN) in the samples are shown.

[0027] Figure 7 UMAP plots showing the expression of smooth muscle cell markers (TAGLN and ACTA2) in the samples are shown.

[0028] Figure 8 UMAP plots showing the expression of fibroblast / stromal markers (LAMB1, COL1A2, and COL1A1) in the samples are shown.

[0029] Fig. 9BHTP (new medium) cardiomyocytes are shown to be characterized by reduced expression of genes involved in redox processes and fatty acid oxidation. A) Single cell RNA sequencing UMAP plot showing the cell clusters present in BHTP (new medium) compared to BHTP (old medium), and the cardiomyocyte populations used for downstream gene set enrichment analysis (GSEA). A subpopulation of proliferating cardiomyocytes, CM (Prlf), is shown in light blue. B) Heat map showing the expression of genes associated with Gene Ontology (GO) biological processes related to redox, fatty acid oxidation, and muscle contraction. Markers of these processes are upregulated during human development (primary cardiomyocytes) from fetal to adult stages. Markers are also elevated in BHTP old medium compared to new medium. C) Dot plot showing example genes of biological processes showing reduced expression of oxidative stress markers in BHTP new medium.

[0030] Fig.10 Shown are relative gene expression of glycolytic markers (PKM2) and redox markers (ALDH1A1, AGMO, DPYD, GPX3, CYP1B1, PLIN5) following pseudo-batch analysis of cardiomyocytes from snRNA-seq data obtained from engineered human cardiac tissue prepared in SF medium or MM (“old”) medium. Data are presented as log2 relative gene expression, normalized to the housekeeping gene TUBA1A.

[0031] Fig.11 It shows that the new medium BHTP retains a higher proportion of proliferative cardiomyocytes. The pie chart shows the percentage of proliferative cardiomyocytes in the total cell population. New medium replicate (rep) 1: 2.66%, new medium replicate 2: 3.98%, old medium replicate 2: 1.12%. The percentage of proliferative cardiomyocytes in the total cardiomyocyte population is: new medium replicate 1: 7.55%, new medium replicate 2: 8.93%, old medium replicate 2: 2.80%.

[0032] Fig.12 Violin plots of single cell RNA-seq of BHTP (new medium) are shown, showing the expression of FGF receptors in different cell populations. FGFR1 is the major FGF receptor expressed in BHTP. FGFR1 is expressed in fibroblasts / stromal cells, cardiomyocytes, endothelial cells, and cardiac progenitor cells.

[0033] Fig.13 Violin plots of single cell RNA-seq of BHTP (new medium) are shown, showing the expression of PDGF receptors in different cell populations. PDGFRB is the major PDGF receptor expressed in BHTP. PDGFRB is primarily expressed in the fibroblast / stromal population.

[0034] Fig.14 The novel serum-free (SF) human heart tissue is shown to be resistant to hypoxia. A) Schematic diagram of the hypoxia test. B) Post-hypoxia staining of control and SF human heart tissue. C) The intensity of nuclear staining is higher in SF human heart tissue. D) The intensity of cardiomyocytes is not changed in human heart tissue. E) Lactate dehydrogenase is lower in SF human heart tissue. F) Lactate is elevated in SF human heart tissue. G) The lactate to pyruvate ratio is highest in SF human heart tissue after hypoxia.

[0035] Fig.15 Figure 2 shows the implantation of a cardiac tissue patch in vivo. A) The patch was sutured to the right ventricle of a 6-month-old sheep heart. B) Image showing 2 BHTPs sutured to the right ventricle of a 6-month-old sheep heart, demonstrating almost complete coverage of the entire surface of the right ventricle. C) The pericardium was sutured with 3 stitches to help protect the patch. D)-F) show the patch intact in situ during the 2.5h acute monitoring period (D and E) and after removal (F).

[0036] Fig.16 The functional validation of the patch in vivo is shown. Steady-state pressure-volume (PV) loops are shown before patch implantation and 15 and 90 minutes after patch implantation.

[0037] Fig.17 A highly linear RV preload-recommendation stroke work-end-diastolic volume relationship was shown before patch implantation and 15 and 90 min after patch implantation.

[0038] Fig.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) output (Panel D), maximum rate of change of RV blood pressure, RV contractility index (RV dP / dtmax, Panel E), and RV preload supplementation stroke work index (PRSWI, Panel F) before (Pre) and after patch implantation at 15-minute intervals are shown. Significance: There were no immediate changes in any variables after patch implantation, and except for slight increases in PA blood pressure (<1 mmHg) and heart rate (10 beats / min), other variables did not change during the subsequent observation period until 120 min after patch implantation.

[0039] Description of the implementation plan

[0040] definition

[0041] Definitions of common terms in cell and molecular biology and biochemistry can be found 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 SpringHarbor 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) Enzymology:DNA,Jon Lorsch(ed.)Elsevier,2013(ISBN 0124199542);Laboratory Methods inEnzymology:RNA,Jon Lorsch(ed.)Elsevier,2013(ISBN:9780124200371,0124200370):Current Protocols in Molecular Biology(CPMB),Frederick M.Ausubel(ed.),JohnWiley and Sons,2014(ISBN 047150338X,9780471503385),Current Protocols inProtein Science(CPPS),John E.Coligan(ed.). Sons,Inc.,2005;andCurrent Protocols in Immunology(CPI)(John E.Coligan,ADA M Kruisbeek,David HMargulies,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)

[0042] As used in this specification and the appended claims, for example, the singular and the singular forms of the terms "a", "an", "the" and "the" optionally include plural references unless the context clearly dictates otherwise. For example, "a" cell includes one cell, one or more cells, and a plurality of cells.

[0043] 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 the amount of the entity, which can be zero but not more than the specified value. For example, "up to about 10%" refers to an amount from 0% to about 10%.

[0044] The term "and / or", such as "X and / or Y", should be understood as "X and Y" or "X or Y", and should be regarded as providing clear support for both meanings or just one of them.

[0045] In this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or a group of elements, integers or steps, but not the exclusion of any other element, integer or step, or a group of elements, integers or steps.

[0046] Numerical ranges include the numbers defining the ranges. It is meant that each maximum numerical limit given in this specification includes each lower numerical limit, as if these lower numerical limits were clearly written in this article. Each minimum numerical limit given in this specification includes each higher numerical limit, as if these higher numerical limits were clearly written in this article. Each numerical range given in this specification includes each narrower numerical range falling within this broad numerical range, as if these narrower numerical ranges were clearly written in this article.

[0047] The headings provided herein are not intended to limit the disclosure.

[0048] Throughout the specification, references to specific genes or proteins can be used interchangeably. One skilled in the art will understand in context that the reference is intended to refer to a specific gene or a protein encoded by the gene.

[0049] The terms "human pluripotent stem cells" and "hPSC" refer to cells derived from, obtainable from, or originating from human tissue that exhibit pluripotency. hPSCs may be human embryonic stem cells or human induced pluripotent stem cells.

[0050] Human pluripotent stem cells can be derived from the inner cell mass or reprogrammed using the Yamanaka factors from many fetal or adult somatic cell types. hPSCs can be generated using somatic cell nuclear transfer.

[0051] The terms "human embryonic stem cells", "hES cells" and "hESCs" refer to cells derived from, obtainable from or originating from human embryos or blastocysts, which have self-renewal and pluripotency or totipotency, and have the ability to generate all cell types present in mature animals. For example, human embryonic stem cells (hESCs) can be isolated from human preimplantation embryos, in vitro fertilized embryos, or human blastocysts obtained from single-cell human embryos expanded to the blastocyst stage.

[0052] The terms "induced pluripotent stem cells" and "iPSCs" refer to cells derived from, obtainable from, or originating from any type of adult somatic cell that is reprogrammed to a pluripotent state by expressing exogenous genes (such as transcription factors, including a preferred combination of OCT4, SOX2, KLF4, and c-MYC). Although hiPSCs exhibit equivalent levels of pluripotency to hESCs, they can be derived from patients to be treated with autologous therapies, with or without simultaneous gene correction prior to differentiation and cell delivery.

[0053] More generally, the methods disclosed herein can be applied to any pluripotent stem cells derived from any patient, or hPSCs that are subsequently modified using gene editing to generate mutant models, or mutant hPSCs that are corrected using gene editing.Gene editing can be performed using CRISPR, TALEN, or ZF nuclease technology.

[0054] As used herein, the term "cell culture" refers to any in vitro cell culture. The term "culture" refers to the process of growing and / or maintaining and / or manipulating cells. The term includes continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell populations maintained in vitro, including 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 tissue specimens or biopsies from animals or humans. These cultures can be from both adults and fetal tissues.

[0055] A "progenitor cell" is a cell that is capable of differentiating along one or more developmental pathways, with or without self-renewal. Typically, a progenitor cell is unipotent or oligopotent, and is capable of at least limited self-renewal.

[0056] The terms "differentiate," "differentiating," and "differentiated" relate to the progression of cells from an early or initial stage of a developmental pathway to a later or more mature stage of a developmental pathway. It should be understood that in this context, "differentiation" does not mean or imply that the cells are completely differentiated and have lost pluripotency or the ability to progress further along a developmental pathway or along other developmental pathways. Differentiation may be accompanied by cell division.

[0057] As is known in the art, the stage or state of cell differentiation can be characterized by the expression and / or non-expression of one of a variety of markers. In this context, "marker" refers to a nucleic acid or protein encoded by the genome of a cell, cell group, pedigree, compartment or subgroup, and its expression or expression pattern changes during the entire development process. Nucleic acid marker expression can be detected or measured by any technology known in the art, including nucleic acid sequence amplification (e.g., polymerase chain reaction) and nucleic acid hybridization (e.g., microarray, RNA hybridization, in situ hybridization), but is not limited thereto. Protein marker expression can be detected or measured by any technology known in the art, including flow cytometry, immunohistochemistry, immunoblotting, protein arrays, protein analysis (e.g., 2D gel electrophoresis), but is not limited thereto.

[0058] When characterizing cell phenotypes, these terms are common and well understood by technicians. By additional guidance, when a cell is considered to be positive or expresses or contains the expression of a given marker (e.g., a given gene or gene product), the technician will infer the presence or evidence of a unique signal of the marker when performing measurements that can detect or quantify the marker in or on the cell. Suitably, the presence or evidence of a unique signal of the marker can be inferred based on the comparison of the measurements obtained for the cell with the same measurements performed on a negative control (e.g., a cell known not to express the marker) and / or a positive control (e.g., a cell known to express the marker). In the case where the measurement method allows for quantitative assessment of markers, a positive cell can produce a signal of a marker that is at least 1.5 times higher than the marker signal produced by a reference cell (e.g., a negative control cell) or than the average marker signal produced by a reference cell or a negative control cell population, for example, at least 2 times, at least 4 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times or even higher. In addition, the positive cells can produce a marker signal that is 3.0 or more standard deviations higher than the mean marker signal produced by a reference cell or negative control cell population, e.g., 3.5 or more, 4.0 or more, 4.5 or more, or 5.0 or more standard deviations higher.

[0059] As used herein, the terms "medium" and "cell culture medium" refer to a medium suitable for supporting the growth of cells in vitro (i.e., cell culture, cell lines, etc.). It is intended that the term is not limited to any particular medium. For example, it is intended that the definition include maintenance medium as well as other medium used for cell differentiation or specialization. In fact, it is intended that the term include any medium suitable for cell culture and growth of cells of interest.

[0060] As used herein, "tissue" refers to an aggregate of cells. In some embodiments, the cells in the tissue are aggregated or fused.

[0061] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy.

[0062] The terms "treatment", "treating", "treat", and the like are used herein to refer generally to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing a disease or symptom from occurring in a subject who may be susceptible to the disease or symptom but has not yet been diagnosed as having the disease or symptom; (b) inhibiting disease symptoms, i.e., preventing their development; or (c) alleviating disease symptoms, i.e., causing the disease or symptom to regress.

[0063] As used herein, the terms "decrease", "reduced", "reduction", "to a lesser extent" or "inhibit" all indicate that a property, level or other parameter is reduced or decreased in a statistically significant amount. In some embodiments, "reduced", "reduction", "reduction" or "inhibit" generally refers to a reduction of at least 10% compared to a reference level (e.g., without a given treatment), and can include, for example, a reduction 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 "inhibit" does not include complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. Preferably, the reduction is to a level within the normal acceptable range for an individual without a given disease.

[0064] As used herein, the terms "increased", "increase", "increases", "enhance", "activate" or "to a greater extent" all refer to an increase in a property, level or other parameter by a statistically significant amount; for the avoidance of any doubt, the terms "increased", "increase", "to a greater extent", "enhance" or "activate" may refer to an increase of at least 10% compared to a reference level, such as 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 an increase of 100% or any increase between 10%-100%, or 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.

[0065] As used herein, "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 point, e.g., a cell, tissue or biological sample obtained from a patient before a disease is diagnosed, or a biological sample not contacted with an agent or composition disclosed herein). Alternatively, a reference level may also refer to the level of a given marker or parameter in a subject, organ, tissue or cell before administering a treatment (e.g., with an agent or by administering a transplant composition).

[0066] As used herein, a "control" or "suitable control" refers to an untreated, 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) relative to a cell, tissue, biological sample, or population contacted or treated with a given treatment. For example, a suitable control may be a cell, tissue, organ, or subject that has not been contacted with an agent or to which a cell as described herein has not been administered. In one example, a control is a cell, tissue, biological sample, or population cultured in a maturation medium, as described in the Examples herein.

[0067] In one or more embodiments described herein, evaluating the expression of various genes includes comparing fold changes. In one embodiment, fold changes are used to measure changes in gene expression levels. In one embodiment, the expression of a gene 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 for quantifying gene expression from RNA sequencing data by normalizing the total read length and sequencing reads. In one embodiment, RPKM calculations provide normalization for comparing gene coverage values. The RPKM value corrects the difference in sample sequencing depth and gene length. In an example, RPKM can be calculated by the following formula: numReads / (geneLength / 1000*totalNumReads / 1000000); wherein, "numReads" refers to the number of reads mapped to a gene sequence; wherein, "geneLength" refers to the length of a gene sequence; and wherein, "totalNumReads" refers to the total number of mapped reads of a sample.

[0068] The terms "agonist" or "activator" are 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 portion of the biological activity of the molecule (e.g., FGF). For example, an FGF agonist or FGF activator refers to a molecule that selectively activates FGF signaling.

[0069] As used herein, the term "inhibitor" 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 activities of the naturally occurring form of the molecule.

[0070] Various embodiments will be described below. It is worth noting that specific embodiments are not intended to be exhaustive descriptions, nor are they limitations on the broader aspects discussed herein. An aspect described in conjunction with a specific embodiment is not necessarily limited to that embodiment, and may be practiced with any other embodiment. "One embodiment", "embodiment" or "exemplary embodiment" mentioned throughout the specification refers to a specific feature, structure or characteristic described in conjunction with that embodiment included in at least one embodiment of the present invention. Therefore, the phrases "in one embodiment", "in an embodiment" or "exemplary embodiment" appearing in various places in this specification do not necessarily all refer to the same embodiment, but may also refer to the same embodiment. In addition, in one or more embodiments, specific features, structures or characteristics may be combined in any suitable manner, which is obvious to those skilled in the art from this disclosure. In addition, although some embodiments described herein include some features included in other embodiments without including other features, the combination of different embodiment features is intended to be within the scope of the present invention. For example, in the appended claims, any of the embodiments claimed for protection may be used in any combination. Unless otherwise specifically stated, any example or embodiment herein shall be deemed to be applicable to any other example or embodiment after making necessary modifications in the details.

[0071] The scope of the present disclosure is not limited by the specific examples described herein, which are provided for illustrative purposes only. As described herein, functionally equivalent methods and systems are clearly within the scope of the present disclosure.

[0072] Throughout the specification, unless specifically stated otherwise or the context requires otherwise, references to individual steps, combinations of materials, groups of steps, or groups of combinations of materials should be understood to include one and more (i.e., one or more) of these steps, combinations of materials, groups of steps, or groups of combinations of materials.

[0073] The present disclosure is illustrated below 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 concerns, the concepts described herein are applicable to other animals. These and other aspects and features of the present disclosure will be more fully reflected from the following description and the appended claims, or may be learned through the practice of the present disclosure as described below.

[0074] The reference herein to a patent document or other matter cited as prior art should not be taken as an admission that the document or matter was known at the priority date of any claim, nor should it be construed as an admission that the information it contains is part of the common general knowledge.

[0075] Methods for generating engineered human cardiac tissue

[0076] The present invention provides a method for producing engineered human cardiac tissue comprising endothelial cells, the method comprising culturing a cell suspension comprising a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocytes in the presence of a FGFR1 agonist and a PDGFR agonist for a period of time and under conditions sufficient to produce engineered human cardiac tissue comprising endothelial cells.

[0077] The present invention also provides a method for producing engineered human cardiac tissue, the method comprising the following steps: i) mixing a cell suspension comprising a population of cardiomyocytes and non-cardiomyocytes derived from a human pluripotent stem cell (hPSC) culture with a flowable hydrogel composition, wherein the cell suspension is in a serum-free culture medium; ii) loading the flowable hydrogel composition comprising the cell suspension into a mold, and incubating the hydrogel for a period of time under conditions sufficient to solidify the hydrogel; and iii) culturing the hydrogel from step ii); wherein the population of cardiomyocytes and non-cardiomyocytes derived from a human pluripotent stem cell (hPSC) culture is contacted with a FGFR1 agonist and a PDGFR agonist, thereby providing engineered human cardiac tissue, wherein the tissue comprises cardiomyocytes, stromal cells / fibroblasts and endothelial cells.

[0078] In one embodiment, before mixing in step i), FGFR1 agonist and / or PDGFR agonist are added to the cell suspension or flowable hydrogel composition in serum-free medium. In another embodiment, before mixing in step i), the group of cardiomyocytes and non-cardiomyocytes derived from human pluripotent stem cells (hPSC) culture are cultivated in the serum-free medium comprising FGFR1 agonist and PDGFR agonist. In an alternative embodiment, after mixing described in step i), FGFR1 agonist and / or PDGFR agonist are added to the flowable hydrogel composition comprising the cell suspension. In another embodiment, in step iii), the solidified hydrogel of step ii) is cultivated in the serum-free medium comprising FGFR1 agonist and PDGFR agonist.

[0079] The present invention provides a method for producing engineered human cardiac tissue, the method comprising the following steps: i) mixing a cell suspension comprising a human pluripotent stem cell (hPSC) culture-derived cardiomyocyte group and a group of non-cardiomyocytes with a flowable hydrogel composition, wherein the cell suspension is in a serum-free medium comprising an FGFR1 agonist and a PDGFR agonist; ii) loading the flowable hydrogel composition comprising the cell suspension into a mold, and incubating the hydrogel for a period of time under conditions sufficient to solidify the hydrogel; and iii) culturing the hydrogel from step ii) in the serum-free medium of step i); thereby providing engineered human cardiac tissue, wherein the tissue comprises cardiomyocytes, stromal cells / fibroblasts and endothelial cells. In yet another embodiment, before mixing in step i), an FGFR1 agonist and / or a PDGFR agonist are also added to the flowable hydrogel composition. In yet another embodiment, after mixing in step ii), an FGFR1 agonist and / or a PDGFR agonist are also added to the flowable hydrogel composition comprising the cell suspension. In yet another embodiment, in step iii), the solidified hydrogel of step ii) is cultured in a serum-free medium comprising a FGFR1 agonist and a PDGFR agonist.

[0080] 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% stromal cells. In a preferred embodiment, the suspension comprises 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% of myocardial cells 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% of stromal cells. In a preferred embodiment, the suspension comprises about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of myocardial cells and about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of stromal cells. In a particularly preferred embodiment, the cell suspension comprises about 70% cardiomyocytes and about 30% stromal cells. In a preferred embodiment, the cardiomyocytes are actin + / CTNT + Cardiomyocytes, and non-cardiomyocytes or stromal cells are CD90 + Stromal cells. In some embodiments, the cell suspension consists primarily of a population of cardiomyocytes and stromal cells derived from a human pluripotent stem cell (hPSC) culture. In one embodiment, a cell suspension consisting primarily of a population of cardiomyocytes and stromal cells derived from a human pluripotent stem cell (hPSC) culture 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.

[0081] In another preferred embodiment, a cell suspension comprising cardiomyocytes and non-cardiomyocytes derived from a hPSC cell culture is obtained from a single differentiation process. That is, it should be understood that the starting population of hPSCs can produce cardiomyocytes and non-cardiomyocytes by directed differentiation to a cardiac lineage, and therefore, in order to further culture the cell suspension and produce cardiac tissue comprising cardiomyocytes, stromal cells / fibroblasts and endothelial cells, it is not necessary to combine, mix or inoculate the cell suspension with, for example, another cell population (e.g., endothelial cells).

[0082] As described herein, the inventors unexpectedly found that by culturing a cell suspension comprising cardiomyocytes and non-cardiomyocytes in the presence of a FGFR1 agonist and a PDGFR agonist combination, human engineered cardiac tissue can be produced, wherein the tissue more appropriately reflects the heterogeneity of the cell types present in natural human cardiac tissue. In other words, human engineered cardiac tissue comprises cardiomyocytes, stromal cells / fibroblasts and endothelial cells. In some embodiments, engineered cardiac tissue may further comprise vascular smooth muscle cells and / or cardiac progenitor cells. In some embodiments, human engineered cardiac tissue comprises cardiomyocytes, stromal cells / fibroblasts, endothelial cells and vascular smooth muscle cells.

[0083] In one aspect, the present invention provides a method for producing a group of cardiomyocytes, stromal cells / fibroblasts and endothelial cells derived from human pluripotent stem cells (hPSC) cultures, the method comprising culturing a cell suspension of a group of cardiomyocytes and non-cardiomyocytes derived from human pluripotent stem cells (hPSC) cultures for a period of time under conditions sufficient to produce a group of endothelial cells derived from human pluripotent stem cells (hPSC) cultures in the presence of a FGFR1 agonist and a PDGFR agonist. On the other hand, the present invention provides the use of FGFR1 agonists and PDGFR agonists in a cell suspension comprising a group of cardiomyocytes and non-cardiomyocytes derived from human pluripotent stem cells (hPSC) cultures. As described above, in some embodiments, the cell suspension comprises about 50% to about 80% of cardiomyocytes and about 20% to about 50% of non-cardiomyocytes, or any other range of cardiomyocytes and non-cardiomyocytes described above. In another embodiment, the cell suspension comprises 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% stromal cells, or any other range of cardiomyocytes and stromal cells as described above. In a preferred embodiment, the cardiomyocytes are actin + / CTNT +Cardiomyocytes, and non-cardiomyocytes or stromal cells are CD90+ stromal cells. In some embodiments, the cell suspension is mainly composed of a group of cardiomyocytes and stromal cells derived from human pluripotent stem cells (hPSC) cultures. In one embodiment, a cell suspension consisting mainly of a group of cardiomyocytes and stromal cells derived from human pluripotent stem cells (hPSC) cultures 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.

[0084] 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 70% 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 65% 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 60% 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 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; about 35% or less stromal cells / fibroblasts; and about 30% or less endothelial cells. In some embodiments, the cardiac tissue also comprises up to about 15% vascular smooth muscle cells. In some embodiments, the cardiac tissue also 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% 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; 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% stromal 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 a more preferred embodiment, 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% stromal 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% 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% stromal 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% stromal 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% stromal 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% stromal cells / fibroblasts; and about 15% to about 25% endothelial cells.

[0085] 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 the 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 the expression of one or more markers associated with cell proliferation. In one embodiment, the proliferative cardiomyocytes express one or more markers selected from ANLN, TOP2A, CDK1 and E2F1.

[0086] Another characteristic of the cardiomyocytes present in the engineered heart tissue is that they express MMP1 + Therefore, in a preferred embodiment, the cardiomyocytes in the engineered heart tissue express MMP1 + .

[0087] Another characteristic of the fibroblasts present in the engineered cardiac tissue is that they express MMP1 +、EMP1 + 、FOXD1 + 、RAB27B + 、NR2F1 + 、F2RL1 + 、SPP1 + 、TMEM158 + PTHLH + 、PHLDA2 + 、MALL + , MYCT1 + ,DUSP4 + 、PLAU + 、TMEM156 + 、CD274 + 、MMP10 + 、ARRDC4 + 、RFX8 + MLPH + , THBD + 、HHEX + 、VGF + 、OTULINL + IL33 + 、CA12 + 、C6orf141 + MFSD2A + and / or CARD10 + Thus, in preferred embodiments, the fibroblasts in the engineered cardiac tissue are positive for one or more of the above markers.

[0088] As described herein, the inventors have determined that FGFR1 is the main FGF receptor expressed in the tissue-engineered cardiac tissue according to the method of the present invention. Therefore, in a preferred embodiment, the method of the present invention includes culturing cells in a basal medium supplemented with a FGFR1 agonist selected from FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, the method of the present invention includes culturing cells in a basal medium supplemented with a FGFR1 agonist selected from FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8, FGF9, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, the method of the present invention includes culturing cells in a basal medium supplemented with a FGFR1 agonist, and the agonist is a member of a FGF1 subfamily, a FGF4 subfamily, a FGF8 subfamily, a FGF9 subfamily, or a FGF19 subfamily. In some embodiments, the method of the present invention includes culturing cells in a basal medium supplemented with a FGFR1 agonist selected from FGF1, FGF2, FGF4, FGF5, FGF6, FGF9, FGF16, and FGF20. More preferably, the FGFR1 agonist is FGF2. In one embodiment, the culture medium includes about 0.5ng / ml-100ng / ml FGF2. Preferably, the concentration of FGF2 is about 1ng / ml-50ng / mL, more preferably about 5ng / ml-20ng / mL, and most preferably about 10ng / mL.

[0089] As described herein, the inventors have also determined that PDGFRβ is the main PDGF receptor expressed in tissue-engineered cardiac tissue according to the method of the present invention. Therefore, in a preferred embodiment, the method of the present invention includes culturing cells in a basal medium supplemented with a PDGFR agonist selected from PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC or PDGF-DD. More preferably, the PDGFR1 agonist is PDGF-BB. In one embodiment, the culture medium comprises about 0.5ng / ml-100ng / ml PDGF-BB. Preferably, the concentration of PDGF-BB is about 1ng / ml-50ng / mL, more preferably about 5ng / ml-20ng / mL, and most preferably about 10ng / mL.

[0090] In one embodiment, the serum-free medium further comprises a basal medium comprising albumin and transferrin. In another preferred embodiment, the basal medium comprises a commercially available supplement comprising albumin and transferrin. In a preferred embodiment, this supplement is Supplements or In a preferred embodiment, 0.1% to 10% or Subtract the amount of insulin administered Supplements or Supplement minus insulin, preferably 0.5%-8%, more preferably 1%-6%, even more preferably 1.5%-4%, and most preferably about 4% or In one embodiment, the B27 supplement comprises biotin, DL alpha tocopheryl acetate, DL alpha-tocopherol, vitamin A (acetate), BSA, fatty acid-free fraction V, catalase, human recombinant insulin, human transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine hydrochloride, glutathione (reduced form), L-carnitine hydrochloride, linoleic acid, linolenic acid, progesterone, putrescine dihydrochloride, sodium selenite, and T3 (triiodothyronine).

[0091] Through detailed research, the inventors have determined that the hydrogel inoculated with cells in the culture medium of low calcium level is important for maintaining the phenotype showing glycolytic metabolism and contractility reduction in engineered cardiac tissue. Therefore, in one embodiment, the basal medium is a low calcium culture medium with a calcium concentration less than physiological calcium concentration (e.g., less than about 2mM). In another embodiment, the basal medium is a low calcium culture medium with a calcium concentration less than about 1.2mM. In another embodiment, the basal medium is a low calcium culture medium with a calcium concentration less than about 1mM. In another preferred embodiment, the basal medium is a low calcium culture medium with a calcium concentration less than about 0.8mM, more preferably less than 0.75mM, even more preferably less than about 0.5mM, most preferably about 0.43mM. Low calcium culture medium. In some embodiments, the basal culture medium is a low calcium culture medium with a calcium concentration of about 0.2mM to about 1mM. It should be understood by technicians that any suitable basal culture medium such as αMEM, DMEM or RPMI with the above-mentioned preferred calcium concentration can be used in the method. In another preferred embodiment, the basal culture medium is an RPMI culture medium.

[0092] Typically, the culturing step is performed for about 5 days to about 7 days. Preferably, the culturing step is performed for about 7 days. In another preferred embodiment, the method comprises replacing the culture medium in the culturing step with a fresh culture medium comprising a FGFR1 agonist and a PDGFR agonist at least every 2 days.

[0093] In one aspect, a culture medium composition suitable for producing cardiomyocytes, stromal cells / fibroblasts and endothelial cell groups derived from human pluripotent stem cells (hPSC) cultures is provided, comprising a basal medium having a calcium concentration lower than a physiological calcium concentration, a FGFR1 agonist and a PDGFR agonist. In some embodiments, the culture medium composition also includes a commercially available supplement comprising albumin and transferrin. The amount and concentration of each component in the culture medium composition are as described herein.

[0094] As shown in the following examples, it has been shown to be advantageous that the engineered cardiac tissue produced by the method of the present invention and the engineered cardiac tissue of the present invention as described below exhibit glycolytic metabolism. This may be advantageous because it provides resistance to hypoxia-induced cell death compared to cells using oxidative phosphorylation. Since implantation in vivo results in a period of hypoxia, this may result in better survival rates. In addition, glycolysis supports cardiomyocyte proliferation (Mills et al. PNAS 2017), which may be beneficial in promoting an increase in the number of cells after implantation.

[0095] In one embodiment, the cardiac tissue produced using the method and the engineered cardiac tissue described herein show a log2 relative gene expression of PKM>2 (normalized to TUBA1A). Another feature of the cardiac tissue produced using the methods described herein is that after 24 hours in SF medium, the culture medium has a lactate concentration of at least 0.5mM or at least 1mM. In another embodiment, after culturing the cardiac tissue produced using the methods described herein in SF medium for 24 hours, the culture medium has a lactate concentration of about 10nmol / million cells / h-20nmol / million cells / h.

[0096] As shown in the following examples, it has been demonstrated that the engineered cardiac tissue produced by the methods of the present invention and the engineered myocardial tissue of the present invention exhibit tolerance to hypoxia. In one embodiment, the cardiac tissue produced using the method and the engineered cardiac tissue described herein show log2 relative gene expression <-4 (normalized for TUBA1A) of ALDH1, AGMO, GPX3, CYP1B1, and PLIN5. Another feature of the cardiac tissue produced using the methods described herein and the engineered cardiac tissue itself (described below) is that after culturing for 20 hours under hypoxic conditions, the cell culture medium from such a culture has a lactate / pyruvate ratio of at least 1:1. However, another feature of the cardiac tissue produced using the methods described herein and the engineered cardiac tissue itself (described below) is that after culturing for 20 hours under hypoxic conditions, cell death is at least 1-fold lower than that of the control cardiac tissue, and cell death may be at least 2-fold, at least 3-fold, or more lower than that of the control cardiac tissue. Cell death can be assessed using conventional methods known to technicians, including but not limited to LDH determination. In a preferred embodiment, after culturing cardiac tissue produced using the methods described herein and engineered cardiac tissue itself (described below) under hypoxic conditions for 20 hours, cell death is at least 1-fold, at least 2-fold, at least 3-fold or more lower than control cardiac tissue as measured by the LDH assay.

[0097] As shown in the following examples, it has been shown that the engineered myocardial tissue produced by the method of the present invention exhibits low contractility. The contractility of the engineered tissue can be evaluated and measured using techniques known to the skilled person (see, for example, Tibucy et al. Circulation. 2017; 135(19): 1832–1847 or Voges et al. Development 2017; 144(6): 1118-1127). In a preferred embodiment, the engineered cardiac tissue exhibits a contractility of less than about 10 mN / mm 2 contractile activity.

[0098] In one embodiment, the hydrogel comprises fibrin, collagen I or matrigel or its arbitrary combination composition.In a preferred embodiment, the hydrogel is a fibrin hydrogel.In a preferred embodiment, fibrin is formed by mixing thrombin and fibrinogen solution.Preferably, in the formation of the fibrin hydrogel, fibrinogen exists with a concentration of about 10mg / mL to about 50mg / mL.More preferably, fibrinogen exists with a concentration of about 20mg / mL.

[0099] In another embodiment, the hydrogel can be functionalized with one or more bioactive agents. For example, bioactive agents (e.g., small molecules, polypeptides including cytokines and chemokines, differentiation factors, signaling pathway inhibitors, etc.) can, for example, promote the viability of cells in the resulting engineered tissue and the further development or differentiation of cells. In one embodiment, one or more bioactive agents can be agents selected from the group consisting of: antiproliferative agents, immunosuppressants, proangiogenic compounds, antibodies or fragments or portions thereof, antibiotics or antimicrobial compounds, antigens or epitopes, aptamers, biopolymers, carbohydrates, cell attachment media (e.g., RGD), cytokines, cytotoxic agents, drugs, enzymes, growth factors or recombinant growth factors and fragments and variants thereof, hormone antagonists, hormones, immune agents, lipids, metals, nanoparticles, nucleic acid analogs, nucleic acids (e.g., DNA, RNA, siRNA, RNAi and microRNA agents), nucleotides, nutrients, oligonucleotides, peptide nucleic acids (PNA), peptides, prodrugs, preventive agents, proteins, small molecules, therapeutic agents, or any combination thereof.

[0100] In one embodiment, in step i), the cell suspension is mixed with the hydrogel to provide about 5×10 6 cells / mL, about 10×10 6 cells / mL, about 15×10 6 cells / mL, about 20×10 6 cells / mL, about 25×10 6 cells / mL, about 30×10 6 cells / mL, about 35×10 6 cells / mL, about 40×10 6 cells / mL, about 45×10 6 cells / mL, about 50×10 6 cells / mL, about 55×10 6 cells / mL, about 60×10 6 cells / mL, about 65×10 6 cells / mL, about 70×10 6 cells / mL, about 75×10 6 cells / mL, about 80×10 6 cells / mL, about 85×10 6 cells / mL, about 90×10 6 cells / mL, about 95×10 6 cells / mL or about 100×10 6 More preferably, in step i), the cell suspension is mixed with the hydrogel to provide a cell concentration of about 5×10 6 cells / mL to about 50×106 Even more preferably, in step i), the cell suspension is mixed with the hydrogel to provide a cell concentration of about 10×10 6 cells / mL, about 15×10 6 cells / mL, about 20×10 6 cells / mL, about 25×10 6 cells / mL, about 30×10 6 cells / mL or about 35×10 6 The cell concentration is cells / mL.

[0101] In one embodiment, upon solidification of the hydrogel, the engineered human cardiac tissue has a 6 pcs, 2×10 6 pcs, 3×10 6 4×10 6 pcs, 5×10 6 pcs, about 10×10 6 pcs, about 15×10 6 pcs, about 20×10 6 pcs, about 25×10 6 pcs, about 30×10 6个 , about 35×10 6 pcs, about 40×10 6 pcs, about 45×10 6 pcs, about 50×10 6 pcs, about 55×10 6 pcs, about 60×10 6 pcs, about 65×10 6 pcs, about 70×10 6个 , about 75×10 6 pcs, about 80×10 6 pcs, about 85×10 6 pcs, about 90×10 6 pcs, about 95×10 6 pcs, about 100×10 6 pcs, about 110×10 6 pcs, about 120×10 6 pcs, about 130×10 6 pcs, about 140×10 6 or about 150×10 6 With reference to the method of the first aspect of the invention, it is the cell density of the patch at the end of step (ii) and before culturing the hydrogel in the serum-free medium defined in step (iii). More preferably, the engineered human cardiac tissue has about 1×10 6 to about 100×10 6Even more preferably, the engineered human cardiac tissue has about 20×10 6 cells to about 150×10 6 cells.

[0102] In one embodiment, upon solidification of the hydrogel, the engineered human cardiac tissue has a 6 cells / cm 2 , about 2×10 6 cells / cm 2 , about 3×10 6 cells / cm 2 , about 4×10 6 cells / cm 2 , about 5×10 6 cells / cm 2 , about 10×10 6 cells / cm 2 , about 15×10 6 cells / cm 2 , about 20×10 6 cells / cm 2 , about 25×10 6 cells / cm 2 , about 30×10 6 cells / cm 2 , about 35×10 6 cells / cm 2 , about 40×10 6 cells / cm 2 , about 45×10 6 cells / cm 2 , about 50×10 6 cells / cm 2 , about 55×10 6 cells / cm 2 , about 60×10 6 cells / cm 2 , about 65×10 6 cells / cm 2 , about 70×10 6 cells / cm 2 , about 75×10 6 cells / cm 2 , about 80×10 6 cells / cm 2 , about 85×10 6 cells / cm 2 , about 90×10 6 cells / cm 2 , about 95×10 6 cells / cm2 or about 100×10 6 cells / cm 2 With reference to the method of the first aspect of the invention, it is the cell density of the patch at the end of step (ii) and before culturing the hydrogel in the serum-free medium defined in step (iii). More preferably, the engineered human cardiac tissue has a cell density of about 1×10 6 cells / cm 2 About 50×10 6 cells / cm 2 Even more preferably, the engineered human cardiac tissue has a cell density of about 1×10 6 cells / cm 2 to about 10×10 6 cells / cm 2 of cell density.

[0103] The engineered human heart patch can be produced in any shape, size, configuration using a suitable mold. In some embodiments, the shape of the tissue reflects the site of application. In one embodiment, the engineered heart tissue has a trapezoidal geometry.

[0104] The skilled person will be aware of various methods in the literature for directed differentiation of hPSCs into cardiac lineages to provide a mixed population of hPSC-derived cardiomyocytes and non-cardiomyocytes. In one embodiment, prior to step i), hPSCs are induced to differentiate into cardiac lineages by the following steps:

[0105] a) culturing hPSCs in a basal medium comprising effective amounts of BMP4, activin A, FGF, and a GSK3 inhibitor for a period of time sufficient to induce mesodermal differentiation of the hPSCs;

[0106] b) culturing the cells obtained in step a) in a basal medium comprising an effective amount of a Wnt signaling pathway inhibitor for a period of time sufficient to induce cardiac differentiation of mesoderm;

[0107] 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.

[0108] 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, the method comprising:

[0109] a) culturing hPSCs in a basal medium comprising effective amounts of BMP4, activin A, FGF, and a GSK3 inhibitor for a period of time sufficient to induce mesodermal differentiation of the hPSCs;

[0110] b) culturing the cells obtained in step a) in a basal medium comprising an effective amount of a Wnt signaling pathway inhibitor for a period of time sufficient to induce cardiac differentiation of mesoderm;

[0111] 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

[0112] d) culturing the cells obtained in step c) in a basal medium comprising an effective amount of a FGFR1 agonist and a PDGFR agonist for a period of time sufficient to generate a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes, stromal cells / fibroblasts, and endothelial cells.

[0113] In a preferred embodiment, prior to step i), hPSCs are induced to differentiate into cardiac lineage by the following steps:

[0114] a) culturing hPSCs in a basal medium comprising an effective amount of BMP4, activin A, FGF, a GSK3 inhibitor, and a serum-free supplement (comprising albumin and transferrin, minus insulin) for about 72 hours, wherein the medium is replaced with fresh medium every day;

[0115] b) culturing the cells obtained in step a) in a basal medium comprising an effective amount of a Wnt signaling pathway inhibitor and a serum-free supplement as in a) for about 72 hours, wherein the medium is replaced with a fresh medium every day;

[0116] c) culturing the cells obtained in step b) in a basal medium comprising an effective amount of a Wnt signaling pathway inhibitor 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 2nd and 4th day of the about 7 days;

[0117] d) culturing the cells obtained in step c) in a basal medium comprising 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;

[0118] Thereby providing a cell suspension comprising a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocytes.

[0119] In one embodiment, in step a), hPSCs are cultured in a basal medium comprising 1 ng / ml-20 ng / ml, preferably 2 ng / ml-15 ng / ml, more preferably 2.5 ng / ml-10 ng / ml, more preferably 3 ng / ml-8 ng / ml, most preferably 4 ng / ml-6 ng / ml, and even most preferably about 5 ng / ml of BMP4; 0.1 ng / ml-10 ng / ml, preferably 1 ng / ml-9 ng / ml, more preferably 2 ng / ml-8 ng / ml, even more preferably 3 ng / ml-7 ng / ml, most preferably 4 ng / ml-6 ng / ml, and even most preferably about 5 ng / ml FGF2; 1ng / ml-20ng / ml, preferably 2.5ng / ml-18ng / ml, more preferably 5ng / ml-16ng / ml, even more preferably 7.5ng / ml-14ng / ml, still more preferably 8ng / ml-12ng / ml, most preferably 8.5ng / ml-10ng / ml, and even most preferably about 9ng / ml Activin A.

[0120] The GSK3 inhibitor in the basal medium of step (a) can be selected from, for example, CHIR99021, CHIR98014, SB216763, TWS119, Tideglusib, SB415286 and LY2090314. However, any GSK3 inhibitor suitable for the method of the present invention can be used. In a preferred embodiment, the GSK3 inhibitor in the basal medium of step (a) is CHIR99021.

[0121] It will be appreciated by the skilled artisan that the concentration of an effective amount of a GSK3 inhibitor varies with the availability and inhibition constant of the inhibitor in question. In the context of the present invention, the term "effective amount" used herein in the context of a GSK3 inhibitor refers to an enzyme inactivation concentration. For example, in the case of CHIR99021, the basal medium in step (a) comprises 0.1 μM-10 μM CHIR99021, preferably 0.2 μM-9 μM, more preferably 0.3 μM-8 μM, even more preferably 0.4 μM-7 μM, still more preferably 0.5 μM-6 μM, more preferably 0.6 μM-5 μM, more preferably 0.7 μM-4 μM, more preferably 0.8 μM-3 μM, most preferably 0.9 μM-2 μM, and even most preferably about 1 μM CHIR99021.

[0122] In one embodiment, the serum-free supplement is Supplements or In a preferred embodiment, 0.1% to 10% or Subtract the amount of insulin administered Supplements or Supplement minus insulin, preferably 0.5%-8%, more preferably 1%-6%, even more preferably 1.5%-4%, and most preferably about 4% or Subtract the amount of insulin.

[0123] In one embodiment, the Wnt pathway inhibitor is selected from C59 (4-(2-methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]phenylacetamide), 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-quinolyl-benzamide), NSC668036 (N-[(1,1-dimethylethoxy)carbonyl]-L-alanyl-(2S)-2-hydroxy-3-methylbutyryl-L-alanine-(1S)-1-carboxy- 2-methylpropyl ester hydrate), 2,4-diaminoquinazoline, quercetin, ICG-001 ((6S,9aS)-hexahydro-6-[(4-hydroxyphenyl)methyl]-8-(1-naphthylmethyl)-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, Wnt and Wnt receptor antibodies and Wnt inhibitory nucleic acids. In a preferred embodiment, the Wnt pathway inhibitor is IWP-4, IWR-1 or ICRT14.

[0124] Any pluripotent stem cell group, including human embryonic stem cell group (hESC) or human induced pluripotent stem cell group (iPSC), can be used as the starting material for obtaining engineered human cardiac tissue using the methods described herein. In one embodiment, the pluripotent progenitor cell group is a human iPSC group. In one embodiment, iPSC cells grow under feeder-free conditions. In another embodiment, the cells obtained from the subject can be used to produce a method for patient-specific iPSC, which can then be differentiated using the methods described herein.

[0125] Engineered cardiac tissue

[0126] The present invention provides engineered human cardiac tissue comprising at least about 30% hPSC-derived cardiomyocytes, about 35% or less hPSC-derived stromal cells / fibroblasts, and about 30% or less hPSC-derived endothelial cells, wherein the above cells are located throughout a hydrogel composition.

[0127] In another embodiment, an 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, wherein the cells are located throughout the hydrogel composition.

[0128] The present invention provides engineered human cardiac tissue produced according to the method described above.

[0129] In some embodiments, human engineered 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% stromal 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% stromal 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% stromal 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% stromal 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 also comprises up to about 15% vascular smooth muscle cells. In some embodiments, the cardiac tissue also 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% stromal 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% stromal 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% stromal 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% stromal 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 a more preferred embodiment, 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 60% cardiomyocytes, about 20% to about 35% 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% stromal 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% stromal 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% stromal cells / fibroblasts; and about 15% to about 30% endothelial cells. In some embodiments, the engineered cardiac tissue comprises about 30% to about 55% cardiomyocytes, about 40% to about 60% 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% stromal 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% stromal 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% stromal cells / fibroblasts; and about 30% endothelial cells.

[0130] 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 a further embodiment, at least about 3% to about 10% of the cardiomyocytes of the engineered cardiac tissue are proliferative, as determined by the expression of one or more markers associated with cell proliferation. In a further embodiment, 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 the expression of one or more markers associated with cell proliferation. In one embodiment, proliferative cardiomyocytes express one or more markers selected from ANLN, TOP2A, CDK1 and E2F1.

[0131] Another characteristic of the cardiomyocytes present in the engineered heart tissue is that they express MMP1 + Therefore, in a preferred embodiment, the cardiomyocytes in the engineered heart tissue express MMP1 + .

[0132] Another characteristic of the fibroblasts present in the engineered cardiac tissue is that they express MMP1 + 、EMP1 + 、FOXD1 + 、RAB27B + 、NR2F1 + 、F2RL1 + 、SPP1 + 、TMEM158 + PTHLH + 、PHLDA2 + 、MALL + , MYCT1 + ,DUSP4 + 、PLAU + 、TMEM156 + 、CD274 + 、MMP10 + 、ARRDC4 + 、RFX8 + MLPH + , THBD + 、HHEX + 、VGF + 、OTULINL + IL33 + 、CA12 + 、C6orf141 + MFSD2A + and / or CARD10 +Thus, in preferred embodiments, the fibroblasts in the engineered cardiac tissue are positive for one or more of the above markers.

[0133] In one embodiment, the cardiac tissue described herein exhibits a log2 relative gene expression of PKM>2 (normalized to TUBA1A). Another feature of the cardiac tissue is that after 24 hours in SF medium, the SF medium has a lactate concentration of at least 0.5 mM or at least 1 mM. In another embodiment, after culturing the cardiac tissue produced using the methods described herein in SF medium for 24 hours, the medium has a lactate concentration of 10-20 nmol / million cells / hour.

[0134] In one embodiment, the engineered cardiac tissue exhibits tolerance to hypoxia. In one embodiment, the cardiac tissue described herein exhibits log2 relative gene expression <-4 (normalized for TUBA1A) for ALDH1, AGMO, GPX3, CYP1B1, and PLIN5. Another feature of the engineered cardiac tissue is that after culturing for 20 hours under hypoxic conditions, the cell culture medium from such culture has a lactate / pyruvate ratio of at least 1:1.

[0135] As shown in the examples below, it has been demonstrated that the engineered cardiac tissue produced by the methods of the present invention exhibits low contractility which is advantageous. In a preferred embodiment, the engineered cardiac tissue exhibits a contractility of less than about 10 mN / mm 2 contractile activity.

[0136] 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 agents. For example, bioactive agents (e.g., small molecules, polypeptides including cytokines and chemokines, differentiation factors, signaling pathway inhibitors, etc.) can, for example, promote the viability of cells in the resulting engineered tissue and the further development or differentiation of cells. In one embodiment, the one or more biologically active 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 antibacterial compound, an antigen or epitope, an aptamer, a biopolymer, a carbohydrate, a cell attachment medium (such as RGD), a cytokine, a cytotoxic agent, a drug, an enzyme, a growth factor or a recombinant growth factor and fragments and variants thereof, a hormone antagonist, a hormone, an immunopharmaceutical, a lipid, a metal, a nanoparticle, a nucleic acid analog, a nucleic acid (such as DNA, RNA, siRNA, RNAi and microRNA agents), a nucleotide, a nutrient, 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.

[0137] In one embodiment, engineered human cardiac tissue has about 1×10 6 pcs, 2×10 6 pcs, 3×10 6 4×10 6 pcs, 5×10 6 pcs, about 10×10 6 pcs, about 15×10 6 pcs, about 20×10 6 pcs, about 25×10 6 pcs, about 30×10 6个 , about 35×10 6 pcs, about 40×10 6 pcs, about 45×10 6 pcs, about 50×10 6 pcs, about 55×10 6 pcs, about 60×10 6 pcs, about 65×10 6 pcs, about 70×10 6个 , about 75×10 6 pcs, about 80×10 6 pcs, about 85×10 6 pcs, about 90×10 6 pcs, about 95×10 6 pcs, about 100×10 6 pcs, about 110×10 6 pcs, about 120×10 6 pcs, about 130×10 6 pcs, about 140×10 6 or about 150×10 6 More preferably, the engineered human cardiac tissue has about 1×10 6 to about 100×10 6 Even more preferably, the engineered human cardiac tissue has about 20×10 6 cells to about 150×10 6 cells.

[0138] In one embodiment, engineered human cardiac tissue has about 1×10 6 cells / cm 2 , about 2×10 6 cells / cm 2 , about 3×10 6 cells / cm 2 , about 4×10 6 cells / cm 2 , about 5×10 6 cells / cm 2, about 10×10 6 cells / cm 2 , about 15×10 6 cells / cm 2 , about 20×10 6 cells / cm 2 , about 25×10 6 cells / cm 2 , about 30×10 6 cells / cm 2 , about 35×10 6 cells / cm 2 , about 40×10 6 cells / cm 2 , about 45×10 6 cells / cm 2 , about 50×10 6 cells / cm 2 , about 55×10 6 cells / cm 2 , about 60×10 6 cells / cm 2 , about 65×10 6 cells / cm 2 , about 70×10 6 cells / cm 2 , about 75×10 6 cells / cm 2 , about 80×10 6 cells / cm 2 , about 85×10 6 cells / cm 2 , about 90×10 6 cells / cm 2 , about 95×10 6 cells / cm 2 or about 100×10 6 cells / cm 2 More preferably, the engineered human cardiac tissue has a cell density of about 1×10 6 cells / cm 2 About 50×10 6 cells / cm 2 Even more preferably, the engineered human cardiac tissue has a cell density of about 1×10 6 cells / cm 2 to about 10×10 6 cells / cm 2 of cell density.

[0139] The engineered human heart patch can be produced in any shape, size, configuration using a suitable mold. In some embodiments, the shape of the tissue reflects the site of application. In one embodiment, the engineered heart tissue has a trapezoidal geometry.

[0140] Engineered human cardiac tissue can be derived from human embryonic stem cell groups (hESC) or human induced pluripotent stem cell groups (iPSC), which can be used as starting materials. In one embodiment, engineered human cardiac tissue is derived from human iPSC groups. In one embodiment, iPSC cells are grown under feeder-free conditions. In another embodiment, engineered human cardiac tissue is derived from cells obtained from patients, and a method for producing patient-specific iPSCs is used, which can then be differentiated using the methods described herein to produce engineered human cardiac tissue.

[0141] Treatment

[0142] In order to engineer human cardiac tissue for transplantation into patients with heart disease and heart failure, it is necessary to provide cardiac tissue patches with biocompatible structures that are durable and suitable for transplantation and can withstand transplantation into hypoxic environments. It is also necessary to provide engineered cardiac tissues with the appropriate variety of cardiomyocytes and non-cardiomyocyte cell types, particularly endothelial cells, so that the transplanted cardiac tissue patches can be vascularized, remain viable, and ultimately functionally integrated into the recipient heart. Therefore, there is a need for better transplant tissues.

[0143] As described herein, the inventors have also unexpectedly discovered that, through specific culture conditions, hPSC-derived cardiac tissue patches are produced, which contain cardiomyocytes, stromal cells / fibroblasts, and an increased number of endothelial cells, which have sufficient transplant durability and enhanced tolerance to hypoxia. Without wishing to be bound by any particular theory, when producing engineered cardiac tissue according to the methods described herein, the differentiation of progenitor cells to endothelial cell fate and pericytes for supporting ECs is promoted, and engineered tissues with primarily glycolytic metabolism accompanied by reduced oxidation (including reduced fatty acid oxidation) and low contractility are provided. This makes the engineered tissue more tolerant to hypoxia and may indicate that such engineered cardiac tissue is more suitable for therapeutic applications, such as transplantation.

[0144] Thus, the present invention provides for the use of engineered human cardiac tissue as described and defined herein as an implant for treating diseased or damaged cardiac tissue in a subject in need thereof.

[0145] The present invention also provides a method of treating diseased or damaged cardiac tissue in a subject in need thereof, comprising implanting in said subject the engineered human cardiac tissue described and defined herein.

[0146] The present invention also provides for the use of the engineered human cardiac tissue described and defined herein in the manufacture of a medicament for treating diseased or damaged cardiac tissue in a subject in need thereof.

[0147] 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 vascular disease, ischemia, myocardial infarction, inflammatory disease, valvular heart disease or myocarditis. In yet another embodiment, the subject suffers from congenital heart disease. In yet another embodiment, the congenital heart disease is selected from the group consisting of single ventricle disease (including hypoplastic left heart syndrome), tetralogy of Fallot, truncus arteriosus, pulmonary atresia, ventricular septal defect, atrial septal defect and endocardial cushion defect.

[0148] Screening methods

[0149] The present invention also provides the use of the engineered human cardiac tissue according to the present invention as an in vitro model for drug toxicity screening or for testing the in vitro method of regulating cardiac function by a candidate pharmacological agent. In one embodiment, the present invention provides a method for analyzing the biological effect of at least one test compound or bioactive agent on cardiomyocytes, comprising contacting the 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.

[0150] Reagent test kit

[0151] Also provided herein are kits comprising one or more cells or tissues generated according to the methods described herein, products or compositions comprising cells or tissues generated according to the methods described herein, optionally comprising an additional therapeutic agent or optionally wherein the cells comprise a reporter system or other modification, a combination of at least two of the agonists, inhibitors, culture media, instruments or other components useful for the methods described herein and in the instructions (e.g., instructions on how to generate cells, perform assays or administer cells, tissues, compositions or products), and a vial or other container for storing one of these aforementioned cells, tissues, compositions, products, agonists, inhibitors, culture media, etc.

[0152] Numbered list of implementations:

[0153] 1. A method for producing engineered human cardiac tissue comprising endothelial cells, the method comprising culturing a cell suspension comprising a population of cardiomyocytes and non-cardiomyocytes derived from a culture of human pluripotent stem cells (hPSCs) for a period of time and under conditions sufficient to produce engineered human cardiac tissue comprising endothelial cells in the presence of a FGFR1 agonist and a PDGFR agonist.

[0154] 2. A method for producing engineered human cardiac tissue, the method comprising the following steps:

[0155] i) mixing a cell suspension comprising a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocytes with a flowable hydrogel composition, wherein the cell suspension is in a serum-free medium;

[0156] ii) loading the flowable hydrogel composition comprising the cell suspension into a mold, and incubating the hydrogel for a period of time and under conditions sufficient to solidify the hydrogel; and

[0157] iii) culturing the hydrogel from step ii) in serum-free culture medium;

[0158] Wherein a population of cardiomyocytes and non-cardiomyocytes derived from a culture of human pluripotent stem cells (hPSCs) are contacted with a FGFR1 agonist and a PDGFR agonist to provide engineered human cardiac tissue, wherein the tissue comprises cardiomyocytes, stromal cells / fibroblasts, and endothelial cells.

[0159] 3. The method according to embodiment 2, wherein the FGFR1 agonist and / or the PDGFR agonist is added to the serum-free culture medium or the flowable hydrogel composition before mixing in step i).

[0160] 4. The method according to embodiment 2, wherein after mixing in step i), the FGFR1 agonist and / or the PDGFR agonist is added to the flowable hydrogel composition comprising the cell suspension.

[0161] 5. A method according to any one of embodiments 2-4, wherein the population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocytes are cultured in a serum-free medium comprising a FGFR1 agonist and a PDGFR agonist prior to mixing in step i).

[0162] 6. The method according to any one of embodiments 2-5, wherein in step iii), the hydrogel of step ii) is cultured in a serum-free medium comprising a FGFR1 agonist and a PDGFR agonist.

[0163] 7. The method according to any one of the preceding embodiments, wherein the cell suspension comprises about 70% cardiomyocytes and about 30% to about 50% non-cardiomyocytes.

[0164] 8. The method according to any one of the preceding embodiments, wherein the cell suspension comprises about 50% to about 75% cardiomyocytes and about 25% to about 50% non-cardiomyocytes.

[0165] 9. The method according to any one of the preceding embodiments, wherein the cell suspension is derived from a single differentiation process.

[0166] 10. The method according to any one of the preceding embodiments, wherein the engineered human cardiac tissue further comprises vascular smooth muscle cells and cardiac progenitor cells.

[0167] 11. The method according to any one of the preceding embodiments, wherein the engineered human cardiac tissue comprises:

[0168] - At least about 30% of myocardial cells;

[0169] - about 35% or less stromal cells / fibroblasts; and

[0170] - About 30% or less endothelial cells.

[0171] 12. The method according to any one of the preceding embodiments, wherein the engineered human cardiac tissue comprises:

[0172] - About 30% to about 70% of myocardial cells;

[0173] - about 10% to about 35% stromal cells / fibroblasts;

[0174] - about 5% to about 30% endothelial cells;

[0175] - Up to about 15% of vascular smooth muscle cells; and

[0176] - Up to about 10% of cardiac progenitor cells.

[0177] 13. A method according to any one of the preceding embodiments, 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.

[0178] 14. The method of embodiment 13, wherein the FGFR1 agonist is FGF2.

[0179] 15. The method according to any one of the preceding embodiments, wherein the PDGFR agonist is selected from PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC or PDGF-DD.

[0180] 16. The method of embodiment 15, wherein the PDGFR agonist is a PDGFRβ agonist.

[0181] 17. The method of embodiment 16, wherein the PDGFR agonist is PDGF-BB.

[0182] 18. The method according to any one of the preceding embodiments, wherein the serum-free medium comprises about 0.5 ng / ml-100 ng / ml of human PDGF-BB and about 0.5 ng / ml-100 ng / ml of human FGF2.

[0183] 19. The method according to any one of the preceding embodiments, wherein the serum-free medium comprises about 10 ng / ml of human PDGF-BB and about 10 ng / ml of human FGF2.

[0184] 20. The method according to any one of the preceding embodiments, wherein the serum-free culture medium comprises a basal culture medium comprising albumin and transferrin.

[0185] 21. The method of embodiment 20, wherein the serum-free culture medium comprises a B27 supplement.

[0186] 22. The method of embodiment 20 or 21, wherein the basal culture medium is a low calcium culture medium having a calcium concentration of less than about 1.2 mM.

[0187] 23. The method according to any one of embodiments 2-22, further comprising replacing the culture medium in step iii) with fresh cell culture medium at least every 2 days.

[0188] 24. The method according to any one of embodiments 2-23, wherein the culturing in step iii) is carried out for at least 5 days.

[0189] 25. The method of any one of the preceding embodiments, wherein about 3% to about 10% of the cardiomyocyte population in the engineered human cardiac tissue are proliferative.

[0190] 26. The method of any one of the preceding embodiments, wherein the engineered human cardiac tissue exhibits glycolytic metabolism.

[0191] 27. A method according to embodiment 26, wherein the cell culture medium obtained after culturing with the engineered human heart tissue for 24 hours has a lactate concentration of 1 mM.

[0192] 28. The method according to any one of the preceding embodiments, wherein the engineered human cardiac tissue exhibits a log2 relative gene expression of PKM >2 when normalized for TUBA1A expression.

[0193] 29. A method according to any of the preceding embodiments, wherein the engineered human cardiac tissue is tolerant to hypoxia.

[0194] 30. The method according to any one of the preceding embodiments, wherein the engineered human cardiac tissue produces a lactate / pyruvate ratio of at least 1:1 in a cell culture medium obtained after culturing for 20 h under hypoxic conditions.

[0195] 31. The method according to any one of the preceding embodiments, wherein the engineered human cardiac tissue exhibits downregulation of ALDH1, AGMO, DPYD, GPX3, CYP1B1, and PLIN5.

[0196] 32. The method according to any one of the preceding embodiments, wherein the cardiomyocytes express MMP1 + .

[0197] 33. The method according to any one of the preceding embodiments, wherein the fibroblasts express MMP1 + 、FOXD1 + 、RAB27B + 、NR2F1 + 、F2RL1 + 、SPP1 + 、TMEM158 + PTHLH + 、PHLDA2 + 、MALL + , MYCT1 + ,DUSP4 + 、PLAU + 、TMEM156 + 、CD274 + 、MMP10 + 、ARRDC4 + 、RFX8 + MLPH + , THBD + 、HHEX + 、VGF + 、OTULINL + IL33 + 、CA12 + 、C6orf141 + MFSD2A + and / or CARD10 + .

[0198] 34. The method according to any one of the preceding embodiments, wherein the engineered human cardiac tissue exhibits a kinetic energy of less than 10 mN / mm 2 contractile activity.

[0199] 35. The method of any one of embodiments 2-34, wherein the hydrogel composition comprises a fibrin hydrogel.

[0200] 36. The method of embodiment 35, wherein fibrin is formed by mixing thrombin and fibrinogen solutions.

[0201] 37. The method of embodiment 36, wherein the fibrinogen is present at a concentration of 10 mg / mL to 50 mg / mL.

[0202] 38. The method of embodiment 36, wherein the fibrinogen is present at a concentration of 20 mg / mL.

[0203] 39. The method according to any one of embodiments 2-38, wherein in step i), the cell suspension is mixed with a hydrogel to provide about 5×10 6 cells / mL to about 100×10 6 The cell concentration is cells / mL.

[0204] 40. The method according to embodiment 39, wherein in step i), the cell suspension is mixed with a hydrogel to provide about 20×10 6 cells / mL to about 50×10 6 The cell concentration is cells / mL.

[0205] 41. The method according to embodiment 40, wherein in step i), the cell suspension is mixed with a hydrogel to provide about 35×10 6 The cell concentration is cells / mL.

[0206] 42. The method according to any one of the preceding embodiments, inducing the differentiation of hPSCs towards the cardiac lineage by the following steps:

[0207] a) culturing hPSCs in a basal medium comprising an effective amount of BMP4, activin A, FGF, a GSK3 inhibitor, a serum-free supplement comprising albumin and transferrin, and minus insulin for about 72 hours, wherein the medium is replaced with fresh medium every day;

[0208] b) culturing the cells obtained in step a) in a basal medium comprising an effective amount of a Wnt signaling pathway inhibitor and the serum-free supplement in a) for about 72 hours, wherein the medium is replaced with fresh medium every day;

[0209] c) culturing the cells obtained in step b) in a basal medium comprising an effective amount of a Wnt signaling pathway inhibitor and a serum-free supplement (comprising albumin and transferrin, including insulin) for about 7 days, wherein the medium is replaced with fresh medium after the 2nd and 4th day of the period of about 7 days;

[0210] d) 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;

[0211] wherein the basal medium used in each of steps a) to d) has a calcium concentration of less than about 1.2 mM;

[0212] Thereby providing a cell suspension comprising a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocytes.

[0213] 43. The method of embodiment 42, wherein:

[0214] - the GSK3 inhibitor is CHIR99021; and / or

[0215] - the inhibitor of the Wnt signaling pathway is IWP-4; and / or

[0216] - The basal culture medium is RPMI or DMEM.

[0217] 44. A method according to any of the preceding embodiments, wherein the engineered human cardiac tissue is in the form of a tissue patch.

[0218] 45. A method according to embodiment 44, wherein the patch is used as an implant in a subject.

[0219] 46. ​​A method according to embodiment 45, wherein the subject suffers from cardiomyopathy or cardiac tissue damage.

[0220] 47. A method according to embodiment 46, wherein the cardiomyopathy or cardiac tissue damage is caused by acute or chronic stress, atherosclerotic vascular disease, ischemia, myocardial infarction, inflammatory disease, heart valve disease or myocarditis.

[0221] 48. A method according to embodiment 45, wherein the subject suffers from congenital heart disease.

[0222] 49. A method according to embodiment 48, wherein the congenital heart disease is selected from single ventricle disease (including hypoplastic left heart syndrome), tetralogy of Fallot, truncus arteriosus, pulmonary atresia, ventricular septal defect, atrial septal defect and endocardial cushion defect.

[0223] 50. The method according to any one of the preceding embodiments, wherein the hPSC is an iPSC.

[0224] 51. A method according to embodiment 50, wherein the iPSCs are derived from a subject suffering from heart disease and / or to be implanted into the engineered human heart tissue.

[0225] 52. The method of any one of the preceding embodiments, wherein the hPSCs are embryonic stem cells.

[0226] 53. An engineered human cardiac tissue comprising at least about 30% hPSC-derived cardiomyocytes, about 35% or less hPSC-derived stromal cells / fibroblasts, and at most about 30% or less hPSC-derived endothelial cells, wherein the above cells are located throughout a hydrogel composition.

[0227] 54. An engineered human cardiac tissue comprising hPSC-derived cardiomyocytes, hPSC-derived stromal cells / fibroblasts, and hPSC-derived endothelial cells, wherein the above cells are derived from a single differentiation process, wherein the above cells are located throughout a hydrogel composition.

[0228] 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.

[0229] 56. The engineered human cardiac tissue of any one of embodiments 53 to 55, wherein the engineered human cardiac tissue comprises:

[0230] - About 30% to about 70% of myocardial cells;

[0231] - about 10% to about 35% stromal cells / fibroblasts; and

[0232] - About 5% to about 30% endothelial cells.

[0233] 57. The engineered human cardiac tissue of any one of embodiments 53 to 56, wherein at most about 10% of the cardiomyocytes in the engineered human cardiac tissue are proliferative.

[0234] 58. The engineered human cardiac tissue of any one of embodiments 53 to 57, wherein the engineered human cardiac tissue exhibits glycolytic metabolism.

[0235] 59. The engineered human heart tissue according to embodiment 53, wherein the cell culture medium obtained after culturing the engineered human heart tissue for 24 hours has a lactate concentration of 1 mM.

[0236] 60. The engineered human cardiac tissue of any one of embodiments 53 to 59, wherein said engineered human cardiac tissue exhibits a log2 relative gene expression of PKM >2 when normalized to TUBA1A.

[0237] 61. The engineered human cardiac tissue of any one of embodiments 53 to 60, wherein the engineered human cardiac tissue is tolerant to hypoxia.

[0238] 62. An engineered human cardiac tissue according to any one of embodiments 53 to 61, wherein the engineered human cardiac tissue produces a lactate / pyruvate ratio of at least 1:1 in the cell culture medium obtained after culturing for 20 hours under hypoxic conditions.

[0239] 63. The engineered human cardiac tissue of any one of embodiments 53 to 62, wherein the engineered human cardiac tissue exhibits a log2 relative gene expression of <-4 for ALDH1, AGMO, GPX3, CYP1B1, and PLIN5 when normalized for TUBA1A expression.

[0240] 64. The engineered human cardiac tissue according to any one of embodiments 53 to 63, wherein the cardiomyocytes express MMP1 + .

[0241] 65. The engineered human cardiac tissue of any one of embodiments 53 to 64, wherein the fibroblasts express MMP1 + 、EMP1 + 、FOXD1 + 、RAB27B + 、NR2F1 + 、F2RL1 + 、SPP1 + 、TMEM158 + PTHLH + 、PHLDA2 + 、MALL + , MYCT1 + ,DUSP4 + 、PLAU + 、TMEM156 + 、CD274 + 、MMP10 + 、ARRDC4 + 、RFX8 + MLPH + , THBD + 、HHEX + 、VGF + 、OTULINL +IL33 + 、CA12 + 、C6orf141 + MFSD2A + and / or CARD10 + .

[0242] 66. The engineered human cardiac tissue of any one of embodiments 53 to 65, comprising:

[0243] - About 30% to about 70% of myocardial cells;

[0244] - about 10% to about 35% stromal cells / fibroblasts;

[0245] - about 5% to about 30% endothelial cells;

[0246] - up to about 15% of vascular smooth muscle cells; and

[0247] - Up to about 10% of cardiac progenitor cells.

[0248] 67. The engineered human cardiac tissue of any one of embodiments 53 to 66, wherein the engineered human cardiac tissue exhibits a kinetic energy of less than 10 mN / mm 2 contractile activity.

[0249] 68. An engineered human cardiac tissue according to any one of embodiments 53 to 67, wherein the hydrogel composition comprises fibrin formed by mixing thrombin and fibrinogen solutions.

[0250] 69. The engineered human cardiac tissue of embodiment 68, wherein the fibrinogen is present at a concentration of 10 mg / mL-50 mg / mL.

[0251] 70. The engineered human cardiac tissue of embodiment 69, wherein the fibrinogen is present at a concentration of 20 mg / mL.

[0252] 71. The engineered human cardiac tissue of any one of embodiments 53 to 70, wherein the total number of cells in the tissue is about 20×10 6 cells to 100×10 6 cells.

[0253] 72. The engineered human cardiac tissue of any one of embodiments 53 to 71, wherein the engineered human cardiac tissue is in the form of a tissue patch.

[0254] 73. An engineered human cardiac tissue according to any one of embodiments 53 to 72, wherein the hPSCs are iPSCs.

[0255] 74. An engineered human cardiac tissue according to any one of embodiments 53 to 73, wherein the hPSCs are embryonic stem cells.

[0256] 75. An engineered human cardiac tissue produced according to the method of any one of embodiment claims 1 to 52.

[0257] 76. A method for analyzing the biological effect of at least one test compound or bioactive agent on cardiomyocytes, comprising contacting the engineered human cardiac tissue of any one of embodiments 53 to 75 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.

[0258] 77. The engineered human cardiac tissue according to any one of embodiments 53 to 75 for use as an implant for treating diseased or damaged cardiac tissue in a subject in need thereof.

[0259] 78. A method of treating diseased or damaged cardiac tissue in a subject in need thereof, comprising implanting in the subject the engineered human cardiac tissue of any one of embodiments 53 to 75.

[0260] 79. Use of the engineered human cardiac tissue of any one of embodiments 53 to 75 in the manufacture of a medicament for treating diseased or damaged cardiac tissue in a subject in need thereof.

[0261] 80. The engineered human cardiac tissue of embodiment 77, the method of embodiment 78, or the use of embodiment 79, wherein the subject suffers from cardiomyopathy or cardiac tissue damage.

[0262] 81. An engineered human cardiac tissue, method or use according to embodiment 80, wherein the cardiomyopathy or cardiac tissue damage is caused by acute or chronic stress, atherosclerotic vascular disease, ischemia, myocardial infarction, inflammatory disease, heart valve disease or myocarditis.

[0263] 82. The engineered human cardiac tissue of embodiment 77, the method of embodiment 78, or the use of embodiment 79, wherein the subject suffers from congenital heart disease.

[0264] 83. An engineered human cardiac tissue, method or use according to embodiment 82, wherein the congenital heart disease is selected from univentricular disease (including hypoplastic left heart syndrome), tetralogy of Fallot, truncus arteriosus, pulmonary atresia, ventricular septal defect, atrial septal defect and endocardial cushion defect. Example

[0265] Materials and methods

[0266] Human pluripotent stem cells

[0267] The hESCs used were female HES3 (WiCell). The following cell lines were obtained from the CIRM hPSC bank funded by the California Institute for Regenerative Medicine: CW30382A (male, named AA) and CW30318C (female, named CC), both obtained from FujiFilm. hPSC cell lines were maintained in mTeSR-1 (Stem Cell Technologies) / Matrigel (Millipore) and passaged using ReLeSR (Stem Cell Technologies). Quality control was performed by karyotyping and mycoplasma testing. PSCs were cultured at 2 × 10 4 cells / cm 2 The cells were seeded in Matrigel-coated flasks and cultured in mTeSR-1 for 4 days.

[0268] Cardiac differentiation

[0269] To induce cardiac mesoderm, hPSCs were cultured in RPMI B27 medium (RPMI 1640 GlutaMAX + 2% B27 supplement (without insulin), 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma) and 1% penicillin / streptomycin (ThermoFisherScientific)) 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). Mesoderm induction required daily culture medium changes for 3 days. Subsequently, RPMI B27 (minus insulin) containing 5 μM IWP-4 (Stem Cell Technologies) was used for another 3 days of cardiac specification, and then 5 μM IWP-4 RPMI B27+ (RPMI1640 glutamate + 2% B27 supplement (with insulin), 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and 1% penicillin / streptomycin) was used for another 7 days, with the medium being changed every 2-3 days. On the last 2 days of differentiation, hPSCs were cultured in RPMI B27+insulin.

[0270] The collection of differentiated cardiomyocytes involved enzymatic digestion to isolate the cells, which were first cultured in 0.2% type I collagenase (Sigma) in PBS (containing Ca2+) containing 20% ​​fetal bovine serum (FBS) at 37°C. 2+ and Mg 2+) digested for 1 h, and then digested in 0.25% trypsin-EDTA at 37°C for 10 minutes. The cells were filtered through a 100 μm mesh cell strainer (BD Biosciences), centrifuged at 300 × g for 3 min, and resuspended in α-MEM glutamine, 10% FBS, 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and 1% penicillin / streptomycin. The inventors also found that Accutase (Sigma) can be used for harvesting for about 15 min, then 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.

[0271] Flow cytometric analysis showed that differentiated cardiomyocytes were approximately 70% α-actin + / CTNT + cardiomyocytes, and about 30% CD90 stromal cells.

[0272] Cardiac tissue patch formation and culture

[0273] Serum-free medium (SF) containing 4% B27 supplement (ThermoFisher Scientific), 10 ng / ml PDGF-BB (RnD Systems), 10 ng / ml bFGF (RnD Systems), 33 ug / mL aprotinin (Sigma), 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma), and 1% penicillin / streptomycin (ThermoFisher Scientific) in RPMI1640 Glutamax (ThermoFisher Scientific) was used for the formation and culture of cardiac tissue patches.

[0274] Prepare a 2X solution of 20mg / mL-100mg / mL of sterile SF solution of fibrinogen. Preheat the SF to 37°C to help dissolve the fibrinogen. Use a P1000 pipette tip with a wide bore to help mix the fibrinogen. Thorough mixing is required and placing it in a 37°C water bath can help. Sterile filter the fibrinogen and keep on ice until needed.

[0275] Prepare a flowable fibrin hydrogel by mixing the following reagents in a polypropylene tube in the order described below. Reagents were thawed on ice and kept on ice throughout the procedure.

[0276] i) Add fibrinogen (Sigma) solution to a final concentration of 10

[0277] mg / ml-50mg / ml.

[0278] ii) Add the same volume of cell suspension in SF as the fibrinogen solution

[0279] Liquid, to reach about 35 million cells per ml, mix well.

[0280] iii) Add thrombin (Sigma) to a final concentration of 2 U / ml and mix well.

[0281] iv) Use a pipette to transfer the solution into the PDMS mold.

[0282] v) Incubate the hydrogel at 37°C for 30-60 minutes to induce gelation of the cell-matrix mixture.

[0283] In this example, human heart tissue was cultured after the cell matrix material was gelled. SF medium was added to cover the human heart tissue, and the medium was changed every 2 to 3 days. At the end of the culture period, the heart tissue was removed from the PDMS mold.

[0284] For control human heart tissue, instead of SF, human heart tissue was cultured in α-MEM GlutaMAX (ThermoFisherScientific), 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 ug / mL aprotinin (Sigma) for 2 days, and then changed to maturation medium (MM), which is glucose-free, contains glutamine and phenol red (ThermoFisher Scientific), and supplemented with 4% B27- (without insulin), 1% GlutaMAX (ThermoFisher Scientific), 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, and 1% penicillin / streptomycin (ThermoFisher Scientific). DMEM containing 1 mM glucose, 100 μM palmitic acid (conjugated to bovine serum albumin in B27 by incubation at 37°C for 2 h, Sigma), and 33 ug / mL aprotinin (Sigma) was replaced every 2-3 days.

[0285] Preparation of PDMS mold for cardiac tissue patch

[0286] A polydimethylsiloxane (PDMS) mold was prepared and the cell suspension in the flowable fibrin hydrogel was seeded onto the mold as described above. To make a mold capable of producing a tissue patch of approximately 3 cm x 5 cm in size, 7.0 g of PDMS base was mixed with 0.7 g of PDMS curing agent ( 184 silicone elastomer PDMS kit) mixed. The polymer is placed in a vacuum desiccator chamber and degassed. Pressure release and degassing are performed every 5 to 10 minutes to remove all bubbles, for a total of about 20 minutes. After degassing, about 7g of polymer is poured into a customized patch mold. The customized patch mold is arranged to have a series of holes, and PDMS can flow into and settle into these holes, thereby forming a series of protrusions or "pillars" in the PDMS mold. When the hydrogel seeded with cells is mounted on the mold, the "pillars" help to form a mesh structure in the cardiac tissue patch.

[0287] The custom patch mold with PDMS was placed in a vacuum desiccator and further degassed for 30 minutes, releasing the pressure every 5-10 minutes to ensure that all bubbles were removed from the "post" holes. The custom patch mold with PDMS was then baked at 65°C for 8 minutes, then rotated 180° and baked for another 27 minutes (35 min in total), and then removed from the oven and cooled to room temperature. The baked PDMS mold was then removed from the custom patch mold and sterilized with 70% ethanol, followed by UV irradiation.

[0288] Isolation of cardiomyocyte nuclei

[0289] According to the previous description (O Bergmann and S Jovinge, J Vis Exp (65) (2012)) but slightly modified, cell nuclei were isolated. In brief, BHTP was electrically homogenized (IKA) in 15 mL 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 inhibitor). The lysate was then homogenized 25 times with a 40 mL dounce tissue grinder (Wheaton). The cell lysate was then filtered through a 100 uM cell strainer, followed by filtering through a 70 uM and 40 uM cell strainer (BD Falcon), and then centrifuged (Beckman Coulter Allegra X-15R) at 1000 x g for 5 min to precipitate the cell nuclei. The nuclear pellet was then resuspended in 3 mL 1M sucrose buffer (1M sucrose, 10 mM Tris-HCl (pH=8), 5 mM magnesium acetate, 1 mM DTT and 1X complete protease inhibitor). The isolated cardiomyocyte nuclear pellet was washed once in PBS (Thermo Fisher Scientific) and centrifuged at 1000 x g to pellet the nuclei before FACS sorting.

[0290] Single nucleus RNA-seq library preparation and sequencing

[0291] The isolated nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific) and then sorted on an inflow cell sorter (BD) with a 70uM nozzle and 60psi pressure setting. The sorted nuclei were counted on a hemocytometer to calculate the nucleus density, and then loaded onto a Chromium Controller 4 (10X Genomics) to form a gel bead emulsion (GEM), with about 5,000 nuclei loaded per sample for library preparation. After GEM formation, Chromium Next GEM Single Cell 3'GEM, Library & GelBead Kit v3.1 was used to prepare the library according to the manufacturer's recommended protocol. The library was sequenced at a resolution of about 100,000 readings per core on NovaSeq 6000 (Illumina). The bioinformatics analysis of single-nuclear RNA sequencing uses Cell Ranger (v3.0.2) to map, process and count the original fastq readings of each sample. Subsequently, the counts were aggregated together to create a table of unique molecular identifier (UMI) counts for each of the 33,939 genes for each sample. All preprocessing and filtering steps for the dataset were subsequently performed using the R statistical programming language (v3.6.0). The cell quality of each sample was independently assessed by examining the total number of cells, the distribution of total UMI counts, the number of unique genes detected in each sample, and the ratio of ribosomal and mitochondrial content in each cell. Briefly, genes with at least one count in at least 20 cells were selected for downstream analysis after removing mitochondrial genes, ribosomal genes, and those that were not annotated, assuming a minimum cluster size of 20 cells. All genes on chromosomes X and Y were removed prior to clustering. For each sample, we used the R package Seurat (v3.0.2) for SC-transformation 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 separately. CCA from the Seurat package with 30 dimensions and 3000 integration anchors was used (T Stuart et al., Cell 177(7), 1888(2019)) The replicate data of each group were integrated and then scaled. Cell clustering was performed using 20 principal components (PCs) and an initial resolution of 0.3.The marker genes used to annotate the clusters were identified as significantly upregulated genes for each cluster by corrected t-test (accounting for mean variance trends and using robust empirical Bayes shrinkage variance), and then TREAT test was performed using the limma R package (v3.40.2), specifying a log-fold change threshold of 0.5 and a false discovery rate (FDR) critical of <0.05. In addition, heat maps showing previously published marker gene expression were used to help interpret the clusters. Visualization of the dataset was mainly performed using nonlinear dimensionality reduction UMAP (Becht et al., Nat Biotechnol (2018)) plots.

[0292] Hypoxia

[0293] Culture medium: MM, SF, MM+HEPES, SF+HEPES

[0294] The culture medium (MM or SF + 10 mM HEPES (Sigma-Aldrich)) was prepared 24 h before the experiment and placed in a modular incubator chamber (MIC-101, Billups-Rothenberg Inc.) for 24 h of degassing and flushing with N2 gas at 50 L / min for 10 min.

[0295] Meanwhile, the culture medium of human cardiac tissue patches prepared according to the above method has been changed to MM or SF (without HEPES).Before the hypoxia experiment, at the 24h mark, control samples of the culture medium were taken from 4 different locations around the tissue and on top of the tissue (for LCMS).

[0296] After sampling, the culture medium was replaced with degassed HEPES containing the culture medium and the patch, and placed in a modular incubator chamber with a humidity reservoir and an anaerobic indicator (thermos Scientific, BR0055B). The chamber was then flushed with N2 pure gas at a rate of 50 L / min for 10 min and placed in a cell culture incubator at 37°C for 20 h.

[0297] After 20 h of hypoxia, tissues were removed from the chambers, and media samples were collected for LCMS analysis (as above), and tissues were then processed for immunostaining.

[0298] Immunostaining

[0299] Human heart tissue patches were fixed with 1% paraformaldehyde (Sigma) at room temperature for 60 min and washed three times with PBS. They were then stained with a primary antibody to α-cardiac actin (Sigma, A-7811, 1:1000) in blocking buffer (5% FBS and 0.2% Triton X-100 (Sigma) PBS on a shaker at 4°C overnight). They were then washed twice in blocking buffer for 2 h and subsequently stained with goat anti-mouse IgG conjugated with Alexa Fluor 555 (Thermofisher, A-21422, 1:400) and Hoescht (1:1000) at 4°C overnight. Human heart tissue patches were washed in blocking buffer for 2 h for 2 times and imaged in situ on a 5X objective using a Leica Thunder microscope, and the entire patch was imaged and stitched together by computer.

[0300] To determine the intensity of nuclear and cardiomyocyte (α-cardiac actin) staining, custom batch files were written in Matlab R2013a (Mathworks). These methods removed background, calculated image intensity, and exported batch data into an Excel (Microsoft) spreadsheet.

[0301] Mass spectrometry

[0302] Metabolites were extracted from conditioned media as described previously (PMID 31690627), but with minor modifications. Specifically, media samples were combined with 4 volumes of extraction buffer containing a 1:1 (v / v) mixture of methanol and acetonitrile, with 2.5 μM D-camphor-10-sulfonic acid (Wako) and 2.5 μM 4-morpholineethanesulfonic acid (Sigma-Aldrich) 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, centrifuged again, and the supernatant was transferred to HPLC vials. Calibration standards were diluted in the original media and then extracted in parallel with the samples.

[0303] Metabolites were separated by LC using a 1290 Infinity II pump (Agilent) with an InfinityLab Poroshell 120 HILIC-Z column (Agilent, 2.7 μm particle size, 2.1 mm id x 100 mm length, PEEK lining). Buffer A was 90:10 (v / v) acetonitrile / water containing 10 mM ammonium acetate and 5 μM methylenephosphonic acid (Sigma-Aldrich), and buffer B was water containing 10 mM ammonium acetate and 5 μM methylenephosphonic acid. Methylenephosphonic acid was included as an additive to increase sensitivity and reduce peak tailing (PMID 29976062). At the same time, the chromatographic 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 6470QQQ with the following parameters: gas temperature of 200°C, gas flow rate of 11 L / min, nebulizer pressure of 40 psi, sheath gas temperature of 400°C, sheath gas flow rate of 12 L / min, capillary voltage of 3000 V (for negative and positive modes), and nozzle voltage of 0 V (for negative mode) and 500 V (for positive mode). Multiple reaction monitoring transitions were calibrated and optimized using metabolite standards. Acquisition was performed with a dwell time of 10 ms.

[0304] LCMS data were extracted using Skyline (version 20.2.0.343-a7a9e8c4f) (PMID 31984744). Metabolite peak areas were normalized to internal standards using calibration standards and converted to absolute quantities.

[0305] LDH assay

[0306] Supernatants from human heart tissue were collected after hypoxia. Lactate dehydrogenase (LDH) assay (Pierce) was performed on these samples according to the manufacturer's instructions to determine the extent of cell death.

[0307] Relative gene expression analysis

[0308] For relative gene expression analysis of major cardiomyocyte cell types between new and old culture media, pseudo-batch samples were created by summing the counts of each technical replicate of a given cell type. To account for differences in sequencing depth used to compare gene expression levels between samples, counts per million (CPM) values ​​were calculated for several housekeeping genes. TUBA1A was chosen as a housekeeping gene because its CPM value was invariant across experimental conditions. Then, to account for differences in gene length, transcripts per million (TPM) values ​​were calculated for several genes of interest and normalized to the TPM value relative to TUBA1A in each condition to calculate relative fold change values ​​(FC), which were scaled using a log2 transformation.

[0309] Animal studies

[0310] Large Animal Model of Right Heart Failure

[0311] Research complied with the guidelines of the National Health and Medical Committee of Australia and was approved by the Murdoch Children's Research Institute Animal Ethics Committee.

[0312] Crossbred sheep of Border-Leicester aged 6-9 months and weighing 27.3±1.3kg (n=6, mean±SD) were anesthetized and prepared for surgery in the lounge of the large animal facility, with a pre-intramuscular injection of ketamine 5mg / kg and xylazine 0.1mg / kg, and then anesthetized with 4% isoflurane delivered by mask. After being transported to the laboratory on a trolley, the animals were transferred to the operating table and placed supine, and the neck and chest areas were then clipped and cleaned with a disinfectant solution. After insertion of a cuffed endotracheal tube, anesthesia was maintained by an electronically controlled ventilator (WATO EX-20Vet, Mindray, Shenzhen, China) delivering isoflurane (2%-3%) and nitrous oxide (10%-20%) in oxygen-enriched air, combined with intravenous infusions of ketamine (1 mg / kg / hr-1.5 mg / kg / hr), midazolam (0.1 mg / kg / hr-0.15 mg / kg / hr), and fentanyl (2 μg / kg / hr-2.5 μg / kg / hr). Transcutaneous arterial O2 saturation was continuously monitored with a pulse oximeter worn on the ear. Through the midline incision of the neck, in the right common carotid artery cannula 7-Fr arterial sheath, for monitoring blood pressure and regular blood gas analysis (ABL800, Radiometer, Copenhagen, Denmark), adjust ventilation to maintain arterial oxygen partial pressure (Pao2) at 100mmHg-120mmHg and carbon dioxide partial pressure (Paco2) at 35mmHg-40mmHg.Through the right external jugular vein, the three-caval cannula is inserted into the superior vena cava, to use liquid (compound sodium lactate, speed is 15ml / kg / hr) and anesthetic drugs (ketamine, midazolam and fentanyl, take by the above dosage).With heating pad and towel covering, body temperature is maintained at 39 degrees Celsius-40 degrees Celsius.

[0313] After intramuscular supplementary injection of buprenorphine (150mg) to relieve pain, the skin of the chest from the base of the neck to the xiphoid process was cut, and a median sternotomy was performed to expose the lungs and heart. After inserting and expanding the chest retractor, an adjustable polyethylene snare was placed around the inferior vena cava just below the heart. The pericardium was then cut between the apex and the ascending aorta, and a pericardial cradle was formed to support the heart. Subsequently, a fluid-filled catheter was inserted into the left atrial appendage and the right atrial appendage to measure left and right heart filling pressures. The fluid-filled catheter and a 3.5-Fr micromanometer catheter (SPR-877, Millar Instruments, Houston, TX, USA) were inserted into the pulmonary artery trunk by a separate purse-string suture to measure mean pulmonary artery blood pressure and high-fidelity pulmonary artery blood pressure, respectively, while a 6-Fr arterial sheath was also inserted into the artery by another purse-string suture. A transit-time flow probe (16 mm or 18 mm PAU, Transonic Systems, Ithaca, NY, USA) was then placed around the pulmonary trunk to measure right ventricular output. Subsequently, the animals were anticoagulated with an intravenous bolus of heparin sodium (40 IU / kg) followed by additional boluses of heparin (20 IU / kg) every hour before a 5-Fr combined micromanometer-conductance catheter (Ventri-Cath 507, Millar Instruments, Houston, TX, USA) was introduced into the right ventricle through a sheath in the pulmonary trunk to obtain high-fidelity pressure and volume signals.

[0314] After instrumental measurement, a model of impaired right ventricular function in the context of right ventricular (RV) volume overload was established by a combination of two interventions. First, a stable degree of pulmonary valve regurgitation was produced by clamping two of the three leaflets that make up the pulmonary valve with 6 or 7 discontinuous 4 / 0 silk sutures on a curved needle that entered and then exited the base of the pulmonary trunk, similar to the method described by Agger et al. (Agger et al., Interact cardiasc Thorac Surg 10(6), 962(2010)). The entrance and exit sides of the suture were knotted, thereby causing a severe degree of pulmonary regurgitation by anchoring the pulmonary valve leaflets to the inner surface of the pulmonary trunk. Subsequently, RV dysfunction was produced by injecting 65,000 polystyrene microbeads (90 μm in diameter, Polysciences, Warrington, PA, USA, Cat. No. 07315) through a catheter inserted into the ascending aorta through a small aortotomy and located in the origin of the right coronary artery according to the method of Huang et al. (Y Huang et al., Asaioj 43(5), M408(1997)). The microbeads were delivered in 2.5 ml of isotonic saline, and the microbead suspension was subjected to brief ultrasonic mixing and subsequent manual shaking before injection. Prior to the administration of the microbeads, a bolus dose of lidocaine (25 mg) was given by intravenous injection to prevent arrhythmias.

[0315] Surgical implantation of BHTP

[0316] The trapezoidal BHTP was carefully secured to the outer surface of the right ventricle, covering the area previously affected by the infusion of microbeads. The patch was secured to the right ventricle using six discontinuous 5 / 0 prolene sutures, four of which were at the corners of the patch, while the remaining sutures were at the midpoints of the base of the trapezoid and its opposite side. The pericardium was then closed over the patch using a combination of discontinuous and continuous 4 / 0 silk sutures. After closure, the pericardium was covered with surgical gauze soaked in isotonic saline, and additional isotonic saline was applied every 15 minutes during the subsequent 2-hour monitoring period to keep the gauze moist.

[0317] Functional evaluation of a large animal model of right heart failure with BHTP implantation

[0318] Experimental protocol

[0319] General hemodynamics (heart rate, aortic and pulmonary artery blood pressures, right ventricular output) and right ventricular pressure-volume measurements were performed just before RV patch application and repeated at 15-minute intervals within 2 hours after RV patch implantation. Two types of data sets were recorded on a computer for each time point. The first was a 20-30-sec steady-state data block. The second was a gold standard assessment of RV function using pressure-volume analysis, which involves briefly (approximately 10 sec) tightening an adjustable snare around the inferior vena cava to temporarily reduce the amount of venous blood returning to the heart and thereby reduce right ventricular preload.

[0320] At the end of the study, animals were humanely killed by injection of an overdose of sodium pentobarbital (100 mg / kg) into the left atrial catheter.

[0321] Processing and analysis of physiological data

[0322] Before each study, the fluid-filled catheter pressures of the aorta, pulmonary artery, left atrium, and right atrium were measured with sensors, referenced to the atmospheric pressure level of the left atrium, and calibrated with a water manometer. Pulmonary trunk 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 the micromanometer-conductance catheter by an interface pressure-volume signal processing system (MV Ultra, Millar Instruments, Houston, TX, USA).

[0323] Analog catheter, micromanometer, and flow probe signals were digitized at a sampling rate of 1 kHz (iNET-100B, GW Instruments, Somerville, MA, USA) and recorded using programmable acquisition and analysis software (Spike2, Cambridge Electronic Design, Cambridge, UK). During physiological data analysis, no filtering was used except for the use of a 48 Hz low-pass filter to remove electrical interference from the signal, and steady-state analysis was performed on ensemble averaged signals typically generated from >30 heart beats.

[0324] Right ventricular pressure and volume data

[0325] RV volume was calculated from the conductance catheter signal using standard methods, combining estimates of blood resistivity, parallel conductance, and a gain constant (4-9). Blood resistivity was measured in small samples (<0.5 ml) of aortic blood (Rho calibration cuvettes, Millar Instruments, Houston, TX, USA) before and after RV patch implantation. Parallel conductance, the shift in the conductance catheter signal related to the conductivity caused by structures surrounding the blood pool in the RV cavity, was obtained by recording changes in RV volume data during injections of 4 ml of hypertonic (10%) saline into the right atrium every 30 min 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); WhitePa and AN Redington, Physiol Meas 21(3), R23 (2000)). The gain constant was calculated as the ratio of the conductance catheter- and flow probe-derived RV stroke volumes under steady-state conditions before each transient occlusion of the IVC.

[0326] A high-fidelity RV pressure rate of change was calculated using a running three-point differential algorithm, and the maximum RV pressure rate of rise (RVdP / dtmax) was used as a measure of RV contractility. The RV end-diastole was defined at the base of the rising stroke of the RV pressure waveform using a curvature-based automatic feature extraction algorithm (JP Mynard et al., Annu Int Conf IEEE Eng Med Biol Soc 2007, 1691 (2007)), and the corresponding RV end-diastolic volume was measured at this time point. RV stroke work was calculated as the area of ​​the RV pressure-volume loop. The RV preload-replenishment stroke work index (PRSWI) 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, and is obtained from a series of pressure-volume loops generated during a transient reduction in RV preload produced by snare tightening around the inferior vena cava. PRSWI represents a robust and sensitive measure of ventricular pumping performance (D Burkhoff et al., Am J Physiol Heart CircPhysiol 289(2), H501 (2005); DD Glower et al., Circulation 71(5), 994 (1985)).

[0327] Statistical analysis

[0328] Results were analyzed using GraphPad Prism version 9 (GraphPad Software Inc., La Jolla, CA, USA). One-way repeated measures analysis of variance (ANOVA) was used to analyze changes in hemodynamic and RV pressure-volume data over time, with specific comparisons assessed by taking the sum of squares within the animal and dividing into each degree of freedom. Data are presented as mean ± SD and were considered significant at P < 0.05.

[0329] Example 1. Characteristics of bioengineered cardiac tissue patches

[0330] Human heart tissue patches and control heart tissue patches were prepared according to the protocol described in the above section entitled Materials and Methods, and were also compared using the above single cell analysis. In brief, after 7 days of culture in SF medium (also referred to as "new medium") or mature medium MM (also referred to as "old medium"), the tissue patches were removed from their molds and frozen. The frozen patches were then cut, homogenized, and cell nuclei were isolated from the cells of each patch. The cell nuclei were then sequenced and analyzed.

[0331] Single-nucleus RNA sequencing (snRNA-seq) of 5889 individual cardiac cell nuclei from human cardiac tissue patches cultured in either SF medium (also called “new medium”) or the maturation medium MM (also called “old medium”) revealed five major cell clusters ( Figure 2 Analysis of marker genes in these five clusters enabled differentiation of the major cardiomyocyte types in 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) ( Figure 2-Figure 8 ). Cultivation of human cardiac tissue patches in the “new medium” was associated with significant changes in cellular composition, characterized by a significant increase in the relative proportions of endothelial cells and smooth muscle cells ( Figure 2 Human cardiac tissue patches cultured in the "old medium" consisted only of cardiomyocytes (about 40%), stromal cells / fibroblasts (about 55%), and cardiac progenitor cells (about 5%), while the "new medium" supported cardiomyocytes (about 40%), stromal cells / fibroblasts (about 25%), cardiac progenitor cells (about 5%), endothelial cells (about 20%), and smooth muscle cells (about 10%).

[0332] Table 1

[0333] The ratio of cell types present in human cardiac tissue patches cultured in new culture medium (New Culture Medium Replicate 1 and New Culture Medium Replicate 2) to the cell types present in human cardiac tissue patches cultured in old culture medium (Old Culture Medium Replicate 2), where the ratio is expressed as a percentage of the total population.

[0334] Cell Type New medium replicate 1 New medium replicate 2 Old medium replicate 2 CM 39.09465 49.47461 39.9776 SMC 11.31687 2.743724 0 Endothelial cells 17.48971 21.45359 0 Fibroblast / stromal cells 28.66941 21.45359 53.7514 Cardiac progenitor cells 3.429355 4.874489 6.270997

[0335] The characteristics of the "new culture" cardiomyocytes are 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 ( Fig. 9 In addition, a subpopulation of cardiomyocytes expressing proliferation markers was identified and was more prevalent in the new medium (approximately 3%) compared to the old medium (approximately 1%). Figure 4 ).

[0336] Fibroblast growth factor receptor FGFR1 is the major FGF receptor expressed in human cardiac tissue patches ( Fig.12 ), while the platelet-derived growth factor (PDGF) receptor PDGFRB is the main PDGF receptor expressed in human cardiac tissue patches ( Fig.13 ). FGFR1 is expressed in cardiomyocytes, stromal cells / fibroblasts, endothelial cells, and smooth muscle cells, whereas PDGRB is highly enriched in stromal cells / fibroblasts in human cardiac tissue patches.

[0337] Example 2. Hypoxia-tolerant cardiac tissue patch

[0338] Human heart tissue patches and control heart tissue patches were prepared according to the protocol described in the above section entitled Materials and Methods and subjected to hypoxia treatment as described above.

[0339] Briefly, after 5 days of culture in SF medium (also called “new medium”) or maturation medium MM, tissue patches were subjected to hypoxia for 20 h. Patches and their respective culture media ( Fig.14 A).

[0340] After 20 h of hypoxia, the nuclear intensity of SF human heart tissue patches was greater, indicating that there were more living cells ( Fig.14 B, Fig.14 C), the myocyte content was unchanged—stained with α-actin, these cells were both viable and nonviable (see Fig.14 B, 14D). There was less cell death in SF human heart tissue patches as reflected by lactate dehydrogenase in the culture medium ( Fig.14 E).

[0341] SF human heart tissue patches also showed enhanced glycolytic metabolism, which was demonstrated by increased lactate production before and after hypoxia ( Fig.14 F) and the ability to adapt to hypoxia (evident by an increase in the lactate to pyruvate ratio) ( Fig.14 G) to prove.

[0342] These data indicate that the cardiac tissue patch of the present invention has enhanced hypoxia tolerance compared to stem cell-derived cardiac tissue prepared according to existing methods, which suggests an increase in the ability to survive transplantation and implantation.

[0343] Example 3. In vivo functional validation of engineered cardiac tissue

[0344] The human cardiac tissue patch was sutured to the right ventricle as described, and cardiac function was monitored for 2 hours after implantation ( Figure 15-18 ). The human cardiac tissue patch was robust and durable and remained intact after implantation ( Fig.15 The shape of the pressure-volume loop did not change after patch implantation, and there was a slight rightward shift 90 minutes after patch implantation, which is consistent with a slight increase in right ventricular size after patch implantation ( Fig.16 After patch implantation, there is a highly linear relationship between right ventricular preload supplementation stroke work and end-diastolic volume ( Fig.17 ). After patch implantation, the slopes of these relationships changed minimally, indicating that right ventricular pumping performance was maintained after patch implantation ( Fig.18 No changes were observed in mean aortic BP (P = 0.71), RV output (P = 0.62), RV dP / dtmax (P = 0.52), or RV PRSWI (P = 0.61). Fig.18 Within 2 hours after patch implantation, heart rate (P = 0.006) and mean pulmonary artery blood pressure (P = 0.0002) showed a slight linear increase ( Fig.18 ), but these changes may be due to the prolonged effects of anesthesia rather than the effect of the patch. These data suggest that the human cardiac tissue patch is well tolerated and is not associated with any adverse acute events after in vivo implantation.

Claims

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

2. A method for producing engineered human cardiac tissue, the method comprising the following steps: i) mixing a cell suspension comprising a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocytes with a flowable hydrogel composition, wherein the cell suspension is in a serum-free medium; ii) loading the flowable hydrogel composition comprising the cell suspension into a mold, and incubating the hydrogel for a period of time and under conditions sufficient to solidify the hydrogel; as well as iii) culturing the hydrogel from step ii) in serum-free culture medium; The population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocytes are contacted with a FGFR1 agonist and a PDGFR agonist to provide engineered human cardiac tissue, wherein the tissue comprises cardiomyocytes, stromal cells / fibroblasts, and endothelial cells.

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

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

5. The method according to any one of claims 2-4, wherein the population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocytes are cultured in a serum-free medium comprising a FGFR1 agonist and a PDGFR agonist prior to mixing in step i).

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 comprising the FGFR1 agonist and the PDGFR agonist.

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

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

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

11. The method of any one of the preceding claims, wherein the engineered human cardiac tissue comprises: - At least about 30% of myocardial cells; - about 35% or less stromal cells / fibroblasts; and - About 30% or less endothelial cells.

12. The method of any one of the preceding claims, wherein the engineered human cardiac tissue comprises: - About 30% to about 70% of myocardial cells; - about 10% to about 35% stromal cells / fibroblasts; - about 5% to about 30% endothelial cells; - Up to about 15% of vascular smooth muscle cells; and - Up to about 10% of cardiac progenitor cells.

13. The method of any one of the preceding claims, 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 the preceding claims, wherein the PDGFR agonist is selected from PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC or PDGF-DD.

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

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

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

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

20. The method according to any one of the preceding claims, wherein the serum-free medium comprises a basal medium comprising albumin and transferrin.

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

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

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

25. The method of any of the preceding claims, wherein about 3% to about 10% of the cardiomyocyte population in the engineered human cardiac tissue are proliferative.

26. The method of any of the preceding claims, wherein the engineered human cardiac tissue exhibits glycolytic metabolism.

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

28. The method of any of the preceding claims, wherein the engineered human cardiac tissue exhibits a log2 relative gene expression of PKM >2 when normalized to TUBA1A expression.

29. The method of any of the preceding claims, wherein the engineered human cardiac tissue is tolerant to hypoxia.

30. The method according to any one of the preceding claims, wherein the engineered human cardiac tissue produces a lactate / pyruvate ratio of at least 1:1 in a cell culture medium obtained after 20 h of culture under hypoxic conditions.

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

32. The method according to any one of the preceding claims, wherein the cardiomyocytes express MMP1 + .

33. The method according to any one of the preceding claims, wherein the fibroblasts express MMP1 + 、EMP1 + 、FOXD1 + 、RAB27B + 、NR2F1 + 、F2RL1 + 、SPP1 + 、TMEM158 + PTHLH + 、PHLDA2 + 、MALL + , MYCT1 + ,DUSP4 + 、PLAU + 、TMEM156 + 、CD274 + 、MMP10 + 、ARRDC4 + 、RFX8 + MLPH + ,THBD + 、HHEX + 、VGF + 、OTULINL + IL33 + 、CA12 + 、C6orf141 + MFSD2A + and / or CARD10 + .

34. The method of any of the preceding claims, wherein the engineered human cardiac tissue exhibits a kinetic energy of less than 10 mN / mm 2 contractile activity.

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

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

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

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

39. The method according to any one of claims 2 to 38, wherein in step i), the cell suspension is mixed with a hydrogel to provide about 5×10 6 Cells / mL to about 100×10 6 Cell concentration in cells / mL.

40. The method of claim 39, wherein in step i), the cell suspension is mixed with a hydrogel to provide about 20×10 6 Cells / mL: about 50×10 6 Cell concentration in cells / mL.

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

42. The method of any one of the preceding claims, wherein the hPSCs are induced to differentiate towards a cardiac lineage by: a) hPSCs were cultured in a medium containing effective amounts of BMP4, activin A, FGF, GSK3 inhibitor Preparation, albumin and transferrin, insulin-minus serum-free basal medium supplemented for about 72 hours, wherein the medium is freshly cultured every day Base replacement; b) culturing the cells obtained in step a) in a basal medium comprising an effective amount of a Wnt signaling pathway inhibitor and the serum-free supplement in a) for about 72 hours, wherein the medium is replaced with fresh medium every day; c) treating the cells obtained in step b) with an effective amount of a Wnt signaling pathway inhibitor and a serum-free supplement containing albumin and transferrin, including insulin Culturing in a basal medium for about 7 days, wherein the medium is replaced with a fresh medium after the 2nd and 4th day of the about 7 days; d) the cells obtained in step c) are placed in a medium containing albumin, transferrin and pancreatic islets Cultured in serum-free basal medium supplemented with 1% glutathione for about 72 h; in, The basal medium used in each of steps a) to d) has a calcium concentration of less than about 1.2 mM; Thereby providing a cell suspension comprising a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocytes.

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

44. The method of any of the preceding claims, wherein the engineered human cardiac tissue is in the form of a tissue patch.

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

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

47. The method of claim 46, wherein the cardiomyopathy or cardiac tissue damage is due to acute or chronic stress, atherosclerotic vascular disease, ischemia, myocardial infarction, inflammatory disease, valvular heart disease, or myocarditis.

48. The method of claim 45, wherein the subject suffers from a congenital heart disease.

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

50. The method of any preceding claim, wherein the hPSC is an iPSC.

51. The method of claim 50, wherein the iPSCs are derived from a subject suffering from heart disease and / or to be implanted into the engineered human heart tissue.

52. The method of any of the preceding claims, wherein the hPSCs are embryonic stem cells.

53. An engineered human cardiac tissue comprising at least about 30% hPSC-derived cardiomyocytes, about 35% or less hPSC-derived stromal cells / fibroblasts, and at most about 30% or less hPSC-derived endothelial cells, wherein the above cells are located throughout a 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, wherein said cells are located throughout a hydrogel composition.

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 of any one of claims 53 to 55, wherein the engineered human cardiac tissue comprises: - About 30% to about 70% of myocardial cells; - about 10% to about 35% stromal cells / fibroblasts; and - About 5% to about 30% endothelial cells.

57. The engineered human cardiac tissue of any one of claims 53 to 56, wherein no more than about 10% of the cardiomyocytes in the engineered human cardiac tissue are proliferative.

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

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

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

61. The engineered human cardiac tissue of any one of claims 53 to 60, wherein the engineered human cardiac tissue is tolerant to hypoxia.

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

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

64. The engineered human cardiac tissue according to any one of claims 53 to 63, wherein the cardiomyocytes express MMP1 + .

65. The engineered human cardiac tissue according to any one of claims 53 to 64, wherein the fibroblasts express MMP1 + 、EMP1 + 、FOXD1 + 、RAB27B + 、NR2F1 + 、F2RL1 + 、SPP1 + 、TMEM158 + PTHLH + 、PHLDA2 + 、MALL + , MYCT1 + ,DUSP4 + 、PLAU + 、TMEM156 + 、CD274 + 、MMP10 + 、ARRDC4 + 、RFX8 + MLPH + ,THBD + 、HHEX + 、VGF + 、OTULINL + IL33 + 、CA12 + 、C6orf141 + MFSD2A + and / or CARD10 + .

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

67. The engineered human cardiac tissue of any one of claims 53 to 66, wherein the engineered human cardiac tissue exhibits a kinetic energy of less than 10 mN / mm 2 contractile activity.

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

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

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

71. The engineered human cardiac tissue of any one of claims 53 to 70, wherein the total number of cells in the tissue is about 20×10 6 cells to 100×10 6 cells.

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

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

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

76. A method for analyzing the biological effect of at least one test compound or bioactive agent on cardiomyocytes, comprising contacting the engineered human cardiac tissue of any one of claims 53 to 75 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.

77. The engineered human cardiac tissue of any one of claims 53 to 75 for use as an implant for treating diseased or damaged cardiac tissue in a subject in need thereof.

78. A method of treating diseased or damaged cardiac tissue in a subject in need thereof, comprising implanting in the subject the engineered human cardiac tissue of any one of claims 53 to 75.

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

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

81. The engineered human cardiac tissue, method or use of claim 80, wherein the cardiomyopathy or cardiac tissue damage is due to acute or chronic stress, atherosclerotic vascular disease, ischemia, myocardial infarction, inflammatory disease, valvular heart disease or myocarditis.

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

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

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