Methods for improving cell viability of cardiomyocytes - Patent application

Abstract

Methods and compositions relate to improving cell viability for in vitro differentiated cardiomyocytes. The addition of ROCK inhibitors to pluripotent stem cells in combination with factors that modulate the differentiation pathway improves the viability and fitness of in vitro differentiated cardiomyocytes. TIFF2025511730000033.tif124170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 63 / 327,650, filed April 5, 2022, and U.S. Provisional Application No. 63 / 413,471, filed October 5, 2022, the contents of each of which are incorporated by reference herein in their entirety.

[0002] Technical Field The technology described herein relates to in vitro differentiated cardiomyocytes and compositions and methods of using same. [Background technology]

[0003] background Methods involving the transplantation of cardiomyocytes to sites of cardiac injury can benefit from improved growth and survival of in vitro differentiated cardiomyocytes. Summary of the Invention

[0004] overview The methods and compositions provided herein relate to the discovery that ROCK inhibitors, such as Y-27632, improve cell viability during in vitro differentiation of cardiomyocytes.Improved cardiomyocyte viability is useful for improving cell and tissue engineering efforts.

[0005] In one aspect, provided herein is an in vitro differentiation method for preparing cardiomyocytes from stem cells, the method comprising contacting the stem cells with an inhibitor of Rho-associated coiled-coil-containing protein kinase (ROCK) throughout the entire period of differentiation of the stem cells into cardiomyocytes.

[0006] In one embodiment of this aspect or any other aspect, the ROCK inhibitor is Y-27632.

[0007] In another embodiment of this aspect or any other aspect, the ROCK inhibitor throughout the entire differentiation period promotes cell viability compared to differentiation where the ROCK inhibitor contact is not throughout the entire differentiation period.

[0008] In another embodiment of this aspect or any other aspect, the stem cells are pluripotent stem cells or cardiomyocyte progenitor cells.

[0009] In another embodiment of this aspect or any other aspect, the stem cells are human.

[0010] In another embodiment of this aspect or any other aspect, the stem cells are induced pluripotent stem cells.

[0011] In another embodiment of this aspect or any other aspect, the stem cell is an embryonic stem cell.

[0012] In another embodiment of this aspect or any other aspect, the stem cell is not an embryonic stem cell.

[0013] In another embodiment of this aspect or any other aspect, the method further includes contacting the stem cell or a cell undergoing differentiation from a stem cell with a small molecule selected from CHIR99021 and WIKI4.

[0014] In another embodiment of this aspect or any other aspect, the method further includes contacting the stem cell or a cell in the process of differentiating from a stem cell with CHIR99021 and WIKI4.

[0015] In another aspect, provided herein is a composition comprising a stem cell, a ROCK inhibitor, and one or both of CHIR99021 and WIKI4.

[0016] In one embodiment of this aspect or any other aspect, the ROCK inhibitor is Y-27632.

[0017] In another embodiment of this aspect or any other aspect, the stem cells are pluripotent stem cells or cardiomyocyte progenitor cells.

[0018] In another embodiment of this aspect or any other aspect, the stem cells are human.

[0019] In another embodiment of this aspect or any other aspect, the stem cells are induced pluripotent stem cells.

[0020] In another embodiment of this aspect or any other aspect, the stem cell is an embryonic stem cell.

[0021] In another embodiment of this aspect or any other aspect, the stem cell is not an embryonic stem cell.

[0022] In another aspect, provided herein is a transplantation composition comprising cardiomyocytes produced by the methods described herein.

[0023] In one embodiment of this aspect or any other aspect, the implant composition further comprises a pharma- ceutically acceptable carrier.

[0024] In another embodiment of this aspect or any other aspect, the implant composition further comprises a gel, scaffold, or matrix.

[0025] In another embodiment of this aspect or any other aspect, the gel, scaffold, or matrix is ​​biodegradable.

[0026] In another embodiment of this or any other aspect, the transplant composition comprises a solubilized basement membrane protein or a preparation thereof, an immunosuppressant, a pan-caspase inhibitor, an anti-apoptotic agent, IGF-1, and K. ATP Further comprising any one or more of the channel openers.

[0027] In another aspect, provided herein is a pharmaceutical composition comprising cardiomyocytes produced by the methods described herein in combination with a pharma- ceutically acceptable carrier.

[0028] In another aspect, provided herein is a pharmaceutical composition comprising an implantation composition described herein in combination with a pharma- ceutically acceptable carrier.

[0029] In another aspect, described herein is a method for improving cardiac function in a subject in need thereof, comprising administering cardiomyocytes produced by the methods described herein, or transplantation or pharmaceutical compositions described herein, to cardiac tissue of a subject in need thereof.

[0030] In one embodiment of this aspect or any other aspect, the subject is a human.

[0031] In another embodiment of this or any other aspect, the subject has damaged cardiac tissue resulting from acute or chronic injury.

[0032] In another embodiment of this aspect and any other aspect, provided herein is a cardiomyocyte produced by a method described herein, or a composition described herein, for use in a method for improving cardiac function in a subject in need thereof, comprising administering the cardiomyocyte produced by the method described herein or the composition described herein to cardiac tissue of the subject in need thereof.

[0033] In one embodiment of this aspect or any other aspect, the subject is a human.

[0034] In one embodiment of this or any other aspect, the subject has damaged cardiac tissue resulting from acute or chronic injury. [Brief description of the drawings]

[0035] [Figure 1A]Figure 1A shows the timeline, basal medium, medium supplements, and small molecules for differentiation of pluripotent stem cells into cardiomyocytes. The addition of the Wnt agonist CHIR is performed on day 3 of differentiation, whereas the Wnt antagonist Wiki is added 48 or 44 hours after CHIR addition. The timing of CHIR to WIKI addition is optimized for each cell line and / or reagent exchange to most efficiently induce cardiomyocyte differentiation.

[0036] [Figure 1B] In Figure 1B, we examine the difference between adding additional WIKI4 on day 6 of differentiation and changing the medium without adding WIKI4 on day 6. Both conditions induced efficient differentiation into mesoderm as indicated by expression of CD56 and PDGFRa analyzed by flow cytometry. When differentiation was continued using the conditions shown in Figure 1A, the condition without WIKI addition on day 6 resulted in efficient differentiation into cardiomyocytes by day 20 of differentiation as measured by intracellular flow cytometry for expression of cardiac troponin T (CTNT). Only a few EPCAM+ cells were detected, demonstrating the lack of epithelial type cells in the culture. Moreover, the absence of WIKI addition resulted in approximately 8E5 cells / mL in the final culture. In contrast, the condition with WIKI addition resulted in a large loss of cells and too few cells to measure either cell number or CTNT expression.

[0037] [Diagram 2] Figure 2 (left) shows cell concentration over time following addition or removal of small molecules and / or medium in cell cultures differentiating with 8uM CHIR without added WIKI (A), or with 8uM CHIR with added WIKI (B). Arrows indicate relevant process steps for orientation. Figure 2 right shows possible mechanisms for cell loss from day 7 onwards in conditions A or B.

[0038] [Diagram 3]Figure 3 (top) shows the current best practice (CBP) differentiation methodology, including the timeline, basal medium, medium supplements, and small molecule addition. Figure 3 (bottom) shows the experimental setup (1) for testing three different methodologies designed to improve cell yield compared to CBP. To improve total cell yield, we tested adding an additional medium change on the day just before significant cell loss begins (2) or adding a ROCK inhibitor at the time of adding the small molecule (CHIR or WIKI, respectively). To monitor cell fate in the experiments, cultures were monitored for cell number (in single cell suspensions and aggregates), glucose, lactate, and pH using a blood gas analyzer (BGA); apoptosis was analyzed by Annexin V staining; and mesoderm formation was further monitored by flow cytometry for CD56 and PDGFRa to determine if the addition of a ROCK inhibitor altered the developmental trajectory of the cells.

[0039] [Figure 4] Figure 4 (top) again shows the experimental design described in Figure 4 for reference. Figure 4 (bottom) shows cell counts at multiple time points from each condition during differentiation for experiment #19. 19-1 is the CBP condition, 19-2 is the condition with a medium change on day 6 (including the addition of 5uM Wiki), and 19-3 is the condition with the addition of ROCK inhibitor at the time of small molecule addition. On day 6 of differentiation, cell numbers in condition 19-3 rose to the point where the medium volume needed to be doubled, so cells were split into 19-3A and 19-3B. As shown in the figure, the CBP condition resulted in cell loss as expected, and the medium change on day 6 resulted in more rapid cell loss, whereas the addition of ROCK inhibitor not only rescued cell numbers / mL, but also resulted in significantly higher cell numbers and the need to double the medium volume.

[0040] [Diagram 5]Figure 5 shows an examination of whether cell loss in CBP conditions was due to cells that had shed from aggregates and were in single cell suspensions as opposed to aggregates, where cells were counted in both aggregates (A) and single cell suspensions (B) during differentiation in CBP conditions. Because there was no accumulation of cells in the single cell suspensions during differentiation, cells that had shed from aggregates could not explain cell loss under CBP conditions.

[0041] [Figure 6-1] Figure 6 shows that apoptosis correlates with cell loss detected in CBP conditions (19-1) and medium exchange (with Wiki) conditions (19-2) compared to ROCK inhibitor conditions (19-3). The figure also includes an additional set of experiments including addition of ROCK inhibitor at the time of addition of small molecules (20-1), or CBP conditions (20-2). As can be seen in the bottom right panel, conditions of cell loss showed elevated Annexin V staining indicative of apoptosis compared to conditions where cell numbers were rescued under ROCK conditions. The flow cytometry dot plot panels show three examples of low (19-3), medium (20-2), and high (19-2) Annexin V staining. [Figure 6-2] A continuation of Figure 6-1 is shown.

[0042] [Figure 7] Figure 7 shows that the purity of cardiomyocytes was similar in each condition tested, even though the cell yield from each experimental condition was significantly different. Using ES cells as a control, there is very little commitment to differentiation into epithelial cells as shown by flow cytometry staining for EPCAM and CD56. Moreover, cardiomyocyte purity as shown by intracellular staining for the cardiac isoform of troponin T (CTNT) and flow cytometry indicates that the purity of cardiomyocytes was >90% in each condition tested. At the end of the culture, all samples from experiments 19 (19-1, 19-3A, 19-3B) that had been completed for differentiation were pooled, resulting in a cell yield of approximately 1.2 billion cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Detailed Description definition The term "differentiate" or "differentiating" is a relative term indicating that a "differentiated cell" is a cell that has progressed further down the developmental pathway than its progenitor cell. Thus, in some embodiments, stem cells, as this term is defined herein, can differentiate into lineage-restricted progenitor cells (e.g., human cardiac progenitor cells or primitive streak mesoderm progenitor cells), which in turn can differentiate further down the pathway into other types of progenitor cells (e.g., tissue-specific precursors such as cardiomyocyte progenitors) and then into terminally differentiated cells (e.g., cardiomyocytes), which may or may not retain the ability to assume a characteristic role in a particular tissue type and to further proliferate.

[0044] The term "pluripotent" or "pluripotent stem cell (PSC)" as used herein refers to cells that have the ability to differentiate under different conditions into cell types characteristic of all three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent cells are primarily characterized by their ability to differentiate into all three germ layers, for example, using nude mice and teratoma formation assays. Although the preferred test for pluripotency is the demonstration of differentiation potential into cells of each of the three germ layers, pluripotency is also demonstrated by the expression of embryonic stem (ES) cell markers.

[0045] As used herein, the terms "induced pluripotent stem cells," "iPSCs," "hPSCs," and "human pluripotent stem cells" are used interchangeably herein and refer to pluripotent cells that are artificially derived (e.g., by reprogramming using one or more methods known in the art) from differentiated somatic cells. iPSCs are capable of self-renewal and differentiation into various types of mature cells, as well as cell fate-committed stem cells, including cells of the cardiac lineage.

[0046] As used herein, "in vitro differentiated cardiomyocytes" refers to cardiomyocytes that are generated in culture, but not necessarily undergo stepwise differentiation from progenitor cells, such as human embryonic stem cells, induced pluripotent stem cells, early mesoderm cells, lateral plate mesoderm cells, or cardiac precursor cells. Thus, while in vivo cardiomyocytes are ultimately derived from stem cells, i.e., during tissue or organ development, the stem cell-derived cardiomyocytes described herein are generated by in vitro differentiation from stem cells. Methods for differentiating stem cells into cardiomyocytes in vitro are known in the art and described elsewhere herein. In one aspect, cardiomyocytes are differentiated from pluripotent stem cells (e.g., PSC-CMs).

[0047] The term "isolated cell" as used herein refers to a cell that has been removed from the organism in which it was originally found, or the progeny of such a cell. Optionally, the cell has been cultured in vitro, for example in the presence of other cells. Optionally, the cell is later introduced into a second organism, or reintroduced into the organism from which it (or its ancestor) was isolated.

[0048] The term "substantially pure" with respect to a particular cell population refers to a cell population that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure with respect to the cells that make up the total cell population. That is, the terms "substantially pure" or "essentially purified" with respect to a cardiomyocyte population refers to a cell population that contains less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, and most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1%, respectively, of cells that are not cardiomyocytes.

[0049] The term "derived from" when used in reference to stem cells means that the stem cells were generated by reprogramming a differentiated cell to a stem cell phenotype. The term "derived from" when used in reference to a differentiated cell means that the cell is the result of differentiation of a stem cell, e.g., differentiation in vitro. As used herein, "iPSC-CM" or "induced pluripotent stem cell-derived cardiomyocytes" are used interchangeably to refer to cardiomyocytes derived from induced pluripotent stem cells. Similarly, "PSC-CM" or "pluripotent stem cell-derived cardiomyocytes" are used interchangeably to refer to cardiomyocytes derived from pluripotent stem cells. In some embodiments, the terms "hPSC-CM" or "human pluripotent stem cell-derived cardiomyocytes" are used interchangeably to refer to cardiomyocytes derived from human pluripotent stem cells.

[0050] As used herein, the term "isoform-selective" refers to the property of an agent, e.g., an inhibitor or activator of a factor, in which the agent inhibits or activates one isoform of the factor, but does not substantially inhibit or activate one or more different isoforms of the factor. In this context, "does not substantially inhibit or activate" means that an isoform selective inhibitor or activator is at least 100-fold more active (whether inhibitory or stimulatory) against a target or reference isoform than against other isoforms of the factor.

[0051] As used herein, the terms "implanting," "administering," or "engraftment" are used in the context of placing cells, e.g., cardiomyocytes produced as described herein, into a subject by a method or route that results in the introduced cells being at least partially localized at a desired site, such as a site of injury or repair, so that a desired effect occurs. Cells, e.g., cardiomyocytes, can be implanted directly into the heart or, alternatively, can be administered by any suitable route that results in delivery to a desired location in the subject, where at least a portion of the implanted cells or components of the cells remain viable. The survival period of the cells after administration to the subject can be as short as a few hours, e.g., 24 hours to a few days, to as long as several years, i.e., long-term engraftment. As one skilled in the art will recognize, long-term engraftment of cardiomyocytes is desirable because cardiomyocytes do not proliferate to an extent that allows the heart to heal from acute injury, including cell death. Thus, grafts can be used to replace cells lost during injury. Grafts can also be used to provide support to cardiac tissue during recovery from cardiac injury. For example, methods for improving engraftment or preventing engraftment arrhythmia, such as those described in US2020-0085880, WO2020 / 190739, or WO2021 / 163037, the contents of each of which are incorporated herein by reference in their entirety, can be combined with the methods and compositions described herein. In one aspect, engraftment is used to refer to cardiomyocytes that have formed functional gap junctions with endogenous cardiomyocytes in a subject.

[0052] As used herein, the term "contacting" when used in reference to a cell includes introducing a cell, agent, surface, scaffold, etc., into the cell in a manner that allows physical contact between the cell, agent, surface, scaffold, etc. and the cell.

[0053] As used herein, the term "cardiac disease" refers to the disease that affects the cardiac tissue of a subject.Non-limiting examples of cardiac disease include cardiomyopathy, cardiac arrhythmia, myocardial infarction, heart failure, cardiac hypertrophy, long QT syndrome, arrhythmogenic right ventricular dysplasia (ARVD), catecholamine-induced polymorphic ventricular tachycardia (CPVT), Barth syndrome, congenital defects, and Duchenne muscular dystrophy.

[0054] The terms "patient", "subject" and "individual" are used interchangeably herein and refer to animals, particularly humans, to which treatment, including preventative treatment, is provided. The term "subject" as used herein refers to humans and non-human animals. The terms "non-human animals" and "non-human mammals" are used interchangeably herein and include all vertebrates, such as mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals, such as chickens, amphibians, reptiles, and others. In one embodiment of any aspect, the subject is a human. In another embodiment of any aspect, the subject is an experimental animal or surrogate animal as a disease model. In another embodiment of any aspect, the subject is a livestock animal, including companion animals (e.g., dogs, cats, rats, guinea pigs, hamsters, and others). The subject may have previously been treated for a disease or has never previously been treated for a disease. The subject may have been previously diagnosed with the disease or may not have been previously diagnosed with the disease. The subject can be of any age, including, for example, a fetus, a newborn, a toddler, a child, an adolescent, an adult, a geriatric subject, etc.

[0055] The terms "reduce", "reduce", "reduction" or "inhibit" are all used herein to mean a statistically significant amount of reduction or decrease in a property, level, or other parameter. In some embodiments, "reduce", "reduce" or "reduce" or "inhibit" typically means a reduction of at least 10% compared to a reference level (e.g., in the absence of 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, "reduce" or "inhibit" does not encompass a complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease can preferably be down to a level that is accepted as within the normal range for individuals without a given disorder.

[0056] The terms "enhanced," "enhancement," "enhancement," or "enhancement" or "activation" are all used herein generally to mean an increase in a property, level, or other parameter by a statistically significant amount; for the avoidance of any doubt, the terms "enhanced," "enhancement," or "enhancement" or "activation" mean an increase of at least 10% compared to a reference level, for example, 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% increase, or an increase up to 100%, or any increase between 10-100% compared to a reference level, 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 increase, at least about 20-fold increase, at least about 50-fold increase, at least about 100-fold increase, or greater increase compared to a reference level.

[0057] As used herein, the terms "comprising" or "comprising" are used in reference to compositions, methods, and their respective components that are essential to the invention, but do not exclude the inclusion of unspecified elements, whether or not essential.

[0058] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic, novel or functional characteristics of that embodiment of the invention.

[0059] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in the description of the embodiment.

[0060] Exemplary apparatus, methods, and systems are described herein. The words "example," "exemplary," and "illustrative" should be understood as used herein to mean "serving as an example, example, or illustration." Any aspect or feature described herein as being "example," "exemplary," or "illustrative" should not necessarily be construed as preferred or advantageous over other aspects or features. The example aspects described herein are not meant to be limiting. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0061] Additionally, the particular arrangements shown in the drawings should not be considered limiting. It should be understood that other embodiments may include more or less of each element shown in a given drawing. Additionally, some of the illustrated elements may be combined or omitted. Still further, example embodiments may include elements not shown in the drawings. As used herein with respect to measurements, "about" means ±5%.

[0062] The details shown in this specification are by way of example and for the sole purpose of illustrative description of preferred embodiments of the invention, and are presented to provide what is believed to be the most useful and easily understood explanation of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, and the description given by way of figures and / or examples will make clear to those skilled in the art how some forms of the invention may be embodied in practice.

[0063] As used herein, and unless otherwise indicated, the terms "a" and "an" are employed to mean "one," "at least one," or "one or more." Unless otherwise required by context, as used herein, singular terms shall include pluralities and plural terms shall include the singular.

[0064] Unless the context requires otherwise, throughout this specification and the claims, the words "comprises," "including," and other terms are to be construed in an inclusive sense, i.e., "including but not limited to," as opposed to an exclusive or exhaustive sense. Words using the singular or plural also include the plural and singular, respectively. Additionally, when used in this application, the words "herein," "on," and "below," and words of similar meaning, shall refer to this application as a whole and not to any particular portions of this application.

[0065] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the art will recognize.

[0066] All references cited herein are incorporated by reference. Aspects of the present disclosure can be modified, as necessary, to employ the systems, functions, and concepts of the above references and applications to provide still further aspects of the present disclosure. These and other changes can be made to the present disclosure in light of the detailed description.

[0067] Particular elements of any of the above-mentioned embodiments can be combined with or substituted for elements of other embodiments. Moreover, the inclusion of particular elements in at least some of these embodiments can be optional, whereby further embodiments can include one or more embodiments that specifically exclude one or more of these particular elements. Moreover, although advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the present disclosure.

[0068] Although specific embodiments of the invention have been described herein for purposes of illustration, it will be appreciated that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

[0069] cardiovascular disease The methods described herein can be used to treat, ameliorate, prevent, or slow the progression of several diseases or symptoms thereof, such as those that result in pathological damage to cardiac structure and / or function (e.g., remodeling in response to heart failure).

[0070] Cardiovascular disease is the disease that affects the heart and / or circulatory system of a subject.Such heart disease or heart-related disease includes, but is not limited to, myocardial infarction, cardiac arrhythmia, heart failure, coronary artery sclerosis, cardiomyopathy, congenital heart defect (e.g., myocardial compaction disorder, septal defect, hypoplasia of the left heart), hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiac hypertrophy, myocarditis, arrhythmogenic right ventricular dysplasia (ARVD), long QT syndrome, catecholamine-induced polymorphic ventricular tachycardia (CPVT), Barth syndrome, valve stenosis, regurgitation, ischemia, fibrillation, polymorphic ventricular tachycardia, and muscular dystrophy or related heart disease such as Duchenne type, and cardiac hypertrophy.In general, the methods and compositions described herein are most useful for treating heart disease or heart disorder with contractile disorder, such as heart failure, myocardial infarction, and cardiomyopathy.

[0071] Symptoms of cardiovascular disease can include, but are not limited to, fainting, fatigue, shortness of breath, chest pain, and palpitations.Cardiovascular disease is generally diagnosed by physical examination, blood test, and / or electrocardiogram (EKG).An abnormal EKG is an indication that a subject has abnormal heart rhythm or cardiac arrhythmia.

[0072] In some embodiments of any of the aspects, the subject has or is at risk of having cardiovascular disease, cardiac damage, or a cardiac event.

[0073] Pluripotent stem cell source The methods and compositions described herein can be used to generate cardiomyocytes by in vitro differentiation, for example, from embryonic stem cells, pluripotent stem cells (e.g., induced pluripotent stem cells), or other stem cells that allow such differentiation and are modified as described herein.Various stem cells that can be used to prepare cardiomyocytes are described below.

[0074] Stem cells are cells that retain the ability to self-renew via mitotic cell division and can differentiate into more specialized cell types. Three broad types of mammalian stem cells include: embryonic stem (ES) cells found in blastocysts, induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells, and adult stem cells found in adult tissues. Other sources of pluripotent stem cells can include amniotic or placenta-derived stem cells. In the developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In the adult organism, stem and progenitor cells replenish specialized cells to act as a repair system for the body, but also maintain normal turnover of regenerative organs such as blood, skin or intestinal tissues. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

[0075] Cardiomyocytes useful for the methods and compositions described herein can be differentiated from both embryonic stem cells and induced pluripotent stem cells, among others.In one aspect, the compositions and methods provided herein use human cardiomyocytes differentiated from embryonic stem cells.Alternatively, in some aspects, the compositions and methods provided herein do not include the generation or use of human cardiogenic cells made from cells taken from living human embryos.

[0076] Embryonic stem cells and their recovery methods are well known in the art and will not be described in detail herein.If a cell possesses one or more of the unique characteristics of embryonic stem cells, thereby allowing the cell to be distinguished from other cells, the cell has the phenotype of embryonic stem cell.Exemplary characteristic embryonic stem cell characteristics include but are not limited to morphology, gene expression or marker profile, proliferation ability, differentiation ability, karyotype, response to specific culture conditions, etc.

[0077] Cells derived from embryonic sources can include embryonic stem cells or stem cell lines obtained from stem cell banks or other recognized depositories. Other means of producing stem cell lines include methods involving the use of blastomere cells from early embryos (approximately the 8-cell stage) prior to blastocyst formation. Such techniques correspond to preimplantation genetic diagnosis techniques routinely practiced in assisted reproduction clinics. Single blastomere cells are co-cultured with established ES cell lines and then separated from them to form fully competent ES cell lines.

[0078] Embryonic stem cells are considered undifferentiated when they are not committed to a specific differentiation lineage. Such cells exhibit morphological characteristics that distinguish them from differentiated cells of embryonic or adult origin. Undifferentiated embryonic stem (ES) cells are easily recognized by those skilled in the art and typically appear as colonies of cells with high nuclear / cytoplasmic ratios and prominent nucleoli in two dimensions under a microscope. In some aspects, the human cardiomyocytes described herein are not derived from embryonic stem cells or any other cells of embryonic origin.

[0079] Adult stem cells are stem cells derived from tissues of postnatal or postneonatal organisms or from adult organisms. Adult stem cells differ structurally from embryonic stem cells by the presence of epigenetic differences, such as differences in DNA methylation patterns, as well as markers that they do or do not express compared to embryonic stem cells.

[0080] In some embodiments, the methods and compositions described herein utilize cardiomyocytes differentiated in vitro from induced pluripotent stem cells. The advantage of using iPSCs to generate cardiomyocytes for the compositions described herein is that the desired human cardiomyocytes are derived from the very subject to which they are administered. That is, somatic cells can be obtained from the subject, reprogrammed into induced pluripotent stem cells, and then redifferentiated into human cardiomyocytes that are administered to the subject (e.g., autologous cells). Since the cardiomyocytes (or their differentiated progeny) are essentially derived from an autologous source, the risk of engraftment rejection or allergic response is reduced compared to the use of cells from another subject or group of subjects.

[0081] In some aspects, cardiomyocytes useful in the compositions described herein are derived from a non-autologous or allogeneic source.

[0082] In some embodiments, iPSCs are cells that have been reprogrammed, which is a process that changes or reverses the differentiation state of differentiated cells (e.g., somatic cells). In other words, reprogramming is a process that drives the differentiation of cells backwards to more undifferentiated or more primitive types of cells. Reprogramming of somatic cells to induced pluripotent stem cells is known in the art and will not be described in detail herein. iPS cells can be generated or derived from terminally differentiated somatic cells as well as adult stem cells or somatic stem cells. That is, non-pluripotent progenitor cells can be made pluripotent or multipotent by reprogramming.

[0083] To confirm the induction of pluripotent stem cells for use by the methods described herein, isolated clones can be tested for the expression of stem cell markers. Such expression in cells derived from somatic cells identifies cells as induced pluripotent stem cells. Stem cell markers can be selected from a non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one embodiment, cells expressing Oct4 or Nanog are identified as pluripotent. Methods for detecting the expression of such markers can include, for example, RT-PCR, and immunological methods such as Western blot or flow cytometry analysis to detect the presence of encoded polypeptides. In some embodiments, detection does not only involve RT-PCR, but also includes detection of protein markers. Although intracellular markers may best be identified via RT-PCR, cell surface markers are readily identified by, for example, immunocytochemistry.

[0084] The reprogrammed somatic cells disclosed herein express alkaline phosphatase (AP); ABCG2; stage-specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49 / 6E; ERas / ECAT5, E-cadherin; beta-III-tubulin; alpha-smooth muscle actin (alpha-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ESC-associated transcript 1 (ECAT1); ESG1 / DPPA5 / ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sal14; dysgerminocyte transcription factor (Utf1); Rex1; p53; G3PDH; Any number of pluripotent cell markers can be expressed, including telomerase including TERT; silent X chromosome genes; Dnmt3a; Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog / ECAT4; Oct3 / 4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; developmental pluripotency associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tcl1); DPPA3 / Stella; DPPA4; other general markers for pluripotency, etc. Other markers can include Dnmt3L; Sox15; Stat3; Grb2; β-catenin, and Bmi1. Such cells can also be characterized by downregulation of markers characteristic of the somatic cell from which the induced pluripotent stem cell was derived.

[0085] Methods for differentiating stem cells into cardiomyocytes in vitro are known in the art and / or described herein below.

[0086] ROCK inhibitors In some aspects of the technology described herein, the stem cells are contacted with at least one ROCK inhibitor during differentiation of the stem cells into cardiomyocytes.

[0087] Rho family small GTP-binding proteins are involved in the regulation of various aspects of cell motility, shape, proliferation, and apoptosis. Rho kinase, also called ROCK, is a serine / threonine kinase that is activated by GTP-binding Rho protein, which phosphorylates downstream targets in the ROCK pathway. Targets of phosphorylation include, but are not limited to, myosin light chain phosphatase, LIM kinase, adducin, and ezrin-radixon-moesin (ERM) proteins. Other or related functions of Rho kinase include, for example, regulating smooth muscle cell contraction, cell migration, and maintaining cell viability and morphology, in part by regulating stress fibers and focal adhesions. ROCK inhibitors are compounds that target ROCK and inhibit ROCK-mediated signal transduction. Examples of ROCK inhibitors specifically contemplated for use in the methods and compositions described herein include, but are not limited to, those in Table 1 shown below. In some embodiments, the ROCK inhibitor is Y-27632. Additional ROCK inhibitors that can be used alone or together, for example, with Y-27632, include those described in Liao et al. 2007. J Cardiovasc Pharmacol. 50(1): 17-24.

[0088] Table 1. Examples of ROCK inhibitors: Isoquinoline and pyridine / pyrrolopyridine-based ROCK inhibitors TIFF2025511730000002.tif253155TIFF2025511730000003.tif229155TIFF2025511730000004.tif21 3155TIFF2025511730000005.tif210155TIFF2025511730000006.tif243155TIFF2025511730000007.t if218155TIFF2025511730000008.tif233155TIFF2025511730000009.tif206155TIFF20255117300000 10.tif241155TIFF2025511730000011.tif224155TIFF2025511730000012.tif217155TIFF20255117300 00013.tif224155TIFF2025511730000014.tif217155TIFF2025511730000015.tif227155TIFF2025511 730000016.tif225155TIFF2025511730000017.tif227155TIFF2025511730000018.tif240155TIFF202 5511730000019.tif213155TIFF2025511730000020.tif243155TIFF2025511730000021.tif240155TIF F2025511730000022.tif244155TIFF2025511730000023.tif231155TIFF2025511730000024.tif159155

[0089] In one embodiment, the ROCK inhibitor Y-27632 has formula (I): It has the structure TIFF2025511730000025.tif41128.

[0090] In some embodiments, the ROCK inhibitor is contacted with stem cells in a differentiation program before differentiation begins, and then maintained in contact with the differentiating cells or cultures throughout the entire program. In vitro differentiation of stem cells into cardiomyocytes, as described further below, generally proceeds in the following order: ESC or iPSC>cardioblast>cardiomyoprogenitor>cardiomyocyte (see, e.g., US20170240861, the contents of which are incorporated herein by reference in their entirety; it is specifically contemplated that including a ROCK inhibitor during differentiation as described herein can benefit the viability of cardiomyocytes differentiated with a different protocol than that described in US20170240861). As described herein, the "day 0" of a differentiation program is the day that the first differentiation promoter contacts the stem cells. Thus, in some embodiments, the ROCK inhibitor can be contacted with the stem cells before the first differentiation promoter is added on day 0. The one or more ROCK inhibitors can be in contact with the differentiating cells for at least 0 days (day 1 of differentiation), at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or more. In some embodiments, the stem cells are in contact with at least one ROCK inhibitor throughout the entire course of differentiation of the stem cells into cardiomyocytes. In some embodiments, contact with at least one ROCK inhibitor is initiated when the Wnt modulator is added during the differentiation protocol and is maintained throughout the entire course of differentiation thereafter. In some embodiments, contact with at least one ROCK inhibitor is initiated when the Wnt modulator is added during the differentiation protocol and is maintained for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more of the time until differentiation into cardiomyocytes is complete. Fully or fully differentiated cardiomyocytes express cardiac troponin T (cTnT). Fully differentiated cardiomyocytes are also typically contractile.

[0091] When differentiation generally proceeds in the order of ESC or iPSC>cardiac mesoderm>cardiomyoprogenitor>cardiomyocyte, ROCK inhibitor can be present, for example, at the stage of differentiation of ESC or iPSC to cardiac mesoderm. In some embodiments, ROCK inhibitor is present for at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, for example 100%, during the differentiation of ESC or iPSC to cardiac mesoderm. Markers of cardiac mesoderm are known to those skilled in the art, and include, for example, mesoderm posterior 1 (Mesp1). ROCK inhibitor can also be present during the transition from cardiac mesoderm to cardiac mesoderm. In some embodiments, the ROCK inhibitor is present for at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, e.g., 100%, of the time during which the cardiogenic mesoderm cells differentiate into cardiac progenitor cells. Markers of cardiac progenitor cells are known to those of skill in the art and include, for example, G protein-coupled receptor 4 (GPR4), see, for example, Toran et al., Scientific Reports 9: 467 (2019). The ROCK inhibitor can also be present during the transition of cardiac progenitor cells to cardiomyocytes. In some embodiments, the ROCK inhibitor is present for at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, e.g., 100%, of the time during which the cardiac progenitor cells differentiate into cardiac myocytes. Markers of cardiac myocytes are known to those of skill in the art and include, for example, cardiac troponin T (cTnT).

[0092] In some embodiments, the ROCK inhibitor is present in the medium at a concentration of at least 0.1 μM to 100 mM, 0.1 μM to 95 mM, 0.1 μM to 90 mM, 0.1 μM to 85 mM, 0.1 μM to 80 mM, 0.1 μM to 75 mM, 0.1 μM to 70 mM, 0.1 μM to 65 mM, 0.1 μM to 60 mM, 0.1 μM to 55 mM, 0.1 μM to 50 mM, 0.1 μM to 45 mM, 0.1 μM to 40 mM, 0.1 μM to 35 mM, 0.1 μM to 30 mM, 0.1 μM to 25 mM, 0.1 μM to 20 mM, 0.1 μM to 15 mM, 0.1 μM to 25 mM, 0.1 μM to 20 ... .1μM~10mM, 0.1μM~5mM, 0.1μM~1mM, 0.1μM~0.9mM, 0.1μM~0.8mM, 0.1μM~0.7mM, 0.1μM~0.6mμM, 0.1μM~0.5mM, 0.1μM~0.4mM, 0.1μM~0.3mM , 0.1μM~0.2mM, 0.5μM~100mM, 1μM~100mM, 5μM~100mM, 10μM~100mM, 15μM~100mM, 20μM~100mM, 25μM~100mM, 30μM~100mM, 35μM~100mM, 40μ M~100mM, 45μM~100mM, 50μM~100mM, 55μM~100mM, 60μM~100mM, 65μM~100mM, 70μM~100mM, 75μM~100mM, 80μM~100mM, 85μM~100mM, 90μM~10 0mM, 95μM~100mM, 0.1mM~100mM, 0.1mM~95mM, 0.1mM~90mM, 0.1mM~85mM, 0.1mM~80mM, 0.1mM~75mM, 0.1mM~70mM, 0.1mM~65mM, 0.1mM~60mM , 0.1mM~55mM, 0.1mM~50mM, 0.1mM~45mM, 0.1mM~40mM, 0.1mM~35mM, 0.1mM~30mM, 0.1mM~25mM, 0.1mM~20mM, 0.1mM~15mM, 0.1mM~10mM, 0.1 mM~5mM, 0.1mM~1mM, 0.1mM~0.9mM, 0.1mM~0.8mM, 0.1mM~0.7mM, 0.1mM~0.6mM, 0.1mM~0.5mM, 0.1mM~0.4mM, 0.1mM~0.3mM, 0.1mM~0.2mM, 0.5mM~100mM, 1mM~100mM, 5mM~100mM, 10mM~100mM, 15mM~100mM, 20mM~100mM, 25mM~100mM, 30mM~100mM, 35mM~100mM, 40mM~100mM, 45mM ~100mM, 50mM~100mM, 55mM~100mM, 60mM~100mM, 65mM~100mM, 70mM~100mM, 75mM~100mM, 80mM~100mM, 85mM~100mM, 90mM~100mM, 95mM~ 100mM, 0.1μM~100μM, 0.1μM~95μM, 0.1μM~90μM, 0.1μM~85μM, 0.1μM~80μM, 0.1μM~75μM, 0.1μM~70μM, 0.1μM~65μM, 0.1μM~60μM, 0.1μM ~55μM, 0.1μM~50μM, 0.1μM~45μM, 0.1μM~40μM, 0.1μM~35μM, 0.1μM~30μM, 0.1μM~25μM, 0.1μM~20μM, 0.1μM~15μM, 0.1μM~10μM, 0.1μM~5 μM, 0.1μM~1μM, 0.1μM~0.9μM, 0.1μM~0.8μM, 0.1μM~0.7μM, 0.1μM~0.6μM, 0.1μM~0.5μM, 0.1μM~0.4μM, 0.1μM~0.3μM, 0.1μM~0.2μM, 0. 5μM~100μM, 1μM~100μM, 5μM~100μM, 10μM~100μM, 15μM~100μM, 20μM~100μM, 25μM~100μM, 30μM~100μM, 35μM~100μM, 40μM~100μM, 45μM~ It is present in concentrations of 100μM, 50μM-100μM, 55μM-100μM, 60μM-100μM, 65μM-100μM, 70μM-100μM, 75μM-100μM, 80μM-100μM, 85μM-100μM, 90μM-100μM, 95μM-100μM, 0.5μM-40μM, 1μM-30μM, 5μM-20μM, 1μM-10μM, 5μM-10μM, 5μM-40μM, 5μM-30μM, 5μM-25μM, 5μM-20μM, 1μM-10μM, 1μM-5μM.

[0093] ROCK inhibitor Y-27632 is commercially available. Exemplary sources include, but are not limited to, Y27632 2HCL (Cat. No. S1049, Selleck Chem, Houston, TX); Y-27632 (Cat. No. B1293, APExBio, Houston, TX); ROCK inhibitor (Y-27632) (Cat. No. SCM075, Sigma Aldrich, St. Louis, MO). Additional examples can be found in J. Med. Chem. 2016, 59, 2269-2300, which is incorporated herein in its entirety.

[0094] ROCK inhibitors can induce stem cell aggregation. When cell concentration and / or ROCK inhibitor concentration are high, ROCK inhibitors may induce the formation of large cell clusters, and cells inside the clusters may experience conditions that are sufficiently different from the conditions outside or nearby that affect cell viability and / or differentiation. Cells inside the large clusters may lose viability, which affects the total yield of differentiated cells. Without wishing to be bound by theory, cells inside such clusters may lose or reduce contact with ROCK inhibitors, and therefore do not benefit from the protective effect of ROCK inhibitors demonstrated herein. On the other hand, when cell concentration is low, cells may not be able to aggregate or adhere to substrates, and lose viability. To alleviate these problems, cell concentration and / or ROCK inhibitor concentration can be titrated. For example, excessive cell aggregation can be addressed by lowering the dose of ROCK inhibitor (e.g., less than 20 μM inhibitor for 5-5000 cells), and insufficient aggregation or adhesion due to low cell numbers (e.g., during single cell cloning) can be addressed with higher doses of ROCK inhibitor (e.g., greater than 0.1 μM inhibitor for 5-5000 cells).

[0095] Wnt Signaling (Activators and Inhibitors) A hallmark of classical Wnt signaling activation is an increase in the levels of the protein β-catenin. β-catenin, a transcription factor, is constitutively produced and exists as a pool of monomeric protein in the cytoplasm. (See, e.g., Papkoff, J. et al., Mol. Cell Biol. 1996; 16: 2128-2134). The main mechanism for controlling the cytoplasmic levels of β-catenin is by direct physical degradation when recruited to a large multiprotein complex (the "degradation complex"). After formation of the complex, it is stabilized by GSK3β-mediated phosphorylation of PP2A as well as the protein components Axin and APC. GSK3β then phosphorylates β-catenin, thereby making it recognizable by β-transducin repeat-containing protein (β-TrCP), which targets β-catenin for ubiquitination and proteosomal degradation. (See, e.g., Aberle et al., EMBO J. 1997; 16: 3797-804; Latres et al., Oncogene 1999; 18: 849-54; Liu et al., Proc. Natl. Acad. Sci. USA 1999; 96: 6273-8). An alternative degradation pathway has been shown to involve ubiquitination induced by complexation with Siah-1 and the C-terminus of APC. (Matsuzawa et al., Mol Cell 2001; 7: 915-926; Liu et al., Mol. Cell 2001; 7: 927-936). In addition to its role as a transcription factor, β-catenin is further involved in cell adhesion. [Nelson et al., Science 2004; 303: 1483-1487; Ilyas et al., J. Pathol. 1997; 182: 128-137.

[0096] β-catenin can be found at cell surface sites of cell-cell contact known as adherens junctions, where β-catenin is complexed with E-cadherin. Thus, disruption of the E-cadherin-catenin complex can increase the cytoplasmic levels of free β-catenin, thereby stimulating transcriptional activity. Activation of the cell surface receptors cRON, epidermal growth factor receptor (EGFR) and c-ErbB2 by liberating β-catenin can also stimulate classical Wnt signaling. Other signaling pathways can either activate or promote the effects of Wnt signaling. For example, signaling via insulin-like growth factor (IGF) can activate Wnt signaling by "soaking up" available GSK3β, thereby preventing the formation of the "degradation complex."

[0097] As used herein, the term "Wnt agonist" refers to any agent that activates the Wnt / β-catenin pathway or inhibits the activity and / or expression of an inhibitor of Wnt / β-catenin signaling, such as an antagonist or inhibitor of GSK-3β activity.As used herein, a Wnt activator can enhance signaling through the Wnt / β-catenin pathway at any point along the pathway, such as, but not limited to, increasing the expression and / or activity of Wnt, or β-catenin or Wnt-dependent genes and / or proteins, and decreasing the expression and / or activity of endogenous inhibitors of Wnt and / or β-catenin, or decreasing the expression and / or activity of endogenous inhibitors of components of the Wnt / β-catenin pathway, such as decreasing the expression of GSK-3β.

[0098] Some non-limiting examples of Wnt pathway agonists include GSK-3β inhibitors (e.g., CHIR99201), TCS2002, TWS119, SB-216763, BIO, and lithium chloride. In some embodiments, exemplary Wnt activators include CHIR99201, TCS2002, TWS119, SB-216763, BIO, and lithium chloride.

[0099] In one aspect, the CHIR99021 GSK-3β inhibitor has formula (II): It has the structure TIFF2025511730000026.tif40128.

[0100] In another embodiment, the GSK-3β inhibitor BIO has formula (III): It has a structure of TIFF2025511730000027.tif32128.

[0101] In another embodiment, the GSK-3β inhibitor SB-216763 has formula (IV): TIFF2025511730000028.tif has a structure of 37128.

[0102] Without wishing to be bound by theory, Wnt proteins and their cognate receptors signal through at least two distinct intracellular pathways. The "classical" Wnt signaling pathway (also referred to herein as the Wnt / β-catenin pathway) involves Wnt signaling through β-catenin to activate transcription via TCF-related proteins (van de Wetering et al. (2002) Cell 109 Suppl: S13-9; Moon et al. (2002) Science 296(5573): 1644-6). There is an alternative non-classical pathway in which Wnt activates protein kinase C (PKC), calcium / calmodulin-dependent kinase II (CaMKII), JNK and Rho-GTPase (Veeman et al. (2003) Dev Cell 5(3): 367-77), and is often involved in the control of cell polarity.

[0103] As used herein, the term "Wnt antagonist" or "Wnt inhibitor" refers to any agent that inhibits the Wnt / β-catenin pathway or enhances the activity and / or expression of the inhibitor of Wnt / β-catenin signaling, for example, an activator or enhancer of GSK-3β activity.As used herein, a Wnt inhibitor can suppress the Wnt / β-catenin pathway at any point along the pathway, for example, but not limited to, by decreasing the expression and / or activity of Wnt or β-catenin or Wnt-dependent genes and / or proteins, and by increasing the expression and / or activity of endogenous inhibitors of Wnt and / or β-catenin, or by increasing the expression and / or activity of endogenous inhibitors of Wnt / β-catenin pathway components, for example, by increasing the expression of GSK-3β or by inhibiting tankyrase enzyme.

[0104] Some non-limiting examples of Wnt antagonists include the Wnt pathway inhibitors WIKI4, XAV939 (tankyrase inhibitor), E7449, AZ6102, and JW55.

[0105] In another embodiment, the WIKI4 tankyrase inhibitor has formula (V): TIFF2025511730000029.tif has a structure of 39128.

[0106] In another embodiment, the XAV-939 tankyrase inhibitor has formula (VI): It has the structure TIFF2025511730000030.tif25128.

[0107] In another embodiment, the IWR tankyrase inhibitor has formula (VII): It has a structure of TIFF2025511730000031.tif31128.

[0108] In another embodiment, the JW55 tankyrase inhibitor has formula (VIII): TIFF2025511730000032.tif has a structure of 33128.

[0109] In some embodiments, useful dose ranges for Wnt antagonists (e.g., WIKI4) are 0.5-5 μM, 0.5-4 μM, 0.5-3 μM, 0.5-2 μM, 0.5-1 μM, 4-5 μM, 3-5 μM, 2-5 μM, 1 μM, 0.5-2 μM, 0.75-2 μM, 0.9 μM-2 μM, or any range therebetween.

[0110] In some embodiments, the dose of the Wnt antagonist is, for example, at least 20 ng / mL, at least 30 ng / mL, at least 40 ng / mL, at least 50 ng / mL, at least 60 ng / mL, at least 70 ng / mL, at least 80 ng / mL, at least 90 ng / mL, at least 100 ng / mL, at least 110 ng / mL, at least 120 ng / mL, at least 130 ng / mL, at least 140 ng / mL, at least 150 ng / mL, at least 160 ng / mL, at least 170 ng / mL, at least 180 ng / mL, at least 190 ng / mL, at least 200 ng / mL, or more.

[0111] It has been demonstrated herein that the cell loss in in vitro cardiomyocyte differentiation associated with the use of Wnt modulators, including Wnt activator CHIR99021 and Wnt inhibitor WIKI4, can be reduced by including one or more ROCK inhibitors during differentiation.It is particularly contemplated that any in vitro cardiomyocyte differentiation program that uses one or more Wnt modulators as described herein or known in the art can benefit from the use of one or more ROCK inhibitors as described herein or known in the art.The in vitro differentiation of stem cells into cardiomyocytes is further described herein below.

[0112] Immunosuppressants As used herein, an "immunosuppressant" is a drug that inhibits or blocks the activity of the immune system. There are various groups of immunosuppressants, including, but not limited to, glucocorticoids, cytostatics, certain antibodies, drugs that act on immunophilins, and other drugs. Immunosuppressants can help limit or avoid acute transplant rejection of organs (e.g., heart), tissues, or cells (e.g., cardiomyocytes). In some embodiments, the immunosuppressant is selected from glucocorticoids and drugs that act on immunophilins. In some embodiments, the immunosuppressant includes cyclosporine A (anti-inflammatory, calcineurin inhibitor), corticosteroids (prednisone, methylprednisolone, etc.), mycophenolate mofetil (MMF), and / or tacrolimus.

[0113] Apoptosis inhibitors As used herein, an "apoptosis inhibitor" is a molecule, drug, or protein that blocks or inhibits programmed cell death or apoptosis. Apoptosis inhibitors include, for example, caspase inhibitors. Caspases are a family of cysteine ​​proteases that play an essential role in apoptosis. Caspase inhibitors can include selective inhibitors that inhibit only one or a limited subset of specific caspases, or broad-spectrum or so-called "pan-caspase" inhibitors that, as the name implies, more broadly inhibit caspase enzymes. Selective caspase inhibitors include, as non-realistic examples, vernacasan (VX-765; caspase 1 inhibitor), Z-DEVD-FMK (caspase 3 inhibitor) and Z-IETD-FMK (caspase 8 inhibitor), among others. Pan-caspase inhibitors include, but are not limited to, Q-VD-Oph, Z-VAD-FMK, emricasan, and Z-VAD(OH)-FMK, among others.Other apoptosis inhibitors target different components of the apoptosis pathway, such as, for example, cytochrome C inhibitors (e.g., minocycline, methazolimid, gamma-tocotrienol, 3-hydroxypropyl-triphenylphosphonium-conjugated imidazole substituted oleic acid (TPP-IOA), 3-hydroxypropyl-triphenylphosphonium-conjugated imidazole substituted stearic acid (TPP-ISA), and TPP-6-ISA, among others), BH3 interacting domain death agonists, BIID inhibitors (e.g., BI-6C9, TC9-305, BI-11A7, 3-o-trilthiazolidine-2,4-dione, among others), Fas inhibitors (e.g., KR-33493, RKTS-33, dichlorovinyl dimethyl phosphate (DDVP), geldanamycin, vitamin D3, cilazapril, M50054, vanillic acid, NCX-1000, among others), tumor necrosis factor (TNF) inhibitors (e.g., infliximab, etanercept, adalimumab, golimumab, and Abdelhafez, among others). et al., chapter "Apoptotic Inhibitors as Therapeutic Targets for Cell Survival" in Cytotoxicity - Definition, Identification, and Cytotoxic Compounds, ISBN 978-1-78984-755-0 (2019)), inhibitors of p53 upregulated regulator of apoptosis (PUMA) (e.g., CLZ-8, among others), and Bax inhibitors (e.g., PD98059, vitamin E, tanshinones, among others).

[0114] K-ATP channel drugs As used herein, "potassium channel opener" or "K-ATP channel drug" is a type of drug that promotes ion permeation through potassium channel. There are 4 classes and 14 subclasses of K-ATP channel drugs. Examples of K-ATP channel drugs include, but are not limited to, pinacidil, diazoxide, nicorandil, and BNS180448.

[0115] In vitro differentiation of cardiomyocytes The methods and compositions described herein can use grafts containing in vitro differentiated cardiomyocytes. Methods for differentiation of cardiomyocytes from ESCs or iPSCs are known in the art. See, for example, LaFlamme et al., Nature Biotech 25:1015-1024 (2007) (describes differentiation of cardiomyocytes from ESCs), and Batalov & Feinberg, Biomarker Insights 10: 71-76 (2015) (reviews various approaches for differentiation of cardiomyocytes from ESCs and iPSCs). Exemplary methods for generating cardiomyocytes or cardiomyocyte progenitors from pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells) are also described, for example, in US2020-0085880, the entire contents of which are incorporated herein by reference.

[0116] Various other methods of differentiating stem cells into cardiomyocytes are known to those of skill in the art, see, e.g., Lyra-Leite et al., STAR Protoc. 3: 101560 (2022), "A review of protocols for human iPSC culture, cardiac differentiation, subtype specification, maturation, and direct reprogramming", which is incorporated herein by reference, and for a review see the entire reference and the references cited therein, in particular Tables 1 and 2 therein.

[0117] These approaches use various elements and conditions to activate and guide the differentiation programs that result in their respective cell types, the pathways and certain elements involved are described below.

[0118] In certain embodiments, the stepwise differentiation of ESCs or iPSCs into cardiomyocytes proceeds in the following order: ESCs or iPSCs>cardiac mesoderm>cardiomyocyte precursor cells>cardiomyocytes (see, e.g., US 20170240861, the entire contents of which are incorporated herein by reference).

[0119] As will be appreciated by those skilled in the art, in vitro differentiation of cardiomyocytes produces as an end result cells having phenotypic and morphological characteristics of cardiomyocytes, but the differentiation steps of in vitro differentiation need not be the same as those that occur naturally in an embryo. That is, during differentiation into cardiomyocytes, it is specifically contemplated herein that the stepwise differentiation approach utilized to produce such cells does not need to proceed through every precursor cell type specified during embryogenesis, but can essentially "skip" certain developmental periods that occur during embryogenesis.

[0120] The technology can be as described in any of the following numbered sections.

[0121] Item 1. An in vitro differentiation method for preparing cardiomyocytes from stem cells, comprising contacting the stem cells with an inhibitor of Rho-associated coiled-coil-containing protein kinase (ROCK) throughout the entire period of differentiation of the stem cells into cardiomyocytes.

[0122] Item 2. The method according to item 1, wherein the ROCK inhibitor is an isoquinoline or pyridine / pyrrolopyridine-based ROCK inhibitor.

[0123] Item 3. The method according to item 1, wherein the ROCK inhibitor is an indazole-based ROCK inhibitor.

[0124] Item 4. The method of item 1, wherein the ROCK inhibitor is a pyridine-, pyrrolopyridine-, or pyrimidine-based ROCK inhibitor.

[0125] Item 5. The method according to item 1, wherein the ROCK inhibitor is a pyrazole-based ROCK inhibitor.

[0126] Item 6. The method according to item 1, wherein the ROCK inhibitor is a methylenephenyl-substituted pyrazole-based ROCK inhibitor.

[0127] Item 7. The method according to item 1, wherein the ROCK inhibitor is a benzimidazole, benzothiazole, indole, or indazole-substituted pyrazole derivative ROCK inhibitor.

[0128] Item 8. The method according to item 1, wherein the ROCK inhibitor is a quinazolinone-substituted pyrazole derivative ROCK inhibitor.

[0129] Item 9. The method of item 1, wherein the ROCK inhibitor is a thiophene-substituted pyrimidine-based or aminofurazan-based ROCK inhibitor.

[0130] Item 10. The method according to item 1, wherein the ROCK inhibitor is a 6- or 7-substituted isoquinoline or isoquinolinone-based ROCK inhibitor.

[0131] Item 11. The method according to item 1, wherein the ROCK inhibitor is a boron derivative ROCK inhibitor.

[0132] Item 12. The method according to item 1, wherein the ROCK inhibitor is a soft ROCK inhibitor.

[0133] Item 13. The method according to item 1, wherein the ROCK inhibitor is an isoform-selective ROCK-II inhibitor.

[0134] Item 14. The method according to item 1, wherein the ROCK inhibitor is Y-27632.

[0135] Item 15. The method of any one of the preceding items, wherein the presence of the ROCK inhibitor throughout the entire differentiation period promotes cell viability compared to differentiation where the ROCK inhibitor contact is not throughout the entire differentiation period.

[0136] Item 16. The method according to any one of the preceding items, wherein the stem cells are pluripotent stem cells or cardiac progenitor cells.

[0137] Item 17. The method of any one of the preceding items, wherein the stem cells are human.

[0138] Item 18. The method according to any one of the preceding items, wherein the stem cells are induced pluripotent stem cells.

[0139] Item 19. The method according to any one of the preceding items, wherein the stem cells are embryonic stem cells.

[0140] Item 20. The method according to any one of items 1 to 19, wherein the stem cells are not embryonic stem cells.

[0141] Item 21. The method according to any one of the preceding items, further comprising contacting the stem cell, or a cell in the process of differentiating from the stem cell, with a small molecule selected from CHIR99021 and WIKI4.

[0142] Item 22. The method according to any one of the preceding items, further comprising contacting the stem cell, or a cell in the process of differentiating from the stem cell, with CHIR99021 and WIKI4.

[0143] Item 23. A composition comprising stem cells, a ROCK inhibitor, and one or both of CHIR99021 and WIKI4.

[0144] Item 24. The composition according to Item 23, wherein the ROCK inhibitor is Y-27632.

[0145] Item 25. The composition according to Item 23 or 24, wherein the stem cells are pluripotent stem cells or cardiac progenitor cells.

[0146] Item 26. The composition according to any one of Items 23 to 25, wherein the stem cells are human.

[0147] Item 27. The composition according to any one of Items 23 to 26, wherein the stem cells are induced pluripotent stem cells.

[0148] Item 28. The composition according to any one of Items 23 to 27, wherein the stem cells are embryonic stem cells.

[0149] Item 29. The composition according to any one of Items 23 to 27, wherein the stem cells are not embryonic stem cells.

[0150] Item 30. A transplant composition comprising cardiomyocytes produced by the method according to any one of items 1 to 22.

[0151] Item 31. The transplant composition according to Item 30, further comprising a pharma- ceutically acceptable carrier.

[0152] Item 32. The implant composition according to item 30 or 31, further comprising a gel, scaffold, or matrix.

[0153] Item 33. The implant composition of item 32, wherein the gel, scaffold, or matrix is ​​biodegradable.

[0154] Item 34. Solubilized basement membrane protein or preparation thereof, immunosuppressant, pan-caspase inhibitor, anti-apoptotic agent, IGF-1, and K ATP Item 34. The transplant composition according to any one of items 30 to 33, further comprising any one or more of the channel opening agents.

[0155] Item 35. A pharmaceutical composition comprising cardiomyocytes produced by the method according to any one of items 1 to 28, or the transplant composition according to any one of items 30 to 34, in combination with a pharma- ceutically acceptable carrier.

[0156] Item 36. A method for improving cardiac function in a subject in need thereof, comprising administering cardiomyocytes produced by the method according to any one of items 1 to 22, or the composition according to any one of items 23 to 35, to cardiac tissue of a subject in need thereof.

[0157] Item 37. The method of item 36, wherein the subject is a human.

[0158] Item 38. The method of item 36 or 37, wherein the subject has damaged cardiac tissue due to acute or chronic injury.

[0159] Item 39. Cardiomyocytes produced by the method according to any one of items 1 to 22, or the composition according to any one of items 23 to 35, for use in a method for improving cardiac function in a subject in need thereof, comprising administering the cardiomyocytes produced by the method according to any one of items 1 to 22, or the composition according to any one of items 23 to 35, to cardiac tissue of a subject in need thereof.

[0160] Item 40. The use according to Item 39, wherein the subject is a human.

[0161] Item 41. The method of item 39 or 40, wherein the subject has damaged cardiac tissue due to acute or chronic injury.

[0162] Clause 42. The composition of clause 23, wherein contact with at least one ROCK inhibitor is initiated when CHIR99021 is added during the differentiation protocol, beginning at least on day 1 of differentiation of the stem cells.

[0163] Clause 43. The composition of clause 23, wherein contact with at least one ROCK inhibitor is initiated when WIKI4 is added during the differentiation protocol, beginning at least on day 3 of differentiation of the stem cells. EXAMPLES

[0164] Example 1: The differentiation of pluripotent stem cells (PSCs) into cardiomyocytes involves multiple steps of expansion and differentiation of PSCs into desired lineages. As presented herein, PSCs are thawed from a suspension PSC bank (PMID: 26318718), and aggregated in suspension culture with the addition of ROCK inhibitor Y-27632, which has been shown to enable the survival of dissociated PSCs (PMID: 17529971). After expansion, cells are induced to differentiate into mesoderm and then into cardiomyocytes by modulating Wnt signaling using CHIR99021 (PMID: 23257984) to induce differentiation into mesoderm, followed by the use of WIKI4, a small molecule Wnt inhibitor, to specify mesoderm into cardiomyocyte lineage (PMID: 23227175) (Figure 3).

[0165] Example 2: A challenge with the methodology described herein is the significant cell loss during the addition of CHIR99021 and WIKI4. The addition of Y-27632 at each stage of small molecule Wnt modulation dramatically improved the cell yield of differentiation cultures. For example, the addition of Y-27632 in combination with CHIR99021 and WIKI4 showed more than double the yield of progenitor cardiomyocytes when measured on day 6 of the differentiation process (1.1E6 cells / mL in the presence of Y-27632 vs. 4.7E5 cells / mL in the absence of Y-27632) (Figure 4). Moreover, on day 13 of differentiation, cultures with the addition of Y-27632 yielded nearly four times as many cardiomyocytes as cultures without the addition. Y-27632 was then added to all cultures during differentiation (Figure 4).

[0166] All heading and section designations are used for clarity and reference purposes only and should not be construed as limiting in any way For example, those skilled in the art will recognize the utility of combining various aspects from different headings and sections as appropriate, consistent with the spirit and scope of the invention(s) described herein.

[0167] All references cited in this specification are hereby incorporated by reference in their entirety in this specification to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety in this specification.

[0168] As will be apparent to those skilled in the art, many modifications and variations of this application can be made without departing from its spirit and scope. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the scope of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. An in vitro differentiation method for preparing cardiomyocytes from stem cells, comprising contacting the stem cells with an inhibitor of Rho-associated coiled-coil-containing protein kinase (ROCK) throughout the entire period of differentiation of the stem cells into cardiomyocytes.

2. 2. The method of claim 1, wherein the ROCK inhibitor is an isoquinoline or a pyridine / pyrrolopyridine based ROCK inhibitor.

3. 2. The method of claim 1, wherein the ROCK inhibitor is an indazole-based ROCK inhibitor.

4. 2. The method of claim 1, wherein the ROCK inhibitor is a pyridine, pyrrolopyridine, or pyrimidine-based ROCK inhibitor.

5. 2. The method of claim 1, wherein the ROCK inhibitor is a pyrazole-based ROCK inhibitor.

6. 2. The method of claim 1, wherein the ROCK inhibitor is a methylenephenyl-substituted pyrazole-based ROCK inhibitor.

7. 2. The method of claim 1, wherein the ROCK inhibitor is a benzimidazole, benzothiazole, indole, or indazole substituted pyrazole derivative ROCK inhibitor.

8. 2. The method of claim 1, wherein the ROCK inhibitor is a quinazolinone substituted pyrazole derivative ROCK inhibitor.

9. 2. The method of claim 1, wherein the ROCK inhibitor is a thiophene-substituted pyrimidine-based or aminofurazan-based ROCK inhibitor.

10. 2. The method of claim 1, wherein the ROCK inhibitor is a 6- or 7-substituted isoquinoline or isoquinolinone based ROCK inhibitor.

11. 2. The method of claim 1, wherein the ROCK inhibitor is a boron derivative ROCK inhibitor.

12. The method of claim 1, wherein the ROCK inhibitor is a soft ROCK inhibitor.

13. The method of claim 1, wherein the ROCK inhibitor is an isoform-selective ROCK-II inhibitor.

14. 2. The method of claim 1, wherein the ROCK inhibitor is Y-27632.

15. The method of any one of the preceding claims, wherein the presence of the ROCK inhibitor throughout the entire differentiation period promotes cell viability compared to differentiation where the ROCK inhibitor contact is not throughout the entire differentiation period.

16. The method of any one of the preceding claims, wherein the stem cells are pluripotent stem cells or cardiac progenitor cells.

17. The method of any one of the preceding claims, wherein the stem cells are human.

18. The method of any one of the preceding claims, wherein the stem cells are induced pluripotent stem cells.

19. The method of any one of the preceding claims, wherein the stem cells are embryonic stem cells.

20. The method of any one of claims 1 to 19, wherein the stem cells are not embryonic stem cells.

21. The method of any one of the preceding claims, further comprising contacting the stem cell, or a cell in the process of differentiating from the stem cell, with a small molecule selected from CHIR99021 and WIKI4.

22. The method of any one of the preceding claims, further comprising contacting the stem cell, or a cell in the process of differentiating from the stem cell, with CHIR99021 and WIKI4.

23. A composition comprising stem cells, a ROCK inhibitor, and one or both of CHIR99021 and WIKI4.

24. The composition of claim 23, wherein the ROCK inhibitor is Y-27632.

25. The composition of claim 23 or 24, wherein the stem cells are pluripotent stem cells or cardiac progenitor cells.

26. The composition of any one of claims 23 to 25, wherein the stem cells are human.

27. The composition of any one of claims 23 to 26, wherein the stem cells are induced pluripotent stem cells.

28. The composition of any one of claims 23 to 27, wherein the stem cells are embryonic stem cells.

29. The composition of any one of claims 23 to 27, wherein the stem cells are not embryonic stem cells.

30. A transplantation composition comprising cardiomyocytes produced by the method of any one of claims 1 to 22.

31. 31. The implant composition of claim 30, further comprising a pharma- ceutically acceptable carrier.

32. 32. The implant composition of claim 30 or 31, further comprising a gel, scaffold, or matrix.

33. 33. The implant composition of claim 32, wherein the gel, scaffold, or matrix is ​​biodegradable.

34. Solubilized basement membrane protein or preparation thereof, immunosuppressant, pan-caspase inhibitor, anti-apoptotic agent, IGF-1, and K ATP The implantable composition of any one of claims 30-33, further comprising any one or more of the channel openers.

35. A pharmaceutical composition comprising cardiomyocytes produced by the method of any one of claims 1 to 28 or a transplant composition of any one of claims 30 to 34 in combination with a pharma- ceutically acceptable carrier.

36. A method for improving cardiac function in a subject in need thereof comprising administering cardiomyocytes produced by the method of any one of claims 1 to 22, or a composition of any one of claims 23 to 35, to cardiac tissue of a subject in need thereof.

37. 37. The method of claim 36, wherein the subject is a human.

38. 38. The method of claim 36 or 37, wherein the subject has damaged cardiac tissue due to acute or chronic injury.

39. A cardiomyocyte produced by the method of any one of claims 1 to 22 or a composition of any one of claims 23 to 35 for use in a method for improving cardiac function in a subject in need thereof, comprising administering the cardiomyocyte produced by the method of any one of claims 1 to 22 or the composition of any one of claims 23 to 35 to cardiac tissue of a subject in need thereof.

40. 40. The use of claim 39, wherein the subject is a human.

41. 41. The method of claim 39 or 40, wherein the subject has damaged cardiac tissue due to acute or chronic injury.

42. The composition of claim 23, wherein contact with at least one ROCK inhibitor is initiated when CHIR99021 is added during the differentiation protocol, beginning at least on day 1 of differentiation of the stem cells.

43. The composition of claim 23, wherein contact with at least one ROCK inhibitor is initiated when WIKI4 is added during the differentiation protocol, beginning at least on day 3 of differentiation of the stem cells.

Images