HK1237366B - Method for differentiation of pluripotent stem cells into cardiomyocytes - Google Patents

Description

Method for differentiating pluripotent stem cells into cardiomyocytes

Technical Field

The present application relates to a method for differentiating pluripotent stem cells (PSCs) into cardiomyocytes. In addition, the present application relates to a method for differentiating human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into proliferating cardiomyocytes, which is based on a ligation step induced by a chemically defined medium.

Background Art

For many years, a variety of cell culture systems have been used in preclinical drug development. However, established cell models only partially reflect the specific physiology of drug-related diseases because they are derived from tumorigenic tissues or from transformed and immortalized cells. Specifically, because terminally differentiated cardiomyocytes have been shown to have limited proliferation potential, they do not have the ability to effectively generate cell models for drug development. Therefore, there is a need for more disease-related human cell types that can be used as reliable cell models in research and drug development.

Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) provide researchers with tremendous opportunities to generate functional human cell types (such as cardiomyocytes, neurons, pancreatic cells, etc.). Robust processes for in vitro differentiation of pure hESC and iPSC-derived human cardiomyocyte (hESCM) cultures will be powerful tools that will not only enhance our understanding of early human cardiogenesis, but also use cardiomyocytes as non-transformed human cell models to test drug efficacy in the preclinical stages of drug development and assess cardiac toxicity before entering the clinic. In addition, hESC-derived human cardiomyocytes open up opportunities to identify pathways that are critical for cardiac regeneration and ultimately lead to support for the clinical application of stem cell-based therapies.

To develop cellular assay models for drug research and development, such differentiation protocols ideally need to produce cells that meet the following criteria: a) be robust, with high levels of reproducibility; b) yield large quantities of highly purified cell types; c) be differentiated in a short time; d) produce cells that can be frozen to ensure batch consistency across multiple screening campaigns; and e) provide functional and physiological relevance for modeling disease-specific readouts. P.W. Burridge et al. reviewed existing approaches for differentiating pluripotent cells into cardiomyocytes (P. Burridge, Keller, Gold, & Wu, 2012). To date, no known protocols meet these criteria. Specifically, cardiomyocytes obtained using known protocols are difficult to freeze and thaw without losing any functional properties.

To address these needs, we have developed a new differentiation method that produces large quantities of highly purified cardiomyocytes (up to 95%). This differentiation process uses defined small molecules to guide differentiation into the cardiomyocyte lineage within a 10-day time span. To further increase their purity, cardiomyocytes are enriched by replating them under conditions preferred by cardiomyocytes. In addition, the cardiomyocytes can then be frozen, stored under liquid nitrogen, and thawed again. The cardiomyocytes have been tested for compliance with several screening formats for drug research and development. The present invention provides an improved method for differentiating pluripotent stem cells into cardiomyocytes in a shorter time and with significantly increased yields compared to prior art processes. This new method alleviates the need to obtain embryoid bodies or small cell clusters from pluripotent stem cells and eliminates the main drawbacks of the low reproducibility and standardization of previously known methods. In addition, the high efficiency allows these defined cardiomyocytes to be used on a large scale in the pharmaceutical industry for drug discovery and safety assessment, for regenerative medicine applications, and for in vitro disease simulations.

SUMMARY OF THE INVENTION

1. Provided herein is a method for differentiating pluripotent stem cells into cardiomyocytes, the method comprising the steps of:

a) providing pluripotent stem cells at a density of 3-7 x 10 ⁵ /cm ² ;

b) incubating the cells in an insulin-free medium containing a compound of the formula:

In one embodiment, the cells are incubated in insulin-free medium containing 0.3-10 μM of the compound.

In one embodiment, step b) comprises incubating the cells for 12-48 hours.

In one embodiment, the method further comprises the step c) incubating the cells in an insulin-free medium containing Wnt-C59.

In one embodiment, step c) comprises incubating the cells in an insulin-free medium containing 1-10 μM Wnt-C59.

In one embodiment, step c) comprises incubating the cells for 24-72 hours.

In one embodiment, the cells are incubated in insulin-free medium for 24-48 hours between steps.

In one embodiment, the method further comprises the step d) incubating the cells in a medium containing insulin.

In one embodiment, the culture medium of steps b), c) and d) comprises vitamin C.

In one embodiment, the pluripotent stem cells are induced pluripotent stem cells.

In one embodiment, the induced pluripotent stem cells are human cells.

In one embodiment, the induced pluripotent stem cells are obtained from an individual suffering from a disease caused by abnormal cardiac cell function.

In one embodiment, there is provided a cardiomyocyte obtained by the method of any of the above embodiments.

In one embodiment, a biobank of cardiomyocytes obtained by the method of any of the above embodiments is provided.

In one embodiment, cardiomyocytes or a biobank of cardiomyocytes obtained by the method of any of the above embodiments are used as an in vitro model of a disease caused by cardiac cell dysfunction.

In one embodiment, a therapeutic composition is provided comprising a cardiomyocyte or a biobank of cardiomyocytes obtained by the method of any of the above embodiments.

Any of the above embodiments may exist alone or in combination.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: FACS analysis identifies a high concentration of cardiomyocytes at day 14 of differentiation. Both hESCs and iPSCs produced similar results. A: Human embryonic stem cell-derived cardiomyocytes. B: Human induced pluripotent stem cell-derived cardiomyocytes.

Figure 2: FACS analysis of multiple cardiomyocyte differentiations demonstrates the robustness of the workflow.

Figure 3: FACS analysis shows that the purification method improves the purity of cardiomyocytes. A: 5.5x10 ⁵ /cm ² cardiomyocytes with 60% purity on day 14. B: 4.4x10 ⁵ /cm ² cardiomyocytes with 98% purity after additional purification steps.

Figure 4: Immunofluorescence staining - Confocal microscopy analysis shows a typical intracellular stripe pattern of α-actinin and troponin T in cardiomyocytes. Green (*): α-actinin; Red (#): troponin; Blue (+): cell nuclei.

Figure 5: xCELLigent analysis – Isoproterenol increases the beating frequency of pluripotent stem cell-derived cardiomyocytes.

Figure 6: Pluripotent stem cell-derived cardiomyocytes show a high percentage of viable cardiomyocytes after thawing on differentiation days 14 and 18. In each experiment, 4 x 10 ⁶ cells were frozen.

Detailed Description of the Invention

The present invention provides an improved method for differentiating pluripotent stem cells into cardiomyocytes in a shorter time and with a significantly increased yield of proliferating cardiomyocytes compared to prior art processes.

The novel method disclosed herein for differentiating human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into definitive cardiomyocytes is based on a chemically defined medium-induced attachment step, generating beating cells after only 10 days (or earlier: 8 days) after initiating differentiation.

In one embodiment, a method is provided for differentiating pluripotent stem cells into cardiomyocytes, the method comprising the steps of:

a) providing pluripotent cells at a density of 3-7 x 10 ⁵ cells/cm ² ;

b) incubating the cells in an insulin-free medium containing a compound of the formula:

3-(3-Amino-phenyl)-4-(1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione (CP21)

The pluripotent stem cells are provided at a density of 3-7 x 10 ⁵ cells/cm ² (i.e., very high density). In one embodiment, the cells are provided at a density of 5.5 x 10 ⁵ cells/cm ^2. Surprisingly, the inventors of the present method have found that providing the cells at a high density improves differentiation efficiency and cardiomyocyte yield.

In one embodiment, prior to initiating differentiation in step a), the cells provided at high density are washed with a suitable buffer or culture medium to remove any dead cells.

The culture medium in step b) is an insulin-free medium. The lack of insulin in the early differentiation medium in step b) is important because previous reports have shown that differentiation medium containing insulin blocks cardiogenesis (Lian u.a., 2013).

To initiate differentiation, cells were incubated in insulin-free medium containing compound 21, (3-(3-amino-phenyl)-4-(1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione, also referred to herein as "Compound 21" or "CP21"; see, e.g., L. Gong et al.; Bioorganic & Medicinal Chemistry Letters 20 (2010), 1693-1696) to activate the wnt pathway. The optimal concentration of compound 21 to induce cardiomyocyte differentiation depends on the cell density of pluripotent cells attached to the cell container. In several parallel experiments using different cell densities ( ^1.8-11x105 /cm2hESC or iPSC) and various CP21 concentrations (0-10μM), it was found that the use of a cell density of ^5.5x105 / ^cm2 and a CP21 concentration of 2μM resulted in the most efficient differentiation of pluripotent stem cells into cardiomyocytes. CP21 concentrations above 5μM showed reduced cell viability. This was surprising because prior art protocols required higher concentrations of other Wnt pathway modulators for efficient differentiation.

In one embodiment, step b) of the differentiation method comprises incubating the cells in a medium containing 0.3-10 μM CP21, preferably 0.5-5 μM CP21. In a preferred embodiment, step b) of the differentiation method comprises incubating the cells in a medium containing 2 μM CP21.

After 24 hours of CP21 incubation, cells showed robust cell death. Testing various CP21 incubation times revealed that 24 hours was optimal for cardiogenesis, while longer or shorter incubation times resulted in inefficient differentiation.

In one embodiment, step b) comprises incubating the cells in an insulin-free medium containing CP21 for 12-48 hours, preferably 18-24 hours.

In a preferred embodiment, step b) comprises incubating the cells in an insulin-free medium containing CP21 for 24 hours.

In one embodiment, the culture medium of step b) comprises vitamin C. Addition of vitamin C to basal culture medium has been shown to improve cardiomyocyte differentiation (Cao u.a., 2012).

Thus, in one embodiment, the culture medium of step b) is an insulin-free culture medium containing CP21 and vitamin C. In one such embodiment, the culture medium comprises 0.5-5 μM CP21 and vitamin C.

In one embodiment, the method further comprises step c) incubating the cells in an insulin-free medium containing Wnt-C59.

Wnt-C59 is a small molecule that blocks the Wnt signaling pathway (WO2010101849, 2-(4-(2-methylpyridin-4-yl)phenyl)-N-(4-(pyridin-3-yl)phenyl)acetamide). Wnt-C59 is a highly potent and selective Wnt signaling antagonist. It prevents palmitoylation of Wnt proteins by porcupine (a membrane-bound O-acyltransferase), thereby blocking Wnt protein secretion and activity.

The use of different concentrations of the wnt repressor Wnt-C59 (1-10 μM) resulted in a significant increase in cardiomyocytes. The optimal concentration was identified as 2 μM. In the absence of added Wnt-C59, differentiation did not produce cardiomyocytes. Concentrations exceeding 5 μM Wnt-C59 showed increased cell death. In one embodiment, step c) of the differentiation method comprises incubating the cells in a medium containing 1-10 μM Wnt-C59. In a preferred embodiment, step c) of the differentiation method comprises incubating the cells in a medium containing 2 μM Wnt-C59.

Because the Wnt pathway is highly complex, other Wnt inhibitors with different modes of action were tested.

The anthelmintic drug niclosamide (Chen et al., Biochemistry. 2009 Nov 3;48(43):10267-74.) promotes Frizzled1 endocytosis, downregulates Dishevelled-2 protein, and inhibits Wnt3A-stimulated β-catenin stabilization and LEF/TCF reporter activity.

Pyrvinium is a potent inhibitor of Wnt signaling that binds to all casein kinase 1 (CK1) family members in vitro and selectively enhances casein kinase 1α (CK1α) kinase activity, leading to stabilization of Axin and increased β-catenin turnover (Thorne et al., Nat Chem Biol. 2010 Nov;6(11):829-36.).

The anti-helminthic drugs niclosamide and pyralidone were tested for their ability to induce cardiomyocyte differentiation. Unlike Wnt-59, neither of the other two Wnt inhibitors resulted in the successful generation of cardiomyocytes. The varying efficacy of the tested Wnt inhibitors on the differentiation of pluripotent stem cells into cardiomyocytes suggests that specific inhibition of the Wnt pathway by blocking Wnt secretion appears to be the key mechanism.

In one embodiment, step c) comprises incubating the cells in an insulin-free medium containing Wnt-C59 for 24-72 hours, preferably 48 hours.

In one embodiment, the insulin-free culture medium of steps b) and c) is a serum-free culture medium. In one embodiment, the insulin-free culture medium is RPMI1680 (Gibco).

In one embodiment, the cells are incubated in an insulin-free medium between each step b) and c) for 24 to 48 hours, preferably 48 hours. In one embodiment, the medium is a serum-free medium. In another embodiment, the medium comprises vitamin C.

In one embodiment, the cells are incubated between each step b) and c) in a serum-free, insulin-free medium containing vitamin C for 24 hours to 48 hours, preferably 48 hours.

In one embodiment, the method described herein for differentiating pluripotent cells into cardiomyocytes according to any of the above embodiments further comprises step d) incubating the cells in a medium containing insulin. In this late stage, insulin promotes the proliferation of cardiomyocytes and their cardiac precursor cells.

In one embodiment, step d) comprises incubating the cells in a medium containing insulin for 36-60 hours, preferably 48 hours. In one embodiment, the medium is a serum-free medium. In another embodiment, the medium contains vitamin C.

Suitable culture media for the expansion step d) are, for example, DMEM, high glucose + L-glutamine + pyruvate and carnitine, taurine, creatine, BSA, vitamin C, or the iCell Cardiomyocyte Maintenance Medium from Cellular Dynamics international.

Preferably, the culture medium is replaced between each step, for example, by removing the culture medium by drawing or centrifuging the cells and discarding the supernatant, and then adding the culture medium for the subsequent step to the cells. In one embodiment, the cells are washed with a suitable buffer or culture medium before adding the culture medium for the subsequent step to remove any dead cells. The buffer or culture medium for washing cells is known in the art. An example of a buffer suitable for washing cells is, for example, phosphate buffered saline (PBS).

In one embodiment, the pluripotent cells used in the differentiation method are cultured under conditions that allow stable growth and/or doubling time. For example, the cells are cultured in a pluripotency medium and passaged several times. As used herein, "pluripotency medium" refers to any chemically defined medium that is used to attach pluripotent stem cells as single cells to a monolayer while maintaining their pluripotency, and is well known in the art. In one embodiment, the pluripotency medium is a serum-free medium containing a small molecule inhibitor of the Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) family of protein kinases (referred to herein as a ROCK kinase inhibitor).

In one embodiment, the ROCK kinase inhibitor is selected from 1-(5-isoquinolinesulfonyl)homopiperazine), N-benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide), and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride.

Examples of ROCK kinase inhibitors used herein are Fasudil (1-(5-isoquinolinesulfonyl)homopiperazine), Thiazovivin (N-benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide), and Y27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, e.g., catalog number 1254 from Tocris bioscience). In a preferred embodiment, the ROCK kinase inhibitor is Y27632. In one embodiment, the pluripotency medium is a serum-free medium containing 2-20 μM Y27632, preferably 5-10 μM Y27632. In another embodiment, the pluripotency medium is a serum-free medium containing 2-20 μM Fasudil. In another embodiment, the pluripotency medium is a serum-free medium containing 0.2-10 μM Thiazovivin.

Using the novel method presented herein, it is now possible to differentiate cardiomyocytes expressing α-actinin and troponin T from pluripotent stem cells with a yield of up to 60-98%.

In one embodiment, the method further comprises step e) reseeding the cells and incubating them in an insulin-free medium. This step further improves the purity of the cardiomyocytes. In one embodiment, the cells are reseeded and incubated in an insulin-free medium supplemented with fetal bovine serum for 18-32 hours, preferably 24 hours. In one such embodiment, the culture medium further comprises a ROCK inhibitor. In one embodiment, the ROCK inhibitor is Y-27632.

Cardiomyocytes obtained by the method described here can be expanded for several passages and maintain their functional properties after freezing and thawing.

The term "differentiation" as used herein refers to one or more steps in which a poorly differentiated cell is transformed into a somatic cell, such as a pluripotent stem cell into a cardiomyocyte. Differentiation of pluripotent stem cells into cardiomyocytes is achieved by the methods described herein.

The term "stem cell" as used herein refers to a cell with self-renewal ability. "Undifferentiated stem cells" as used herein refer to stem cells with the ability to differentiate into a variety of cell types. As used herein, "pluripotent stem cells" as used herein refer to stem cells that can produce cells of a variety of cell types. Pluripotent stem cells (PSC) include human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC). Human induced pluripotent stem cells can be derived from reprogrammed somatic cells, for example, by transduction of four determined factors (Sox2, Oct4, Klf4, c-Myc) by methods known in the art. Human somatic cells can be obtained from healthy individuals or from patients. These donor cells can be easily obtained from any suitable source. It is preferred herein to allow separation of donor cells without performing invasive procedures on the human body, such as human skin cells, blood cells, or cells that can be obtained from urine samples. Although human pluripotent stem cells are preferred, this method is also applicable to non-human pluripotent stem cells, such as primates, rodents (e.g., rats, mice, rabbits) and dog pluripotent stem cells.

As used herein, "cardiomyocytes" are cells that express at least the cell marker troponin T (troponin T type 2 (cardiac), gene symbol TNNT2, Entrez Gene: 7139, UniProtKB: P45379), and in a preferred embodiment, also express the cell marker α-actinin (ACTN2 actinin, α2, gene symbol ACTN2, Entrez Gene: 88, UniProtKB: P35609). The expression of troponin T and/or α-actinin can be assessed by methods known in the art, such as by FACS analysis as described in the Examples section. Cardiomyocytes can exhibit spontaneous cyclic contractile activity ("beating"). This means that when cardiomyocytes obtained by the methods of the present invention are cultured in a suitable tissue culture environment with appropriate Ca++ concentration and electrolyte balance, the cells can be observed to contract in a cyclic manner across one axis of the cell and then release from contraction without the need to add any additional components to the culture medium. In addition, cells obtained by the methods disclosed herein can exhibit other characteristics of cardiomyocytes, such as ion channels or appropriate electrophysiology.

As used herein, "proliferating cardiomyocytes" are cells that express α-actinin and troponin T and proliferate by cell division.

"Marker expression" refers to the transcription of a gene into mRNA, which is then usually translated into a protein (its gene product) that performs a function in the cell. Marker expression can be detected and quantified at the RNA level or at the protein level by methods known in the art. It is preferred herein to detect marker expression at the protein level, for example, by testing for the presence of a protein with an antibody that binds to the marker.

Any of the above embodiments may exist alone or in combination.

In one embodiment of the present invention, a method for producing patient-specific or healthy individual-specific cardiomyocytes is provided. To this end, the methods described herein are used to differentiate human induced pluripotent stem cells (iPSCs) obtained from patients or healthy individuals into cardiomyocytes. Somatic cells obtained from patients or healthy individuals can be reprogrammed into pluripotent stem cells by methods known in the art to obtain patient-specific human iPSCs. For example, fibroblasts, keratinocytes, or adipocytes can be obtained from individuals in need of treatment or from healthy individuals through skin biopsy and reprogrammed into induced pluripotent stem cells by methods known in the art. Other somatic cells suitable as sources of induced pluripotent stem cells are leukocytes, epithelial cells, or other cells obtained from urine samples obtained from blood samples. Patient-specific induced pluripotent stem cells can then be differentiated into patient-specific or healthy individual-specific cardiomyocytes by the methods described herein. In another aspect of the present invention, a cardiomyocyte colony produced by any of the aforementioned methods is provided. Preferably, the cardiomyocyte colony is patient-specific, i.e., derived from iPSCs obtained from diseased individuals. In another embodiment, the cardiomyocyte colony is obtained from healthy individuals.

Patient-derived cardiomyocytes are disease-related in vitro models for studying the pathophysiology of diseases such as dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and coronary heart disease. In one embodiment, the cardiomyocytes obtained by this method are used to screen for compounds that reverse, inhibit, or prevent diseases (such as cardiac hypertrophy, reduced beating efficiency, cardiomyocyte stripe malformation, and insufficient calcium treatment) caused by cardiac cell dysfunction. Preferably, the cardiomyocytes obtained by the methods of the present invention as described herein are derived from diseased individuals. In another embodiment, the cardiomyocytes obtained by this method are used to screen and evaluate new targets and compounds for treating heart disease (such as those mentioned above). Preferably, the cardiomyocytes obtained by the methods of the present invention as described herein are derived from individuals with diseases (such as dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and coronary heart disease). Differentiating cardiomyocytes from diseased individuals is the only opportunity to evaluate drug safety in early stages in human background models. In another embodiment, the cardiomyocytes obtained by this method are used as in vitro models of the heart.

The present invention provides an efficient method for providing patient-specific cardiomyocytes or compatible cells from healthy individuals with the same HLA type suitable for transplantation, both of which are derived under xeno-free conditions. "Xeno-free culture conditions" refers to a culture medium and substrate for attachment that only contains components of human and recombinant origin. Therefore, the risk of contamination by xenobiotics is avoided, and kidney cells can be safely used in regenerative medicine. Differentiating patient-specific induced pluripotent stem cells (iPSCs) into patient-specific cardiomyocytes using the methods described herein is a technique for producing cardiomyocytes of autologous origin that can be easily performed and reproduced. The use of autologous and/or compatible cells in cell therapy provides a huge advantage over the use of non-autologous cells, which may be subject to immunological rejection. In contrast, autologous cells are unlikely to elicit a significant immunological response.

In another preferred aspect of the present invention, it is envisaged to generate a biobank of patient-specific cardiomyocytes. In one embodiment, a biobank is generated comprising different populations of cardiomyocytes obtained from healthy individuals and/or patients. The term "biobank" as used herein refers to a library of biological samples collected from different individuals or species. The archived compilation of samples and related data is intended for research purposes, with the goal of studying diseases associated with dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and coronary artery disease. In another embodiment, the biobank is used in vascular regenerative medicine methods.

On the other hand, the present invention provides a therapeutic composition comprising cardiomyocytes produced by any of the aforementioned methods or comprising any of the aforementioned cell colonies. Preferably, the therapeutic composition further comprises a physiologically compatible solution, including, for example, a phosphate-buffered saline solution containing 5% human serum albumin. The therapeutic composition can be used to treat, prevent or stabilize diseases such as dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and coronary heart disease. For example, fibroblasts, keratinocytes or adipocytes can be obtained from individuals in need of treatment or from healthy individuals by skin biopsy and reprogrammed into induced pluripotent stem cells by methods known in the art ("Induction of pluripotent stem cells from adult human fibroblasts by defined factors." Takahashi et al., 2007, Cell 131, 861-72). Other somatic cells suitable as a source of induced pluripotent stem cells are leukocytes, epithelial cells obtained from a blood sample, or other cells obtained from a urine sample. The patient-specific induced pluripotent stem cells are then differentiated into cardiomyocytes by the methods described herein, collected, and introduced into the individual to treat the condition. Cardiomyocytes produced by the methods of the present invention can be used to replace or assist in the normal function of diseased or damaged tissue.

Another embodiment of the present invention is the use of a biobank of cardiomyocytes in the treatment of diseases associated with dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and coronary heart disease. The biobank preferably comprises cardiomyocytes obtained from patients or healthy individuals with several HLA types. Transplanting cells obtained from healthy donors to individuals in need of treatment with compatible HLA types avoids the problem of rejection reactions typically associated with allogeneic cell transplantation. Typically, rejection is prevented or alleviated by administering immunosuppressants or anti-rejection drugs such as cyclosporine. However, such drugs have significant side effects, such as immunosuppression, carcinogenic properties, nephrotoxicity, and are very expensive. The present invention eliminates or at least significantly reduces the need for anti-rejection drugs (such as cyclosporine, imulan, FK-506, glucocorticoids, and rapamycin, and derivatives thereof).

For the treatment methods of the present invention, administration of cardiomyocytes to a mammal is not intended to be limited to a specific route of administration, dosage, or frequency of administration; the present invention contemplates all routes of administration, including intramuscular, intravenous, intraarticular, intralesional, subcutaneous, or any other route sufficient to provide a dose sufficient to prevent or treat the disease. Cardiomyocytes can be administered to a mammal in a single dose or in multiple doses. When multiple doses are administered, the doses can be spaced apart from each other by, for example, one week, one month, one year, or ten years. One or more growth factors, hormones, interleukins, cytokines, small molecules, or other cells can also be used before, during, or after administration of the cells to further bias them toward a specific cell type.

Example

Materials and methods

CP21R7: 3-(3-Amino-phenyl)-4-(1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione (also referred to herein as "Compound 21" or "CP21"; see, e.g., L. Gong et al.; Bioorganic & Medicinal Chemistry Letters 20 (2010), 1693-1696)

CP21R7

Wnt-C59: 2-(4-(2-methylpyridin-4-yl)phenyl)-N-(4-(pyridin-3-yl)phenyl)acetamide (Cellagen Technology, catalog number C7641-2s, WO2010101849):

Human ESCs: SA001 and LOT CA001 were isolated on March 20, 2001, at the University and Cellartis AB, Arvid Wallgrens Backe 20, SE-413 46, Sweden, in accordance with all applicable Swedish laws and approved by the Local Research Ethics Committees of the University and Uppsala University. Embryo Source: Frozen, from IVF surplus. Donor Confidentiality: To protect the privacy and confidentiality of the donors, all identification related to the embryo donors was removed. Therefore, no information about the donors is available. It should be noted that the donations did not result in any financial benefit to the donors. We are authorized to conduct hESC research and derive different cell lines. The Ethikkommission beider Basel and the Federal Office of Public Health have approved our research project (Ref-No: R-FP-S-1-0002-0000).

Human iPSCs: Cat. No. SC101A-1 Lot No. 110218-FF from SBI System Biosciences / Cat. No. A13777 from Life technologies Episomal hiPSC Line.

Human pluripotent stem cells were routinely cultured on hESC-qualified Matrigel (BD Bioscience) in TeSR1 medium (Stem Cell Technologies). Cultures were passaged every 4-6 days using StemPro Accutase (Invitrogen). To improve viability, TeSR1 medium included 10 μM ROCK inhibitor 1 hour prior to enzymatic dissociation.

500 ml differentiation medium

RPMI1680+Glutamax 481ml GIBCO#61870

Vitamin C (10mg/ml) 4ml Sigma#A4544

(Final concentration: 80 μg/ml)

B27-Insulin (50x) 10ml Invitrogen #05-0129SA

Penicillin/Streptomycin 5ml GIBCO#15140-122

(Final concentration: 50U/ml)

500 ml expansion medium

RPMI1680+Glutamax 481ml GIBCO#61870

Vitamin C (10mg/ml) 4ml Sigma#A4544

(Final concentration: 80 μg/ml)

B27 + Insulin (50x) 10ml Invitrogen #12587-01

Penicillin/Streptomycin 5ml GIBCO#15140-122

(Final concentration: 50U/ml)

Other reagents and materials used in this article:

Matrigel (BD Bioscience, catalog number 354277)

mTeSR1 medium (Stemcell Technologies, catalog number 05850)

Accutase (Innovative Cell Technologies, catalog number AT-104)

Rock inhibitor Y-27632 (Millipore, catalog number SCM075)

RPMI medium (Gibco from Life Technologies, catalog number 61870)

Vitamin C (Sigma, catalog number A4544)

Supplement does not contain insulin (Gibco from Life Technologies, catalog number 0050129SA)

Penicillin-Streptomycin (Gibco from Life Technologies, catalog number 15070)

50xB27 plus insulin without vitamin A (Gibco from Life Technologies, catalog number 12587)

0.05% Trypsin/EDTA, 1x (Gibco from Life Technologies, cat. no. 25300)

autoMACS running buffer (Miltenyi, cat. no. 130-091-221)

Inside Perm + Inside Fix (Miltenyi, Inside Stain Kit, catalog number 130-090-477)

0.1% gelatin (Millipore, Cat. ES-006-B)

Cryogenic tubes (Corning #430659)

Mr. Frosty Freezing Container (Thermo Scientific #5100-0001)

DMSO (Sigma#D2438)

Fetal bovine serum (Invitrogen #16000044)

Falcon cell culture dish 35x10mm (BD#353001)

Falcon cell culture dish 100x20mm (BD#353003)

6-well plate Corning Costar (Sigma #CLS3516)

Anti-sarcomeric α-actinin [EA-53] antibody (Abcam, catalog number ab9465)

Anti-cardiac troponin T antibody (Abcam, catalog number ab45932)

Alexa 488 and donkey anti-mouse IgG (H+L) (Invitrogen, cat. no. A21202)

Alexa 647 donkey anti-rabbit IgG (H+L) (Invitrogen, catalog number A31573)

Alexa 555 donkey anti-rabbit IgG (H+L) (Invitrogen, cat. no. A31572)

Hoechst 33258, pentahydrate (bis-Benzimide) (Molecular Probes, catalog number H3569)

Differentiation of cardiomyocytes from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs)

Human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) were cultured in 10 ml of mTeSR1 medium (Stemcell Technologies, catalog number 05850) in Matrigel (BD Bioscience, catalog number 354277)-coated 56 cm ² dishes at 37° C. and 5% CO 2 .

Before initiating cardiomyocyte differentiation, passage the cells 3-4 times to ensure that the pluripotent stem cells display stable growth and doubling time.

To propagate pluripotent stem cells while maintaining their pluripotent state, hESCs or iPSCs were treated as follows: washed once with 10 ml PBS-/- and then incubated with 3 ml Accutase (Innovative Cell Technologies, catalog number AT-104) at 37°C and 5% CO2 for 2-3 minutes to detach the cells.

The Accutase enzymatic reaction was terminated with 7 ml of mTeSR1, and the cells were centrifuged at 500 x g for 3 minutes.

Resuspend the cells in 10 ml of mTeSR1 and count them. For further culture, seed 2 x 10 ⁶ cells onto a 56 cm ² dish coated with Matrigel. Additionally, culture hESCs or iPSCs in 10 ml of mTeSR1 and 10 μM Rock inhibitor Y-27632 (Millipore, catalog number SCM075) at 37°C and 5% CO 2 . Subsequently, replace 10 ml of mTeSR1 medium daily, culture pluripotent stem cells to 80% density, and then passage.

To successfully differentiate into cardiomyocytes, pluripotent stem cells were seeded at a high density of 5.5 x 10 ⁵ /cm ² with hESCs or iPSCs. Passaging and culture were performed as described above for pluripotent stem cells.

After 24 hours (1 day), hESCs or iPSCs were washed once with 180 μl/cm ² PBS-/-, and the culture medium was replaced with 180 μl/cm ² differentiation medium.

To initiate differentiation of pluripotent stem cells toward the cardiac lineage, the culture medium contained 2 μM Compound 21 (CP21), a small molecule, highly selective inhibitor of glycogen synthase kinase 3 (GSK3β).

After incubation with CP21 for 24 h (day 2), cells were washed with PBS-/- as described above and cultured in 220 μl/ ^cm2 differentiation medium for 48 h.

After 48 hours (day 4), cells were washed with PBS-/- as described above and cultured for 48 hours in 220 μl/ ^cm2 differentiation medium containing 2 μM Wnt-C59 (Cellagen Technology, catalog number C7641-2s, WO2010101849), a potent Wnt signaling inhibitor that blocks Wnt secretion.

After 48 hours (day 6), cells were washed with PBS-/- as described above and cultured in 220 μl/ ^cm2 differentiation medium for 48 hours.

After 48 hours (day 8), cells were washed with PBS-/- as described above and cultured for 48 hours in 220 μl/ ^cm2 RPMI medium containing vitamin C, penicillin-streptomycin but now including B27 without vitamin A plus insulin (= expansion medium).

The first cardiomyocytes, visible as beating cells, were observed on day 8 of differentiation and further increased until day 14.

The medium was subsequently changed every 48 hours with 220 μl/cm ² of expansion medium.

Cell characterization

To test the efficiency of the differentiation method, cardiomyocytes were characterized by cell immunohistochemistry and fluorescence activated cell sorting (FACS) on day 14 of differentiation using antigens specific for cardiomyocytes.

Fluorescence-activated cell sorting (FACS) analysis

Cells were washed with 180 μl/cm ² PBS-/- and dissociated with 100 μl/cm ² 0.05% 1× Trypsin/EDTA (Gibco by Life Technologies, Cat. No. 25300) at 37° C. and 5% CO ₂ for 5-10 minutes.

As needed, gently scrape the cells from the culture vessel by pipetting up and down, then incubate at 37°C and 5% _CO2 for 5-10 minutes.

Then, triple expansion medium and 10% fetal bovine serum (FBS) were added.

Then, cells were filtered through a 100 μm cell strainer and counted.

For analysis, 1x10 ⁶ cell suspension was transferred to a 1.5 ml tube. After centrifugation at 500xg for 3 minutes, the supernatant was discarded and the cells were fixed with 50 μl Inside Fix (Miltenyi, Inside Stain Kit, catalog number 130-090-477) and 50 μl PBS-/- for 15 minutes at room temperature in the dark.

Then, 100 μl of autoMACS running buffer (Miltenyi, catalog number 130-091-221) was added and the cells were centrifuged. The supernatant was discarded, and the cells were washed with 100 μl of Inside Perm (Miltenyi, Inside Stain Kit, catalog number 130-090-477), centrifuged, and the supernatant discarded. The cells were incubated with anti-sarcomeric α-actinin [EA-53] antibody (Abcam, catalog number ab9465) and anti-cardiac troponin T antibody (Abcam, catalog number ab45932) diluted 1:100 in Inside Perm for 1 hour at 4°C.

The cells were then washed with 500 μl of running buffer, centrifuged, and the supernatant discarded. The cells were incubated with secondary antibodies (1:1000 in InsidePerm) for 10 minutes at room temperature. The following secondary antibodies were used: Alexa 488 donkey anti-mouse IgG (H+L) (Invitrogen, catalog number A21202) and Alexa 647 donkey anti-rabbit IgG (H+L) (Invitrogen, catalog number A31573).

Subsequently, the cells were washed with 500 μl of running buffer, and after centrifugation, the cells were resuspended in 500 μl of running buffer and measured by a fluorescence-activated cell sorting (FACS) system.

Differentiation of cardiomyocytes from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) using different CP21 concentrations

The above procedure was repeated using different CP21 concentrations. The results are shown in the table below: (-): no cardiomyocytes were obtained, (+)-(+++): amount of cardiomyocytes obtained.

Cp21 concentration μM 0 0.3 1 2 3 5 10 Experiment I - - + ++ ++ + - Experiment II - - + +++ + - - Experiment III - - + ++ ++ - -

purification

To increase the purity of cardiomyocytes, an enrichment step was developed.

Cells were washed with 180 μl/cm ² PBS-/- and dissociated with 100 μl/cm ² 0.05% 1x Trypsin/EDTA (Gibco by Life Technologies, Cat. No. 25300) at 37° C. and 5% CO ₂ for 5-10 minutes as described above.

As needed, gently scrape the cells from the culture vessel by pipetting up and down, then incubate at 37°C and 5% _CO2 for 5-10 minutes.

Then, triple expansion medium and 10% fetal bovine serum (FBS) were added.

Then, cells were filtered through a 100 μm cell strainer and counted.

The plate coated with 130 μl/cm ² of 0.1% gelatin (Millipore, catalog number ES-006-B) was incubated at 37° C. for 1 hour.

2.7 x 10 ⁵ /cm ² cells were seeded in 180 μl/cm ² of expansion medium containing 10% fetal bovine serum (FBS). In addition, 10 μM Rock inhibitor was added. After 24 hours, the medium was replaced with expansion medium without FBS and Rock inhibitor. The medium was changed every 48 hours.

On days 18-21, cells were analyzed by FACS and replated again in different formats as described above for the following analyses: immunofluorescence staining, xCELLigence to examine beating rhythm and pro-arrhythmic effects of compounds in stem cell-derived cardiomyocytes.

Transfer cells to a plate format suitable for the assay. Allow cells to attach for 24 hours in 200 μl/ ^cm² of expansion medium supplemented with 10% fetal bovine serum (FBS). Additionally, add 10 μM Rock inhibitor. After 24 hours, replace the plate with 220 μl/ ^cm² of expansion medium without FBS or Rock inhibitor. Change the medium every 48 hours.

Freezing and thawing of cardiomyocytes

On day 14, cardiomyocytes were replated as described above for purification. On day 18, cells were dissociated as described above and analyzed by FACS for α-actinin and troponin T expression. Cultures containing 80% or more cardiomyocytes were frozen. Cultures containing less than 80% cardiomyocytes were discarded.

Count the cells and freeze 4 x 10 ⁶ cells per cryogenic vial using 1 ml of cold FBS containing 10% DMSO and 10 μM Y-27632.

The cells were centrifuged at 500 x g for 3 minutes and then carefully resuspended in FBS supplemented with 10% DMSO and 10 μM Y-27632. 1 ml aliquots of the cardiomyocyte suspension were placed into cryovials pre-chilled at 4°C and frozen at -80°C for 24 hours. The cryovials were then stored in liquid nitrogen.

To thaw cardiomyocytes, incubate the tube in a 37°C water bath for 1-2 minutes. Carefully transfer the cells to 10 ml of expansion medium plus 10% fetal bovine serum. Centrifuge the cells at 300 x g for 2 minutes. Resuspend the pellet in 6 ml of expansion medium plus 10% fetal bovine serum and 10 μM Y-27632 and plate in three wells of a 0.1% gelatin-coated 6-well plate. After 24 hours, replace the cells with 220 μl/ ^cm² of expansion medium without FBS and Y-27632. The medium is then changed every three days, and after 5-7 days, the cells are plated in a plate format suitable for the assay (e.g., 96-well format for assays detecting cardiac streak anomalies; 96-well format for assays recording beating frequency).

xCELLigent Cardiomyocyte Beating Assay

Isoproterenol increases heart rate and myocardial contractility by stimulating cardiac β-1 receptors. To examine this proarrhythmic effect in stem cell-derived cardiomyocytes, 7 x 10 ⁴ /cm ² cells were seeded on a special E-Plate Cardio 96 (Roche, catalog number 05232368001) coated with 130 μl/cm ² of 0.1% gelatin at 37°C for 1 hour. After allowing the cells to attach to the plate and recover for 2 days as described above, the medium was changed to iCell Cardiomyocyte Maintenance Medium (Cellular Dynamics, catalog number CMM-100-120-005). Cells were measured using the xCELLigence RTCA Cardio System (Roche Applied Science). Seven days after seeding, cells were treated with 3 μM isoproterenol and directly measured. Each 96-well plate was measured at a resolution of 12.9 ms. Measurements were continuous for the first 3 minutes, followed by 1-minute intervals every 15 minutes for the next 24 hours.

Immunofluorescence staining

For immunofluorescence staining, cells were fixed with 4% paraformaldehyde for 15 min at room temperature.

After washing with PBS-/-, cells were blocked and permeabilized with PBS-/- containing 10% donkey serum and 0.1% Triton (blocking buffer) at room temperature for 20 minutes. Cells were then stained with primary antibodies anti-sarcomeric α-actinin [EA-53] (Abcam, catalog number ab9465) and anti-cardiac troponin T (Abcam, catalog number ab45932) at a dilution of 1:100 in blocking buffer overnight at 4°C.

Cells were washed with PBS-/- and stained with secondary antibodies Alexa488 and donkey anti-mouse IgG (H+L) (Invitrogen, catalog number A21202) and Alexa555 donkey anti-rabbit IgG (H+L) (Invitrogen, catalog number A31572) diluted 1:1000 in blocking buffer at room temperature. After several PBS-/- wash steps, cell nuclei were stained with Hoechst 33258 pentahydrate (bis-Benzimide) (Molecular Probes, catalog number H3569) diluted 1:1000 in PBS-/-.

result

After differentiation, cells were analyzed for their cardiomyocyte content. Figure 1 shows FACS analysis of quantified cardiomyocytes at day 14 of differentiation.

An average of 80-90% of cardiomyocytes were characterized as double-positive cells for α-actinin and troponin T. In Figure 1, a subpopulation of cells stained single-positive for α-actinin (5-10%). This indicates more immature cardiomyocytes, and for this reason, this population was not included in the scoring. This result was independent of whether hESCs (Figure 1A) or iPSCs (Figure 1B) were used as the source of pluripotent stem cells. Starting with ^5.5x105 / ^cm2 pluripotent stem cells, the differentiation process produced an average of ^4-5x105 / ^cm2 α-actinin and troponin T-positive cardiomyocytes.

To demonstrate the robustness of the differentiation process, we performed several experiments and analyzed the cardiomyocyte content of each culture. Figure 2 shows 10 independent experiments, demonstrating that the differentiation efficiency into cardiomyocytes ranged from 95% to 40%. However, the majority of experiments (7 of 10 experiments) showed a cardiomyocyte content exceeding 75%, which is an acceptable proportion. Experiments that yielded 60% or more cardiomyocytes were continued. Differentiations that yielded less than 60% cardiomyocytes were discarded. The inter-experimental variability is most likely due to the quality of the pluripotent stem cells and the culture conditions at the start of differentiation.

To further improve the purity of the cardiomyocytes, an additional purification step was established. On day 14 of differentiation, the cells were detached and analyzed by FACS. Figure 3A shows that a culture counting 9x10 ⁵ /cm ² cells contained 60% (5.4x10 ⁵ /cm ² ) cardiomyocytes on day 14. For the purification method to be successful, the minimum percentage of α-actinin-positive cells should be 60% or greater. The dissociated cells were reseeded (2.7x10 ⁵ /cm ² ) and cultured in expansion medium. After 7 days, the cells were harvested, 4.5x10 ⁵ /cm ² cells were counted, and analyzed. Figure 3B shows that after the purification step, the cardiomyocyte content in the culture increased from 60% to 98%, demonstrating that a highly enriched population of 4.4x10 ⁵ /cm ² of cardiomyocytes was effectively generated using this method. The cells were then transferred to a culture format that met the assay conditions.

Cardiomyocytes were further characterized by immunofluorescence analysis. Figure 4 shows immunofluorescence staining of cardiomyocytes at day 27 using antibodies against α-actinin (green) and troponin T (red), along with a nucleus-specific Hoechst dye (blue). The resulting immunofluorescence staining in Figure 4 shows characteristic α-actinin and troponin T-specific striations in cardiomyocytes.

Activation of beta receptors on the heart induces a positive chronotropic effect in cardiomyocytes. To confirm that pluripotent stem cell-derived cardiomyocytes respond to beta receptor activation, cardiomyocytes were incubated with the beta receptor agonist isoproterenol and then analyzed using the xCELLigence system. Figure 5 shows that after incubation of pluripotent stem cell-derived cardiomyocytes with 3 μM isoproterenol, the beating rate increased from 45 to 60 beats per minute compared to untreated controls. This experiment further demonstrates that the pluripotent stem cell-derived cardiomyocytes generated by this differentiation process are similar to functional human cardiomyocytes.

Freezing and thawing of cardiomyocytes is often difficult due to low levels of cell recovery after thawing.

Since having a large number of identical cells is important for assay development, we tested whether pluripotent stem cell-derived cardiomyocytes can be stored in a refrigerator and then thawed. We tried freezing differentiated cardiomyocytes at different times (days 14, 18, and 32). As shown in Figure 6, cardiomyocytes frozen in the earlier differentiation stages showed higher cell viability after thawing. However, cells thawed after purification on day 18 of differentiation provided the best conditions for using cells for drug assays. At this stage, cells showed much higher purity after thawing, and cardiomyocytes could be directly transferred to a cell culture vessel that met the assay format.

When thawing cardiomyocytes frozen at differentiation day 32, the survival rate was very low, with many cells lost. This is due to the low proliferation rate of cells at this stage, resulting in a low recovery of cardiomyocytes after thawing.

We determined that the optimal time to freeze pluripotent cell-derived cardiomyocytes is after purification on differentiation day 18. At this stage, the recovery rate averages over 85% of α-actinin and troponin T-positive cells, and the cardiomyocytes are still proliferating, providing optimal conditions for further use of the cells in assay development.

Claims (6)

1. An in vitro culture method for differentiating pluripotent stem cells into cardiomyocytes, the method comprising the steps of: a) Provide pluripotent stem cells at a density of 3-7 x ^10⁵ / ^cm² ; b) Incubate the cells in insulin-free medium containing 0.5–5 μM of the following compound for 12–48 hours: c) Incubate the cells in insulin-free medium containing 1-10 μM Wnt-C59 for 24-72 hours. Between steps b) and c), the cells are incubated in insulin-free medium for 24-48 hours. 2. The method of claim 1, further comprising step d) incubating the cells in a culture medium containing insulin. 3. The method of claim 1 or 2, wherein the culture medium in steps b), c) and d) contains vitamin C. 4. The method of any one of claims 1 to 3, wherein the pluripotent stem cells are induced pluripotent stem cells. 5. The method of claim 4, wherein the induced pluripotent stem cells are human cells. 6. The method of claim 4 or 5, wherein the induced pluripotent stem cells are obtained from an individual suffering from a disease caused by cardiac cell dysfunction.