Method of differentiation of pluripotent stem cells to cardiomyocytes

Abstract

The present invention discloses a method for enhancing cardiomyocyte production from induced pluripotent stem cells (iPSCs) by adjusting the culture medium exchange process during the transition from WNT activation to inhibition for differentiation. Instead of completely replacing the culture medium, this innovative technique involves partially changing the medium, which allows for a controlled exposure to WNT activators concurrent with the introduction of WNT inhibitors. This key modification in the protocol markedly increases the consistency and efficiency of cardiomyocyte generation, reducing variability between batches. By fine-tuning the medium exchange process, this method streamlines the creation of cardiomyocytes for use in regenerative medicine, emphasizing the critical adjustment phase within the culture conditions.

Description

PATENT ATTORNEY DOCKET NO.: N2041-03401 METHOD OF DIFFERENTIATION OF PLURIPOTENT STEM CELLS TO CARDIOMYOCYTES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 555,818, filed February 20, 2024, the disclosure of the prior application is considered to be part of and is herein incorporated by reference in the disclosure of this application in its entirety. BACKGROUND FIELD OF THE INVENTION

[0002] The present invention relates generally to cardiomyocytes and more specifically to methods of generating cardiomyocytes from pluripotent stem cells (PSCs). BACKGROUND INFORMATION

[0003] Cell therapeutic approaches are broadly classified according to their mechanism of action: transplantation of cells such as mesenchymal stromal cells (MSCs), which may provide benefits through immunomodulation in a mostly undefined and paracrine manner; the use of therapeutic cells for the specific replacement of degenerated or damaged tissue, thereby restoring its function; and the engineering of cells to actively combat diseases by secreting specific factors or targeting and eliminating unwanted cells, as seen in cancer immunotherapy. Induced pluripotent stem cells (iPSCs) have emerged as a versatile and universal starting material for generating cells to meet these diverse therapeutic needs. The promise of iPSCs lies in their defining characteristics: the ability for self-renewal, which is crucial for clonal isolation and expansion, and pluripotency, which provides the basis for generating virtually any cell type.

[0004] Over the years, dedicated differentiation protocols based on iPSCs have been developed, leading to their application in clinical trials. One notable application is the differentiation of iPSCs into cardiomyocytes, a process that mimics early in vivo cardiogenesis. This involves directing pluripotent stem cells (PSCs) into the mesoderm lineage, followed by cardiac specification to generate cardiomyocytes. Various strategies have been employed to achieve this, including WNT pathway stimulation using small molecules like CHIR99021 and BMP-driven protocols. These pathways may also act synergistically, enhancing the robustness of cardiomyocyte differentiation. For example, the addition of BMP4 can work in concert with WNT signaling, regulating critical target genes in the early differentiation stages. This cooperative action allows for lower effective doses of both WNT and BMP agonists, improving efficiency and consistency across experiments.PATENT ATTORNEY DOCKET NO.: N2041-03401

[0005] However, the transition from bench to bedside faces significant challenges, particularly in meeting the stringent requirements of good manufacturing practice (GMP) for advanced therapeutic medicinal products (ATMPs). Many current iPSC manipulation protocols, primarily developed in academic settings, struggle to comply with GMP standards, which demand rigorous qualification of suppliers, devices, and critical reagents, preferably in GMP quality. These standards aim to simplify ATMP manufacturing workflows and ensure inter-experimental consistency, as failures in GMP compliance can lead to extensive investigations and corrective actions.

[0006] Most existing differentiation protocols exhibit complexities, such as being technically inefficient, inconsistent, or reliant on undefined, complex, or animal-derived components. These protocols may also involve hard-to-qualify or GMP-incompatible equipment and are often inefficient in terms of cost and process duration.

[0007] Given these challenges, there is a pressing need to develop methodologies for generating cardiomyocytes from PSCs that are robust, efficient, and compatible with GMP standards. Such methodologies would enable the reliable production of cardiomyocytes for therapeutic applications, overcoming the current limitations and fulfilling the promise of PSC technology in regenerative medicine. SUMMARY OF THE INVENTION

[0008] The present invention identifies a critical advancement in the differentiation of PSCs into cardiomyocytes through the novel approach of conducting a partial medium exchange during the transition from WNT signaling activation to WNT signaling inhibition. This counterintuitive method, which diverges from the traditional practice of fully replacing the culture medium to reverse signaling pathway activities, has been found to significantly enhance the reproducibility and robustness of cardiomyocyte generation. By maintaining a degree of exposure to WNT activators while commencing WNT inhibition, this technique facilitates a more consistent and efficient cardiac specification process. The invention posits that this partial medium exchange strategy is vital for overcoming batch-to-batch variations, thereby laying the groundwork for a truly reliable methodology in the field of regenerative medicine and therapeutic applications.

[0009] In one aspect, the present invention provides a method of inducing cardiomyocyte differentiation from pluripotent stem cells (PSCs) comprising: a) contacting PSCs in a culture medium for one or more days wherein the culture medium comprises a WNT signaling pathway activator and: (i) a bone morphogenetic protein (BMP) signaling pathway activator; (ii) optionally an fibroblast growth factor (FGF) / extracellular signaling-regulated kinase (ERK) signaling pathway activator; and (iii) optionally a TGF-β signaling pathway activator; and b) partiallyPATENT ATTORNEY DOCKET NO.: N2041-03401 exchanging the culture medium from a) with a culture medium comprising a WNT signaling pathway inhibitor and further culturing for one or more days, thereby inducing the PSCs to differentiate into cardiomyocytes.

[0010] As featured and disclosed herein, activators and inhibitors of the FGF / ERK signaling pathway include, as non-limiting examples, signaling pathway ligands, small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the FGF signaling pathway, resulting in a corresponding activation or inhibition in cellular FGF signaling. In some aspects of this disclosure, activation of the FGF signaling pathway and / or the mitogen- activated protein kinase (MAPK) / ERK signaling pathway is achieved through repression of a FGF signaling pathway inhibitor and / or a MAPK / ERK signaling pathway inhibitor, including, as non- limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the FGF signaling pathway and / or the MAPK / ERK signaling pathway or an antibody or small molecule directed to a FGF signaling pathway inhibitor and / or MAPK / ERK signaling pathway inhibitor. In some embodiments, the FGF / ERK signaling pathway activator is FGF2. In some aspects, the activator of the FGF signaling pathway may also include an activator or inhibitor of related signal transduction pathways including, as a non-limiting example, the MAPK / ERK signal transduction pathway.

[0011] In some embodiments, the concentration of FGF / ERK signaling pathway activator or inhibitor ranges from between about 2.5 ng / ml to 20 ng / ml. In some embodiments, the FGF / ERK signaling pathway activator is FGF2 at a concentration ranging from between about 2.5 ng / ml to 20 ng / ml. In some embodiments, the FGF / ERK signaling pathway activator is at a concentration that ranges from 2.5 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 11 ng / ml, from 11 to 12 ng / ml, from 12 to 13 ng / ml, from 13 to 14 ng / ml, from 14 to 15 ng / ml, from 15 to 16 ng / ml, from 16 to 17 ng / ml, from 17 to 18 ng / ml, from 18 to 19 ng / ml, from 19 to 20 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, FGF2 is at a concentration of about 7.5 ng / ml. As featured and disclosed herein, activators and inhibitors of the transforming growth factor beta (TGF-β) superfamily include, as non-limiting examples, signaling pathway ligands, small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the TGF-β signaling pathway, resulting in a corresponding activation or inhibition in cellular TGF-β signaling. In some aspects of this disclosure, activation of the TGF-β signalingPATENT ATTORNEY DOCKET NO.: N2041-03401 pathway is achieved through repression of a TGF-β signaling pathway inhibitor, including, as non- limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the TGF-β signaling pathway or an antibody or small molecule directed to a TGF-β signaling pathway inhibitor. In some aspects, the activator of the TGF-β signaling pathway may also include an activator or inhibitor of related signal transduction pathways, including, as non-limiting examples, the bone morphogenetic protein (BMP), ERK / MAPK, phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt), and non-receptor tyrosine kinase 1 (SRC) signaling pathways. In some embodiments, the TGF-β signaling pathway activator comprises Activin A. In some embodiments, the concentration of the TGF-β signaling pathway activator or inhibitor ranges between about 2.5 ng / ml to 20 ng / ml. In some embodiments, the TGF-β signaling pathway activator is at a concentration ranging from between about 2.5 ng / ml to 20 ng / ml. In some embodiments, the TGF-β signaling pathway activator is at a concentration that ranges from 2.5 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 11 ng / ml, from 11 to 12 ng / ml, from 12 to 13 ng / ml, from 13 to 14 ng / ml, from 14 to 15 ng / ml, from 15 to 16 ng / ml, from 16 to 17 ng / ml, from 17 to 18 ng / ml, from 18 to 19 ng / ml, from 19 to 20 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, Activin A is at a concentration of about 7.5 ng / ml.

[0012] As featured and disclosed herein, activators of the BMP pathway include, as non-limiting examples, BMP family ligands, e.g., BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP8b, BMP10, BMP11, and BMP 15; Alantolactone; FK506; isoliquiritigenin; and 4’- hydroxychalcone. In some embodiments, activation of the BMP pathway may be achieved through repression of a BMP pathway inhibitor, including, as a non-limiting example, the use of an inhibitory nucleic acid targeting an inhibitor of the BMP pathway or an antibody or small molecule directed to a BMP pathway inhibitor. In some embodiments, the BMP pathway activator comprises BMP4. In some embodiments, the BMP pathway activator is at a concentration ranging from between about 1 ng / ml to 10 ng / ml. In some embodiments, the BMP pathway activator is at a concentration that ranges from 1 to 2 ng / ml, from 2 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, BMP4 is at a concentration of about 5 ng / ml. In some embodiments, BMP4 is at a concentration of about 7.5 ng / ml.

[0013] In some embodiments, the volume of the partial culture medium exchange as featured and described herein is in a volume ranging from between about 5% to 90%. In some embodiments,PATENT ATTORNEY DOCKET NO.: N2041-03401 the volume of the culture medium exchanged in b) is about 10%, 25%, 50%, or 75% of the culture medium in a). In some embodiments, the volume of the culture medium exchanged in step b) is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90%, or any percentage of the culture medium in a) ranging between any two percentages referred to above or herein). In some embodiments, the volume of the culture medium exchanged in step b) is about 25% of the culture medium in a).

[0014] As featured and disclosed herein activators of the WNT signaling pathway include, as non- limiting examples, CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)- 2pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile); WNT family ligands; RSPO co- agonists; lithium chloride; TDZD8 (4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione); BIO- Acetoxime ((2’Z,3’E)-6-Bromoindirubin-3′-acetoxime); A1070722 (1-(7-Methoxyquinolin- 4-yl)-3-[6 (trifluoromethyl)pyridin-2-yl]urea); HLY78 (4-Ethyl-5,6-Dihydro-5-methyl- [1,3]dioxolo[4,5-j]phenanthridine); CID 11210285 hydrochloride (2-Amino-4-(3,4- (methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride); WAY-316606; (hetero) arylpyrimidines; IQ1; QS11; SB-216763; and / or DCA. In some embodiments, activation of the WNT signaling pathway may be achieved through repression of a WNT signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the WNT signaling pathway or an antibody or small molecule directed to a WNT signaling pathway inhibitor. In some aspects, the WNT signaling pathway activator is added to the PSC culture at a concentration that ranges between about 0.1 µM to 10 µM. In some embodiments, the WNT signaling pathway activator is added to the PSC culture at a concentration that ranges from 0.1 to 0.5 µM, from 0.5 to 1.0 µM, from 1.0 to 2.0 µM, from 2.0 to 3.0 µM, from 3.0 to 4.0 µM, from 4.0 to 5.0 µM, from 5.0 to 6.0 µM, from 6.0 to 7.0 µM, from 7.0 to 8.0 µM, from 8.0 to 9.0 µM, from 9.0 to 10 µM, or ranging between any two concentrations referred to above or herein. In some embodiments, the WNT signaling pathway activator comprises CHIR99021. In some embodiments, the WNT signaling pathway activator is CHIR99021 at a concentration ranging from between about 0.5 µM to about 1 µM. In some embodiments, CHIR99021 is at a concentration of about 0.5 µM.

[0015] As featured and disclosed herein are culture medium supplements including or excluding vitamins, proteins, and amino acids. In some embodiments, the vitamin supplements include or exclude vitamin C as ascorbate, ascorbic acid, salts thereof, or derivatives thereof. In some embodiments, the culture medium comprises ascorbate. In some embodiments, the ascorbate is 2- phospho-L-ascorbate. In some embodiments, the culture medium optionally comprises one or more salts or derivatives of 2-phospho-L-ascorbate. In some embodiments, protein supplementsPATENT ATTORNEY DOCKET NO.: N2041-03401 include or exclude albumin, transferrin, fibronectin, aprotinin, and fetuin. In some embodiments, the protein supplement is albumin. In some embodiments, the protein supplement is human serum albumin (HSA). In some embodiments, the amino acid supplements include or exclude glutamine. In some embodiments, the glutamine is L-glutamine. In some embodiments, the vitamin supplement is ascorbate or 2-phospho-L-ascorbate, or any salt or any derivative thereof, at a concentration ranging from between about 50 µM to 500 µM. In some embodiments, the vitamin supplement is at a concentration that ranges from 50 to 100 µM, 100 to 150 µM, from 150 to 200 µM, from 200 to 250 µM, from 250 to 300 µM, from 300 to 350 µM, from 350 to 400 µM, from 450 to 500 µM, or ranging between any two concentrations referred to above or herein. In some embodiments, the protein supplement is albumin at a concentration ranging from between about 0.5 mg / ml to 10 mg / ml. In some embodiments, the protein supplement is at a concentration that ranges from 0.5 to 1.0 mg / ml, 1.0 to 2.0 mg / ml, from 2.0 to 3.0 mg / ml, from 3.0 to 4.0 mg / ml, from 4.0 to 5.0 mg / ml, from 5.0 to 6.0 mg / ml, from 6.0 to 7.0 mg / ml, from 7.0 to 8.0 mg / ml, from 8.0 to 9.0 mg / ml, from 9.0 to 10 mg / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, the amino acid supplement is glutamine at a concentration ranging from between about 0.2 mM to 4 mM. In some embodiments, the amino acid supplement is at a concentration that ranges from 0.2 to 0.5 mM, 0.5 to 1.0 mM, 1.0 to 2.0 mM, from 2.0 to 3.0 mM, from 3.0 to 4.0 mM, or ranging between any two concentrations referred to above or herein. In some embodiments, the culture medium from a) and / or b) further comprises 2-phospho- L-ascorbate and / or albumin, and optionally L-glutamine. In some embodiments, the culture medium is supplemented with about 250 µM 2-phospho-ascorbate and / or about 5 mg / ml albumin, and optionally about 2 mM L-glutamine.

[0016] In some embodiments, the contacting in a), that is, the PSCs are grown in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator, is for at least 2 days. In some embodiments, the contacting in a) is for at least 1, 2, 3, 4, 5, 6, 7, or 8, or more days, or for any length of time ranging between any two of the recited number of days referred to above or herein. In some embodiments, the contacting in a) is for 1 day. In some embodiments, the contacting in a) is for 2 days. In some embodiments, the contacting in a) is for 4 days. In some embodiments, the contacting in a) is for 5 days. In some embodiments, the contacting in a) is for 6 days. In some embodiments, the contacting in a) is for 7 days. In some embodiments, the contacting in a) is for 8 days. In some embodiments, the contacting in a) is for more than 8 days.

[0017] In some embodiments, the incubation in b) follows the culture medium from contacting PSCs in a) being partially exchanged with a culture medium comprising a WNT signaling pathway inhibitor for 2 to 5 days. In some embodiments, the incubation in b) is for 1 day, 2 days, 3 days, 4,PATENT ATTORNEY DOCKET NO.: N2041-03401 days, or 5 days. In some embodiments, the incubation in b) is for more than 5 days. In some embodiments, the incubation in b) is for at least 1, 2, 3, 4, 5, 6, 7, or 8, or more days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the incubation in b) is for 1 day. In some embodiments, the incubation in b) is for 2 days. In some embodiments, the incubation in b) is for 4 days. In some embodiments, the incubation in b) is for 5 days. In some embodiments, the incubation in b) is for 6 days. In some embodiments, the incubation in b) is for 7 days. In some embodiments, the incubation in b) is for 8 days. In some embodiments, the incubation in b) is for more than 8 days. In some embodiments, the incubation in b) is for 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein.

[0018] As featured and disclosed herein, inhibitors of the WNT signaling pathway include, as non- limiting examples, C-59 (4-(2-Methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide), DKK1, IWP2 (N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2- d]pyrimidin-2-yl)thio]-acetamide), Ant1.4Br, Ant1.4CI, Niclosamide, apicularen, bafilomycin, XAV939 (3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4- one), IWR1 (4-(1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8- quinolinyl-Benzamide), NSC668036 (N-[(1,1-Dimethylethoxy)carbonyl]-L-alanyl-(2S)-2- hydroxy-3-methylbutanoyl-L-Alanine-(1S)-1-carboxy-2-methylpropyl ester hydrate), 2,4- diamino-quinazoline, Quercetin, ICG-001 ((6S,9aS)-Hexahydro-6-[(4-hydroxyphenyl)methyl]-8- (1-naphthalenylmethyl)-4,7-dioxo-N-(phenylmethyl)-2H-pyrazino[1,2-a]pyrimidine-1(6H)- carboxamide), PKF115-584, BML-284 (2-Amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3- methoxyphenyl)pyrimidine), FH-535, iCRT-14, JW-55, JW-67, antibodies to WNTs and WNT receptors, WNT inhibitory nucleic acids. In some embodiments, inhibition of the WNT signaling pathway may be achieved through repression of a WNT signaling pathway activator, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an activator of the WNT signaling pathway or an antibody or small molecule directed to a WNT signaling pathway activator. In some embodiments, the WNT signaling pathway inhibitor is selected from IWP2, IWR1, or C-59. In some embodiments, the concentration of the WNT signaling pathway inhibitor is at least 0.1 µM. In some embodiments, the WNT signaling pathway inhibitor is added to the PSC culture at a concentration that ranges from 0.05 to 0.06 µM, 0.06 to 0.07 µM, 0.07 to 0.08 µM, 0.08 to 0.09 µM, 0.09 to 0.1 µM, 0.1 to 0.2 µM, 0.2 to 0.3 µM, 0.3 to 0.4 µM, 0.4 to 0.5 µM, from 0.5 to 0.6 µM, 0.6 to 0.7 µM, 0.7 to 0.8 µM, 0.8 to 0.9 µM, 0.9 to 1 µM from 1.0 to 2.0 µM, from 2.0 to 3.0 µM, from 3.0 to 4.0 µM, from 4.0 to 5.0 µM, from 5.0 to 6.0 µM, from 6.0 to 7.0PATENT ATTORNEY DOCKET NO.: N2041-03401 µM, from 7.0 to 8.0 µM, from 8.0 to 9.0 µM, from 9.0 to 10 µM, or ranging between any two concentrations referred to above or herein. In some embodiments, the concentration of WNT signaling pathway inhibitor is about 2 µM. In some embodiments, C-59, IWP2, IWR1, or a combination thereof is about 2 µM.

[0019] In some embodiments, the cardiomyocytes express maturation markers after about 4 weeks of differentiation. In some embodiments, the cardiomyocytes express maturation markers after about 5 weeks of differentiation. In some embodiments, the cardiomyocytes express maturation markers after about 6 weeks of differentiation. In some embodiments, the cardiomyocytes express maturation markers after more than about 6 weeks of differentiation.

[0020] As featured and disclosed herein, cardiomyocyte maturation markers include, as non- limiting examples, CAS12, S100A4, CASQ1, ADRB1, ADRB2, ATP2A2, RYR2, PLN, JPH2, CAMK2D, ATP1A3, KCNJ2, CACNA2D1, KCNQ1, SCN5A, KCNE1, SLC8A1, KCNH2, SCN4B, KCNE5, MYH6, MYL2, TNNI3, MYM2, MYH7, TPM1, MYL3, TNNC1, TNNT2, ACTC1, LPL, ACOX1, PPARA, PPARD, ACAT1, ACADVL, DGAT1, PPARGC1A, HADHB, AND ACSL1. In some embodiments, the maturation markers comprise MYL2, MYH7, and / or KCNQ1.

[0021] In some embodiments, the cardiomyocyte differentiation is performed in a culture selected from: a two-dimensional culture, a three-dimensional culture, a suspension culture, and an adherent culture.

[0022] In some embodiments, the culture is a plate, a microcarrier bead or a mesh substrate.

[0023] In some embodiments, cardiac troponin T (cTnT) positive cells are observed after 8 days of differentiation. In some embodiments, cTnT positive cells comprise at least 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of PSCs after at least 8 days of differentiation. In some embodiments, cardiac troponin T (cTnT) positive cells comprise at least 80% of the PSCs after 9 days of differentiation.

[0024] In some embodiments, the culture medium from a) and b) is Knockout-DMEM or IMDM.

[0025] In some embodiments, the PSCs are human PSCs (hPSCs).

[0026] In some embodiments, the hPSCs are human induced pluripotent stem cells (hiPSCs) or human embryonic stems cells (hESCs).

[0027] In one aspect, the present invention provides a method of inducing cardiomyocyte differentiation from pluripotent stem cells (PSCs) comprising: a) contacting PSCs with an iPSC culture medium for at least one day, wherein the iPSC culture medium comprises a Rho-associated kinase (ROCK) inhibitor, which can be any ROCK inhibitor featured in this disclosure, to obtain ROCK-inhibitor cultured cells; b) contacting the ROCK-inhibitor cultured cells with a mesodermPATENT ATTORNEY DOCKET NO.: N2041-03401 induction medium for one or more days, wherein the mesoderm induction medium comprises: (i) a WNT signaling pathway activator, which can be any WNT signaling pathway activator featured in this disclosure; (ii) a bone morphogenetic protein (BMP) signaling pathway activator, which can be any BMP signaling pathway activator featured in this disclosure; (iii) optionally an FGF / ERK signaling pathway activator, which can be any FGF / ERK signaling pathway activator featured in this disclosure; and (iv) optionally a TGF-β signaling pathway activator, which can be any TGF-β signaling pathway activator featured in this disclosure; and c) partially exchanging the culture medium from a) with a culture medium comprising a WNT signaling pathway inhibitor and further culturing for one or more days, thereby inducing the PSCs to differentiate into cardiomyocytes.

[0028] As featured and disclosed herein, ROCK inhibitors include, as non-limiting examples, Y- 27632, ripasudil, netarsudil, chroman 1, Y39983, Wf-536, SLx-2119, Azabenzimidazole- aminofurazans, DE-104 Olefins, Isoquinolines, Indazoles, pyridinealkene derivatives, H-1152P, XD-4000, HMN-1152, ROKα 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, rhostatin, BA-210, BA-207, BA-285, BA-1037, BA-215, Ki-23095, VAS-012, quinazoline,GSK- 269962, and salts thereof, including Y-27632 dihydrochloride, H-1152 dihydrochloride, fasudil hydrochloride, SR-3677 dihydrochloride, glycyl-H-1152 dihydrochloride, SB-772077B dihydrochloride, TC-S-7001, RKI-1447 dihydrochloride, and OXA-06 dihydrochloride.

[0029] In some embodiments, the concentration of ROCK inhibitor is in a range between about 1 µM to about 15 µM. In some embodiments, the ROCK inhibitor is Y-27632 at concentration of about 10 µM. In some embodiments, the ROCK inhibitor is at a concentration that ranges from 1.0 to 2.0 µM, from 2.0 to 3.0 µM, from 3.0 to 4.0 µM, from 4.0 to 5.0 µM, from 5.0 to 6.0 µM, from 6.0 to 7.0 µM, from 7.0 to 8.0 µM, from 8.0 to 9.0 µM, from 9.0 to 10 µM, from 10 to 11 µM, from 11 to 12 µM, from 12 to 13 µM, from 13 to 14 µM, from 14 to 15 µM, or ranging between any two concentrations referred to above or herein. INCORPORATION BY REFERENCE

[0030] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document wasPATENT ATTORNEY DOCKET NO.: N2041-03401 specifically and individually indicated to be incorporated by reference. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Incorporated by reference as if set forth in full herein is U.S. Provisional Patent Application 63 / 555,818. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0032] FIG.1A schematically illustrates an optimized cardiac induction protocol based on initial co-stimulation of the FGF, TGFβ, BMP, and WNT pathways. Differentiation commenced upon, not after, EB formation. F: FGF / ERK pathway stimulation; T: TGFβ pathway stimulation; B: BMP pathway stimulation; W: stimulation of the canonical WNT pathway; αW: inhibition of canonical WNT signaling. ITS: Insulin / transferrin / selenium supplementation, Y: Rho-associated kinase (ROCK) inhibitor Y-27632 (promoting cell survival of hPSCs), AscP: 2-phospho-L-ascorbate, HSA: Human serum albumin, Gln: L-Glutamine, KO-DMEM: Knockout™-DMEM.

[0033] FIG. 1B illustrates a cardiac differentiation assessment over 10 independent experiments using an optimized protocol (open dots) and a semi-optimized protocol (black dots). The difference between the two is that a partial medium change (< 75%) is applied from the pathway stimulation to inhibition stage in the left panel, whereas the media exchange was >75% in the semi-optimized case.

[0034] FIG.1C shows the titration of 2-phospho-L-ascorbate supplementation.

[0035] FIG. 2 illustrates the significance of the partial medium change from the WNT pathway stimulation stage to the WNT pathway inhibition stage. Comparative flow cytometry data from various independent experiments comparing different amounts of media exchanges. Note that the most robust differentiation outcomes with high and consistent cardiomyocyte yield between independent experiments are obtained at 25% media exchange or below.

[0036] FIG. 3A illustrates that BMP and WNT co-stimulation (without additional FGF / TGFβ activation) enables superior cardiomyocytes differentiation as compared to WNT stimulation alone (n=2-10 per data point).

[0037] FIG. 3B illustrates the dependency of the BMP+WNT protocol on cell titer (flow cytometry data).PATENT ATTORNEY DOCKET NO.: N2041-03401

[0038] FIG. 3C shows a numerical representation of a heat map of grid-type titration of BMP4 and CHIR99021 concentrations for optimizing cardiac mesoderm induction. Note that WNT activation alone was inferior to co-stimulation of the BMP and WNT pathways.

[0039] FIG. 4A illustrates the titration of the optimal combined dose of FGF2 and Activin A supplementation.

[0040] FIG.4B shows that adding more FGF and TGFβ pathway stimulation makes the protocol more reliable when it comes to cell titer dependence (n = 6–8; dropouts were scored at 0%).

[0041] FIG. 5 illustrates the comparison of different protocol variants. Numbers show % cTnT values obtained by flow cytometry, indicating the purity of the iPSC-cardiomyocytes obtained. Note the flexibility in terms of the duration of the pathway stimulation period (1 or 2 days tested). As an alternative to treating dissociated hPSCs upon 3D aggregate formation (first 4 rows), these may also be pre-formed in hPSC media. Overall, the data show that WNT stimulation alone may be enough to start the heart beating, even if the pathway is stimulated for a longer time or 3D aggregates are not formed first. However, stimulation with both BMP and WNT together gives more consistent results across separate experiments and, on average, leads to higher differentiation efficiencies.

[0042] FIGS. 6A and 6B illustrate the independence of cardiac differentiation from the basal medium. FIG.6A schematically illustrates a cardiac induction protocol using IMDM as the basal medium. B: BMP pathway stimulation; W: stimulation of the canonical WNT pathway; Wi: inhibition of canonical WNT signaling. ITS: Insulin / transferrin / selenium supplementation; AscP: 2-phospho-L-ascorbate; HSA: Human serum albumin; Gln: L-Glutamine. FIG. 6B shows flow cytometry analysis at day 9 of cardiac differentiation using IMDM as the basal medium for cTnT as a proof-of-concept experiment.

[0043] FIG. 7 illustrates the importance of WNT inhibition for cardiac induction following signaling pathway activation. Flow cytometry analysis for cardiac troponin T (cTnT) after 9 days of differentiation with different amounts of C-59, a WNT inhibitor. Results indicate a wide working range of concentrations without apparent cytotoxicity (morphological observations). Note that no WNT inhibition leads to a poor percentage of cardiomyocytes (upper panel). The treatment time for WNT inhibition is flexible and can be extended to at least day 7 (based on representative cTnT flow cytometry data) (bottom panel).

[0044] FIG. 8 shows the importance of the additives L-ascorbate and albumin (HSA). Gene expression analysis after cardiac induction supplemented with the additives at the indicated stages. The synergistic effect of ASC and HSA was shown to be that neither compound alone was able toPATENT ATTORNEY DOCKET NO.: N2041-03401 support robust cardiac induction (a single experiment conducted under semi-optimized conditions of the protocol).

[0045] FIG.9A shows the results of the RNA-Seq analysis. RNA-seq analysis shows that markers and annotations for the cardiac differentiation cluster are much more abundant for important cardiac genes than for other differentiated cell types.

[0046] FIG. 9B illustrates RT-qPCR analysis confirming expected temporal patterns of early / immature (pink), pan / stable (grey), and maturation markers of iPSC-CMs in 3D (n=3). Note the exchange of myosin light and heavy chain-encoding genes by alternative variants.

[0047] FIG. 9C shows an immunostaining of iPSC-CMs under adherent conditions. Immunostaining of iPSC-CMs matured under adherent conditions, showing a pronounced sarcomeric structure. DETAILED DESCRIPTION OF THE INVENTION

[0048] The essence of the present invention lies in the innovative method of partially, rather than completely, exchanging the culture medium when transitioning PSCs from WNT activation to inhibition conditions for cardiomyocyte differentiation. This nuanced approach, moving away from conventional full medium changes, significantly improves the consistency and efficiency of producing cardiomyocytes by maintaining limited exposure to WNT activators alongside the initiation of WNT inhibition. This method crucially minimizes batch-to-batch variations, establishing a more reliable and effective protocol for cardiomyocyte generation, thereby advancing the field of regenerative medicine and its therapeutic applications.

[0049] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only to the appended claims.

[0050] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” include one or more methods and / or steps of the type described herein, which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0051] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.PATENT ATTORNEY DOCKET NO.: N2041-03401

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skills in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0053] In one aspect, the present invention provides a method of inducing cardiomyocyte differentiation from pluripotent stem cells (PSCs) comprising: a) contacting PSCs in a culture medium for one or more days wherein the culture medium comprises a WNT signaling pathway activator and: (i) a bone morphogenetic protein (BMP) signaling pathway activator; (ii) optionally an FGF / ERK signaling pathway activator; and (iii) optionally a TGF-β signaling pathway activator; and b) partially exchanging the culture medium from a) with a culture medium comprising a WNT signaling pathway inhibitor and further culturing for one or more days, thereby inducing the PSCs to differentiate into cardiomyocytes.

[0054] The methods described herein offer valuable applications in the economical and reproducible generation of human cardiomyocyte progenitors or cardiomyocytes. Specifically, these methods facilitate the generation of cardiomyocyte progenitors or cardiomyocytes by employing a partial exchange of culture medium when transitioning induced pluripotent stem cells (iPSCs) from WNT activation to inhibition conditions, potentially advancing their application in regenerative therapies.

[0055] By “contacting” it is meant that the cells are cultured with one or more agents of interest, added to the defined basal or supplemented medium. That is, the cells are cultured in their regular culture basal or supplemented medium, in which a desired concentration of one or more agents of interest is added. For example, the cells are cultured with a BMP signaling pathway activator, a WNT signaling pathway activator and / or inhibitor, an FGF / ERK signaling pathway activator, and a TGF-β signaling pathway activator. As described herein, the cells are cultured with a Rho- associated kinase (ROCK) inhibitor are referred to as ROCK-inhibitor cultured cells.

[0056] A “signaling pathway activator,” as used herein, refers to any molecule that is capable of activating, enhancing, or inducing a signaling pathway of interest. A signaling pathway is a series of chemical reactions in which a group of molecules in a cell work together to control a cell function, such as cell differentiation. A cell receives signals from its environment when a molecule, such as a hormone or growth factor, binds to a specific protein receptor on or in the cell. After the first molecule in the pathway receives a signal, it activates another molecule. This process is repeated through the entire signaling pathway until the last molecule is activated and cell functionPATENT ATTORNEY DOCKET NO.: N2041-03401 is carried out. Abnormal activation of signaling pathways or inhibition of a signaling pathway may lead to diseases or, in the case of pluripotent cells, to an alteration of the pluripotent state and therefore to differentiation. The term “molecule” includes, as non-limiting examples, small molecules (including small molecules that do not have optimal cell permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers, and other polymers, for example proteins, peptides, hormones, carbohydrates, or polyamines. Non-limiting examples of polynucleotides include short interfering nucleic acid (siNA), antisense, enzymatic nucleic acid molecules, 2',5'-oligoadenylate, triplex forming oligonucleotides, aptamers, and decoys. Biologically active molecules include antibodies (e.g., monoclonal, chimeric, humanized, etc.), cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys, and analogs thereof, and small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), antagomirs, and short hairpin RNA (shRNA) molecules.

[0057] In some embodiments, the disclosure pertains to deriving cardiomyocytes by contacting pluripotent stem cells (PSCs) with a cardiomyocyte induction composition. As detailed herein, the composition of cardiomyocyte induction may vary and generally includes effective amounts of one or more of the following: a BMP signaling pathway activator, a WNT signaling pathway activator and / or inhibitor, an FGF / ERK signaling pathway activator, and a TGF-β signaling pathway activator. In some embodiments, an inducing agent useful for the induction of cardiomyocytes may include an activator or inhibitor of the BMP signaling pathway. Activators and inhibitors of the BMP signaling pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like, that activate or inhibit at least one component of the BMP signaling pathway, resulting in a corresponding activation or inhibition of cellular BMP signaling.

[0058] The transforming growth factor beta (TGF-β) superfamily includes TGF-β proteins, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), glial-derived neurotrophic factors (GDNFs), Activins, Inhibins, Nodal, Lefty, and Mülllerian inhibiting substance (MIS). Bone morphogenetic proteins (BMPs) are a group of growth factors also known as cytokines and metabologens. Originally discovered for their ability to induce the formation of bone and cartilage, BMPs are now considered to constitute a group of pivotal morphogenetic signals, orchestratingPATENT ATTORNEY DOCKET NO.: N2041-03401 tissue architecture throughout the body. The important functioning of BMP signals in physiology is emphasized by the multitude of roles for dysregulated BMP signaling in pathological processes.

[0059] BMPs interact with specific receptors on the cell surface, referred to as bone morphogenetic protein receptors (BMPRs). Signal transduction through BMPRs results in the mobilization of members of the SMAD family of proteins. The signaling pathways involving BMPs, BMPRs, and SMADs are important in the development of the heart, central nervous system, and cartilage, as well as postnatal bone development. They have an important role during embryonic development in embryonic patterning and early skeletal formation. As such, disruption of BMP signaling can affect the body plan of the developing embryo. For example, BMP4 and its inhibitors, noggin and chordin, help regulate the polarity of the embryo (i.e., back-to-front patterning). Specifically, BMP4 and its inhibitors play a major role in neurulation and the development of the neural plate. BMP4 signals ectoderm cells to develop into skin cells, but the secretion of inhibitors by the underlying mesoderm blocks the action of BMP4 to allow the ectoderm to continue on its normal course of neural cell development.

[0060] Activators of the BMP pathway include, as non-limiting examples, BMP family ligands, e.g., BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP8b, BMP10, BMP11, and BMP 15; Alantolactone; FK506; isoliquiritigenin; and 4’-hydroxychalcone. In some embodiments, activation of the BMP pathway may be achieved through repression of a BMP pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the BMP pathway, or an antibody or small molecule directed to a BMP pathway inhibitor. In some embodiments, the BMP pathway activator comprises BMP4.

[0061] The WNT signaling pathways are a group of signal transduction pathways that begin with proteins that pass signals into a cell through cell surface receptors. WNT signaling pathways use either nearby cell-cell communication (paracrine) or same-cell communication (autocrine). Three WNT signaling pathways have been characterized: the canonical WNT pathway, the noncanonical planar cell polarity pathway, and the noncanonical WNT / calcium pathway. All three pathways are activated by the binding of a WNT-protein ligand to a Frizzled family receptor, which passes the biological signal to the Dishevelled protein inside the cell. The canonical WNT pathway leads to regulation of gene transcription and is thought to be negatively regulated in part by the SPATS1 gene. The noncanonical planar cell polarity pathway regulates the cytoskeleton, which is responsible for the shape of the cell. The noncanonical WNT / calcium pathway regulates calcium inside the cell. WNT signaling was first identified for its role in carcinogenesis, then for its function in embryonic development. The embryonic processes controlled by WNT signaling include body axis patterning, cell fate specification, cell proliferation, and cell migration. ThesePATENT ATTORNEY DOCKET NO.: N2041-03401 processes are necessary for the proper formation of important tissues, including bone, heart, and muscle. Its role in embryonic development was discovered when genetic mutations in WNT pathway proteins produced abnormal fruit fly embryos. Later research found that the genes responsible for these abnormalities also influenced breast cancer development in mice. WNT signaling also controls tissue regeneration in adult bone marrow, skin, and intestine.

[0062] In some embodiments, an inducing agent useful for the induction of cardiomyocytes may include an activator or inhibitor of the WNT signaling pathway. Activators and inhibitors of the WNT signaling pathway include, as non-limiting examples, small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the WNT signaling pathway, resulting in a corresponding activation or inhibition of cellular WNT signaling.

[0063] Activators of the WNT signaling pathway include, as non-limiting examples, CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)- 2pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile); WNT family ligands including, as non- limiting examples, Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt- 7a / b, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-10a, Wnt-10b, Wnt-11, and Wnt-16b; RSPO co-agonists (e.g., RSPO2); lithium chloride; TDZD8 (4-Benzyl-2-methyl-1,2,4- thiadiazolidine-3,5-dione); BIO-Acetoxime ((2’Z,3’E)-6-Bromoindirubin-3′-acetoxime); A1070722 (1-(7-Methoxyquinolin-4-yl)-3-[6 (trifluoromethyl)pyridin-2-yl]urea); HLY78 (4- Ethyl-5,6-Dihydro-5-methyl-[1,3]dioxolo[4,5-j]phenanthridine); CID 11210285 hydrochloride (2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride); WAY-316606; (hetero) arylpyrimidines; IQ1; QS11; SB-216763; DCA; and the like. In some embodiments, activation of the WNT signaling pathway may be achieved through repression of a WNT signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the WNT signaling pathway or an antibody or small molecule directed to a WNT signaling pathway inhibitor.

[0064] In some embodiments, the WNT signaling pathway activator comprises CHIR99021. In some embodiments, the WNT signaling pathway activator comprises a derivative of CHIR99021. In some embodiments, one or more WNT signaling pathway activators is used.

[0065] Inhibitors of the WNT signaling pathway include, as non-limiting examples, C-59 (4-(2- Methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide), DKK1, IWP2 (N-(6-Methyl- 2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]- acetamide), Ant1.4Br, Ant1.4CI, Niclosamide, apicularen, bafilomycin, XAV939 (3,5,7,8- Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), IWR1 (4-PATENT ATTORNEY DOCKET NO.: N2041-03401 (1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-Benzamide), NSC668036 (N-[(1,1-Dimethylethoxy)carbonyl]-L-alanyl-(2S)-2-hydroxy-3-methylbutanoyl-L- Alanine-(1S)-1-carboxy-2-methylpropyl ester hydrate), 2,4-diamino-quinazoline, Quercetin, ICG- 001 ((6S,9aS)-Hexahydro-6-[(4-hydroxyphenyl)methyl]-8-(1-naphthalenylmethyl)-4,7-dioxo-N- (phenylmethyl)-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxamide), PKF115-584, BML-284 (2- Amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine), FH-535, iCRT- 14, JW-55, JW-67, antibodies to WNTs and WNT receptors, WNT inhibitory nucleic acids, and the like.

[0066] In some embodiments, the WNT signaling pathway inhibitor is selected from IWP2, IWR1, or C-59. In some embodiments, the WNT signaling pathway inhibitor comprises a derivative of IWP2, IWR1, or C-59. In some embodiments, one or more WNT signaling pathway inhibitors is used.

[0067] In some embodiments, an inducing agent useful for the induction of cardiomyocytes may include an activator or inhibitor of the FGF signaling pathway. In some instances, an activator or inhibitor of the FGF signaling pathway may also include activators or inhibitors of related signal transduction pathways, including, as a non-limiting example, the MAPK / ERK signal transduction pathway. Activators and inhibitors of the FGF signaling pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the FGF signaling pathway, resulting in a corresponding activation or inhibition in cellular FGF signaling.

[0068] Activators of the FGF signaling pathway and / or the MAPK / ERK signaling pathway include, as non-limiting examples: FGF family ligands including, as non-limiting examples, FGF1, FGF2, FGF-3, FGF-4, FGF-5, FGF-6, KGF / FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-15, FGF-16, FGF-17, FGF-19, FGF-20, FGF-21, FGF-22, and FGF-23; SUN 11602 (4-[[4-[[2-[(4-Amino-2,3,5,6-tetramethylphenyl)amino]acetyl]methylamino]-1- piperidinyl]methypenzamide); t-Butylhydroquinone; U-46619; C2 Ceramide; Lactosyl Ceramide; Angiotensin II; Baicalin; and the like. In some embodiments, activation of the FGF signaling pathway and / or the MAPK / ERK signaling pathway may be achieved through repression of a FGF signaling pathway inhibitor and / or the MAPK / ERK signaling pathway inhibitor, including, as non- limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the FGF signaling pathway and / or the MAPK / ERK signaling pathway or an antibody or small molecule directed to a FGF signaling pathway inhibitor and / or MAPK / ERK signaling pathway inhibitor.PATENT ATTORNEY DOCKET NO.: N2041-03401

[0069] In some embodiments, an inducing agent useful for the induction of cardiomyocytes may include an activator or inhibitor of the TGF-β signaling pathway. Activators and inhibitors of the TGF-β signaling pathway include, as non-limiting examples, small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like, that activate or inhibit at least one component of the TGF-β signaling pathway, resulting in a corresponding activation or inhibition of cellular TGF-β signaling.

[0070] Activators of the TGF-β signaling pathway include, as non-limiting examples, TGF-β family ligands (e.g., TGF-β proteins and other activators of TGF-β receptors) and portions thereof, Activin A, TGF-β1, TGF-β2, TGF-β3, IDE1 / 2 (IDE1 [1-[2-[(2- Carboxyphenyl)methylene]hydrazide]heptanoic acid], IDE2 [Heptanedioic acid-1-(2- cyclopentylidenehydrazide)]), Nodal, and the like. In some embodiments, activation of the TGF- β signaling pathway may be achieved through repression of a TGF-β signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the TGF-β signaling pathway or an antibody or small molecule directed to a TGF-β signaling pathway inhibitor. In some embodiments, the TGF-β signaling pathway activator comprises Activin A. In some embodiments, the TGF-β signaling pathway activator comprises a derivative Activin A. In some embodiments, one or more TGF-β signaling pathway activators is used.

[0071] In some embodiments, the chemical culture conditions of the presently described methods include a mixture of agents, including a BMP signaling pathway activator which can include any BMP signaling pathway activator featured in this disclosure, a WNT signaling pathway activator and / or inhibitor which can include any WNT signaling pathway activator or WNT signaling pathway inhibitor featured in this disclosure, an FGF / ERK signaling pathway activator which can include any FGF / ERK signaling pathway activator featured in this disclosure, and a TGF-β signaling pathway activator which can include any TGF-β signaling pathway activator featured in this disclosure.

[0072] For example, the mixture of agents includes a BMP signaling pathway activator and a WNT signaling pathway activator and / or inhibitor. In an additional example, the mixture of agents includes a BMP signaling pathway activator, a WNT signaling pathway activator and / or inhibitor, or an FGF / ERK signaling pathway activator alone. In another example, the mixture of agents includes a WNT signaling pathway activator and / or inhibitor, an FGF / ERK signaling pathway activator, or a TGF-β signaling pathway activator.

[0073] In some embodiments, the PSCs are grown in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator for about one day, for 1 to 2 days, 1 to 3 days, 1PATENT ATTORNEY DOCKET NO.: N2041-03401 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, more than 8 days, or for any length of time ranging any two of the number of days referred to above or herein. For example, the cells are grown for about 1, 2, 3, 4, 5, 6, 7, 8, or more days, or for any length of time ranging between any two of the number of days referred to above or herein in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator. In some embodiments, the PSCs are grown in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator for at least 2 days. In some embodiments, the PSCs are grown in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator for 1 day. In some embodiments, the PSCs are grown in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator for 2 days. In some embodiments, the PSCs are grown in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator for 3 days. In some embodiments, the PSCs are grown in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator for 4 days. In some embodiments, the PSCs are grown in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator for 5 days. In some embodiments, the PSCs are grown in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator for 6 days. In some embodiments, the PSCs are grown in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator for 7 days. In some embodiments, the PSCs are grown in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator for 8 days. In some embodiments, the PSCs are grown in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator for more than 8 days.

[0074] In some embodiments, following the initial culture in the presence of a WNT signaling pathway activator and a BMP signaling pathway activator, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for about one day, for 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the number of days referred to above or herein . For example, following the initial culture, the cells are grown for about 1, 2, 3, 4, 5, 6, 7, 8, or more days, or for any length of time ranging between any two of the recited number of days referred to above or herein in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for 1 day. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for 2 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathwayPATENT ATTORNEY DOCKET NO.: N2041-03401 activator and / or a TGF-β signaling pathway activator for 3 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for 4 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for 5 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for 6 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for 7 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for 8 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for more than 8 days.

[0075] In some embodiments, following the initial culture in the presence of a WNT signaling pathway activator and a BMP signaling activator, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator, a WNT signaling activator, and a BMP signaling activator for about one day, for 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the recited number of days. For example, following the initial culture, the cells are grown for about 1, 2, 3, 4, 5, 6, 7, 8, or more days, or for any length of time ranging between any two of the number of days referred to above or herein in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator, a WNT signaling activator, and a BMP signaling pathway activator. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator, a WNT signaling activator, and a BMP signaling activator for 1 day. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator, a WNT signaling activator, and a BMP signaling activator for 2 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator, a WNT signaling activator, and a BMP signaling activator for 3 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator, a WNT signaling activator, and a BMP signaling activator for 4 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator, a WNT signaling activator, and a BMP signaling activator for 5 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator, a WNT signalingPATENT ATTORNEY DOCKET NO.: N2041-03401 activator, and a BMP signaling activator for 6 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator, a WNT signaling activator, and a BMP signaling activator for 7 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator, a WNT signaling activator, and a BMP signaling activator for 8 days. In some embodiments, the PSCs are grown in the presence of an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator, a WNT signaling activator, and a BMP signaling activator for more than 8 days.

[0076] In some embodiments, the culture of PSCs is contacted with a WNT signaling pathway activator and a BMP signaling pathway activator for about 3 days and then with an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for about 4 additional days.

[0077] In some embodiments, contacting the culture of PSCs includes one or more agents selected from about 0.1-10 µM WNT signaling pathway activators, including any of the WNT signaling pathway activators disclosed herein. In some embodiments, contacting the culture of PSCs includes one or more agents selected from about 5-100 ng / ml of any of the BMP signaling pathway activators disclosed herein. In some embodiments, contacting the culture of PSCs includes one or more agents selected from about 2.5 ng / ml to 20 ng / ml of the FGF / ERK signaling pathway activators disclosed herein. In some embodiments, contacting the culture of PSCs includes one or more agents selected from about 2.5 ng / ml to 20 ng / ml TGF-β signaling pathway activators disclosed herein. In some embodiments, contacting the culture of PSCs includes one or more agents selected from: about 0.1-10 µM WNT signaling pathway activators, about 5-100 ng / ml BMP signaling pathway activators, about 2.5 ng / ml to 20 ng / ml FGF / ERK signaling pathway activators, and about 2.5 ng / ml to 20 ng / ml TGF-β signaling pathway activators. In some embodiments, contacting the culture of PSCs includes one or more agents selected from: about 0.5 µM or about 1 µM WNT signaling pathway activators, about 5 ng / ml BMP signaling pathway activators, about 5 ng / ml or about 7.5 ng / ml FGF / ERK signaling pathway activators, and about 5 ng / ml or about 7.5 ng / ml TGF-β signaling pathway activators.

[0078] The WNT signaling pathway activator is added to the PSC culture at a concentration that ranges from between about 0.1 µM to 10 µM. For example, the PSC are grown in a culture medium that includes about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10.5 µM or more of the WNT signaling pathway activator, including any of the WNT signaling pathway activators disclosed herein. In some embodiments, the WNT signaling pathway activator is added to the PSC culture at a concentration that ranges from 0.05 to 0.06 µM,PATENT ATTORNEY DOCKET NO.: N2041-03401 0.06 to 0.07 µM, 0.07 to 0.08 µM, 0.08 to 0.09 µM, 0.09 to 0.1 µM, from 0.1 µM to 0.5 µM, from 0.5 µM to 1.0 µM, from 1.0 to 2.0 µM, from 2.0 to 3.0 µM, from 3.0 to 4.0 µM, from 4.0 to 5.0 µM, from 5.0 to 6.0 µM, from 6.0 to 7.0 µM, from 7.0 to 8.0 µM, from 8.0 to 9.0 µM, from 9.0 to 10 µM, from 10.0 to 10.5 µM or ranging between any two concentrations referred to above or herein. In some embodiments, the mixture includes about 1 µM WNT signaling pathway activator. In some embodiments, the mixture includes about 0.5 µM WNT signaling pathway activator. In some embodiments, the mixture includes about 0.5 µM or 1 µM CHIR99021.

[0079] The BMP signaling pathway activator is added to the PSC culture at a concentration that ranges from between about 1 ng / ml to about 10 ng / ml. For example, the PSC are grown in a culture medium that includes about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10.5 ng / ml or more of the BMP signaling pathway activator, including any of the BMP signaling pathway activators disclosed herein. In some embodiments, the BMP pathway activator is at a concentration ranging from between about 1 ng / ml to 10 ng / ml. In some embodiments, the BMP pathway activator is at a concentration that ranges from 0.5 to 0.6 ng / ml, 0.6 to 0.7 ng / ml, 0.7 to 0.8 ng / ml, 0.8 to 0.9 ng / ml, 0.9 to 1 ng / ml, 1 to 2 ng / ml, from 2 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 10.5 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, the mixture includes about 5 ng / ml BMP4. In some embodiments, the mixture includes about 7.5 ng / ml BMP4.

[0080] As further described in the EXAMPLES, BMP4 can be prepared by resuspension in various solutions. For example, dry BMP4 can be resuspended with PBS / 0.01% HSA or in citric acid (as recommended by the manufacturer). PBS / 0.01% HSA may reduce the biological activity of BMP4, as compared to its activity when prepared in citric acid. One of skilled in the art would easily recognize that a concentration of 1-10 ng / ml of BMP4 could be significantly lowered if the BMP4 is resuspended in citric acid and therefore has greater biological activity.

[0081] The FGF / ERK signaling pathway activator is added to the PSC culture at a concentration that ranges from between about 2.5 ng / ml to about 20 ng / ml. For example, the PSC are grown in a culture medium that includes about 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25 ng / ml or more of the FGF / ERK signaling pathway activator, including any of the FGF / ERK signaling pathway activators disclosed herein. In some embodiments, the FGF / ERK signaling pathway activator is at a concentration that ranges from 0.25 to 0.5 ng / ml, 0.5 to 0.75 ng / ml, 0.75 to 1 ng / ml, 1 to 1.5 ng / ml, 1.5 to 2 ng / ml, 2 to 2.5 ng / ml, 2.5 to 3 ng / ml, from 3 to 3.5 ng / ml, from 3.5 to 4 ng / ml, from 4 to 4.5 ng / ml, fromPATENT ATTORNEY DOCKET NO.: N2041-03401 4.5 to 5 ng / ml, from 5 to 5.5 ng / ml, from 5.5 to 6 ng / ml, from 6 to 6.5 ng / ml, from 6.5 to 7 ng / ml, from 7 to 7.5 ng / ml, from 7.5 to 8 ng / ml, from 8 to 8.5 ng / ml, from 8.5 to 9 ng / ml, from 9 to 9.5 ng / ml, from 9.5 to 10 ng / ml, from 10 to 10.5 ng / ml, from 10.5 to 11 ng / ml, from 11 to 11.5 ng / ml, from 11.5 to 12 ng / ml, from 12 to 12.5 ng / ml, from 12.5 to 13 ng / ml, from 13 to 13.5 ng / ml, from 13.5 to 14 ng / ml, from 14 to 14.5 ng / ml, from 14.5 to 15 ng / ml, from 15 to 15.5 ng / ml, from 15.5 to 16 ng / ml, from 16 to 16.5 ng / ml, from 16.5 to 17 ng / ml, from 17 to 17.5 ng / ml, from 17.5 to 18 ng / ml, from 18 to 18.5 ng / ml, from 18.5 to 19 ng / ml, from 19 to 19.5 ng / ml, from 19.5 to 20 ng / ml, from 20 to 20.5 ng / ml, from 20.5 to 21 ng / ml, from 21 to 21.5 ng / ml, from 21.5 to 22 ng / ml, from 22 to 22.5 ng / ml, from 22.5 to 23 ng / ml, from 23 to 23.5 ng / ml, from 23.5 to 24 ng / ml, from 24 to 24.5 ng / ml, from 24.5 to 25 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, the mixture includes about 5 ng / ml of FGF / ERK signaling pathway activator. In some embodiments, the mixture includes about 7.5 ng / ml of FGF / ERK signaling pathway activator. In some embodiments, the mixture includes about 5 or about 7.5 ng / ml of FGF2.

[0082] The TGF-β signaling pathway activator is added to the PSC culture at a concentration that ranges from between about 2.5 ng / ml to about 20 ng / ml. For example, the PSC are grown in a culture medium that includes about 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25 ng / ml or more of the TGF-β signaling pathway activator, including any of the TGF-β signaling pathway activators disclosed herein. In some embodiments, the TGF-β signaling pathway activator is at a concentration that ranges from 0.25 to 0.5 ng / ml, 0.5 to 0.75 ng / ml, 0.75 to 1 ng / ml, 1 to 1.5 ng / ml, 1.5 to 2 ng / ml, 2 to 2.5 ng / ml, 2.5 to 3 ng / ml, from 3 to 3.5 ng / ml, from 3.5 to 4 ng / ml, from 4 to 4.5 ng / ml, from 4.5 to 5 ng / ml, from 5 to 5.5 ng / ml, from 5.5 to 6 ng / ml, from 6 to 6.5 ng / ml, from 6.5 to 7 ng / ml, from 7 to 7.5 ng / ml, from 7.5 to 8 ng / ml, from 8 to 8.5 ng / ml, from 8.5 to 9 ng / ml, from 9 to 9.5 ng / ml, from 9.5 to 10 ng / ml, from 10 to 10.5 ng / ml, from 10.5 to 11 ng / ml, from 11 to 11.5 ng / ml, from 11.5 to 12 ng / ml, from 12 to 12.5 ng / ml, from 12.5 to 13 ng / ml, from 13 to 13.5 ng / ml, from 13.5 to 14 ng / ml, from 14 to 14.5 ng / ml, from 14.5 to 15 ng / ml, from 15 to 15.5 ng / ml, from 15.5 to 16 ng / ml, from 16 to 16.5 ng / ml, from 16.5 to 17 ng / ml, from 17 to 17.5 ng / ml, from 17.5 to 18 ng / ml, from 18 to 18.5 ng / ml, from 18.5 to 19 ng / ml, from 19 to 19.5 ng / ml, from 19.5 to 20 ng / ml, from 20 to 20.5 ng / ml, from 20.5 to 21 ng / ml, from 21 to 21.5 ng / ml, from 21.5 to 22 ng / ml, from 22 to 22.5 ng / ml, from 22.5 to 23 ng / ml, from 23 to 23.5 ng / ml, from 23.5 to 24 ng / ml, from 24 to 24.5 ng / ml, from 24.5 to 25 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, the mixture includes about 5 ng / ml of TGF-β signaling pathwayPATENT ATTORNEY DOCKET NO.: N2041-03401 activator. In some embodiments, the mixture includes about 7.5 ng / ml of TGF-β signaling pathway activator. In some embodiments, the mixture includes about 5 or about 7.5 ng / ml of Activin A.

[0083] In some embodiments, contacting the culture of PSCs includes about 0.5 µM CHIR99021, and about 5 ng / ml BMP4.

[0084] In some embodiments, contacting the culture of PSCs includes about 0.5 µM CHIR99021, about 5 ng / ml BMP4, and about 7.5 ng / ml FGF2.

[0085] In some embodiments, contacting the culture of PSCs includes about 0.5 µM CHIR99021, about 5 ng / ml BMP4, and about 7.5 ng / ml Activin A.

[0086] In some embodiments, contacting the culture of PSCs includes about 0.5 µM CHIR99021, about 5 ng / ml BMP4, about 7.5 ng / ml FGF2, and about 7.5 ng / ml Activin A.

[0087] In some embodiments, contacting the culture of PSCs includes about 0.5 µM CHIR99021, about 5 ng / ml BMP4, and about 5 ng / ml FGF2.

[0088] In some embodiments, contacting the culture of PSCs includes about 0.5 µM CHIR99021, about 5 ng / ml BMP4, and about 5 ng / ml Activin A.

[0089] In some embodiments, contacting the culture of PSCs includes about 0.5 µM CHIR99021, about 5 ng / ml BMP4, about 5 ng / ml FGF2, and about 5 ng / ml Activin A.

[0090] In some embodiments, contacting the culture of PSCs includes about 1 µM CHIR99021.

[0091] In some embodiments, contacting the culture of PSCs includes about 1 µM CHIR99021, and about 5 ng / ml BMP4.

[0092] In some embodiments, contacting the culture of PSCs includes about 1 µM CHIR99021, about 5 ng / ml BMP4, and about 7.5 ng / ml FGF2.

[0093] In some embodiments, contacting the culture of PSCs includes about 1 µM CHIR99021, about 5 ng / ml BMP4, about 7.5 ng / ml FGF2, and about 7.5 ng / ml Activin A.

[0094] In some embodiments, contacting the culture of PSCs includes about 1 µM CHIR99021, about 5 ng / ml BMP4, and about 5 ng / ml FGF2.

[0095] In some embodiments, contacting the culture of PSCs includes about 1 µM CHIR99021, about 5 ng / ml BMP4, about 5 ng / ml FGF2, and about 5 ng / ml Activin A.

[0096] In some embodiments, contacting the culture of PSCs with a WNT signaling pathway activator and a BMP signaling pathway activator is for about 2 to 5 days.

[0097] In some embodiments, contacting the culture of PSCs with a WNT signaling pathway activator and a BMP signaling pathway activator is for about 3 to 5 days.

[0098] In some embodiments, contacting the culture of PSCs with a WNT signaling pathway activator and a BMP signaling pathway activator is for about 4 to 5 days.PATENT ATTORNEY DOCKET NO.: N2041-03401

[0099] In some embodiments, contacting the culture of PSCs with a WNT signaling pathway activator and a BMP signaling pathway activator is for about 2, 3, 4, 5 days, or for any length of time ranging between any two of the recited number of days.

[0100] In some embodiments, the PSCs are subsequently contacted with an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for about 2 to 5 days.

[0101] In some embodiments, the PSCs are subsequently contacted with an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for about 3 to 5 days.

[0102] In some embodiments, the PSCs are subsequently contacted with an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for about 4 to 5 days.

[0103] In some embodiments, the PSCs are subsequently contacted with an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for about 2, 3, 4, 5 days, or for any length of time ranging between any two of the recited number of days.

[0104] The culture medium from contacting PSCs is partially exchanged with a culture medium comprising a WNT signaling pathway inhibitor for one or more days.

[0105] In some embodiments, the culture medium from contacting PSCs is partially exchanged in a volume ranging from between about 5% to 90% with a culture medium a WNT signaling pathway inhibitor for one or more days. In some embodiments, the volume of the culture medium exchanged is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90%, or any percentage of the culture medium from contacting PSCs ranging between any two percentages referred to above or herein.

[0106] In some embodiments, the volume of the culture medium exchanged is about 10%, 25%, 50%, or 75% of the culture medium from contacting PSCs.

[0107] In some embodiments, the volume of culture medium exchanged is about 25% of the culture medium from contacting PSCs.

[0108] In some embodiments, the culture medium from contacting PSCs is partially exchanged with a culture medium comprising about at least 0.1 µM WNT signaling pathway inhibitor. For example, the culture medium is exchanged with a culture medium comprising about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5 µM or more of the WNT signaling pathway inhibitor, including any of the WNT signaling pathway inhibitors disclosed herein. In some embodiments, the WNT signaling pathway inhibitor is added to the PSC culture at a concentration that ranges from 0.05 to 0.06 µM, 0.06 to 0.07 µM, 0.07 to 0.08 µM, 0.08 to 0.09 µM, 0.09 to 0.1 µM, 0.1 to 0.2 µM, 0.2 to 0.3 µM, 0.3 to 0.4 µM, 0.4 to 0.5 µM, from 0.5 to 0.6 µM, 0.6 to 0.7 µM, 0.7 to 0.8 µM, 0.8 to 0.9 µM, 0.9 to 1 µM from 1.0 to 2.0 µM, from 2.0 to 3.0 µM, from 3.0 to 4.0 µM, from 4.0 to 5.0 µM, fromPATENT ATTORNEY DOCKET NO.: N2041-03401 5.0 to 6.0 µM, from 6.0 to 7.0 µM, from 7.0 to 8.0 µM, from 8.0 to 9.0 µM, from 9.0 to 10 µM, or ranging between any two concentrations referred to above or herein. In some embodiments, the culture medium comprises about 2 µM of the WNT signaling pathway inhibitor. In some embodiments, the culture medium comprises about 2 µM of a combination of WNT signaling pathway inhibitors including C-59, IPW2, or IWR1. In some embodiments, the culture medium comprises about 2 µM of C-59, 2 µM of IPW2, or 2 µM of IWR1.

[0109] In some embodiments, the culture medium from contacting PSCs is partially exchanged with a culture medium comprising a WNT signaling pathway inhibitor for one or more days. For example, the culture medium from contacting PSCs is partially exchanged with a culture medium comprising a WNT signaling pathway inhibitor for about 1, 2, 3, 4, 5, 6, 7, 8, or more days, or any range of time falling between any two of the recited number of days above or herein. In some embodiments, the culture medium from contacting PSCs is partially exchanged with a culture medium comprising a WNT signaling pathway inhibitor for about 2 to 5 days. In some embodiments, the culture medium from contacting PSCs is partially exchanged with a culture medium comprising a WNT signaling pathway inhibitor for about 3 to 5 days. In some embodiments, the culture medium from contacting PSCs is partially exchanged with a culture medium comprising a WNT signaling pathway inhibitor for about 4 to 5 days. In some embodiments, the culture medium from contacting PSCs is partially exchanged with a culture medium comprising a WNT signaling pathway inhibitor for about 2, 3, 4, or 5 days. In some embodiments, the culture medium from contacting PSCs is partially exchanged with a culture medium comprising a WNT signaling pathway inhibitor for more than 5 days.

[0110] In some embodiments, about 25% of the culture medium from contacting PSCs is exchanged with a culture medium comprising 2 µM C-59 for about 2 to 5 days. In some embodiments, about 25% of the culture medium from contacting PSCs is exchanged with a culture medium comprising 2 µM C-59 for about 3 to 5 days. In some embodiments, about 25% of the culture medium from contacting PSCs is exchanged with a culture medium comprising 2 µM C- 59 for about 4 to 5 days. In some embodiments, about 25% of the culture medium from contacting PSCs is exchanged with a culture medium comprising 2 µM C-59 for about 2, 3, 4, or 5 days, or any range of time falling between any two of the recited number of days above or herein.

[0111] In some embodiments, culture medium supplements include or exclude vitamins, proteins, and amino acids are added to the culture medium. In some embodiments, the vitamin supplement is vitamin C as ascorbate, ascorbic acid, any salt thereof, or any derivative thereof. In some embodiments, the culture medium supplement comprises vitamin C, which can be any vitamin C featured in this disclosure. In some embodiments, the culture medium supplement comprisesPATENT ATTORNEY DOCKET NO.: N2041-03401 vitamin C or a salt thereof, which can include, as non-limiting examples, sodium, potassium, calcium, magnesium, lithium, and ammonium vitamin C salts. In some embodiments, the vitamin C salt can be sodium ascorbate (also known as “mineral ascorbate”), calcium ascorbate, potassium ascorbate, magnesium ascorbate, zinc ascorbate, molybdenum ascorbate, chromium ascorbate, manganese ascorbate, or any combination thereof. In some embodiments, vitamin C can be vitamin C with bioflavonoids, ascorbate and vitamin C metabolites, ascorbyl palmitate, D- erythorbic acid, or any combination thereof. In some embodiments, the culture medium comprises ascorbate. In some embodiments, ascorbate is 2-phospho-L-ascorbate, a salt thereof, or a derivative thereof. In some embodiments, the culture medium supplement comprises 2-phospho- L-ascorbate or a salt thereof, which can include, as non-limiting examples, sodium, potassium, calcium, magnesium, lithium, and ammonium 2-phospho-L-ascorbate salts, e.g., 2-phospho-L- ascorbic acid trisodium salt, L-ascorbic acid sesquimagnesium salt (also known as “2-Phospho-L- ascorbic acid magnesium”), and 2-O-a-D-glucopyranosyl-L-ascorbic Acid (also known as “ascorbyl glucoside”). In some embodiments, vitamin C, ascorbate, 2-phospho-L-ascorbate, ascorbic acid, and other forms of vitamin C are a salt form, which can include, as non-limiting examples, monosodium, disodium, trisodium, monopotassium, dipotassium, tripotassium, mono- ammonium, di-ammonium, or tri-ammonium salts. In some embodiments, one or more derivatives of 2-phospho-L-ascorbate are optionally added to the culture medium. In some embodiments, one or more salts of ascorbate or of 2-phospho-L-ascorbate are optionally added to the culture medium. In some embodiments, the protein supplement is albumin. In some embodiments, the albumin is HSA. In some embodiments, the amino acid supplement is glutamine. In some embodiments, ascorbate and / or albumin, and optionally L-glutamine, are added to the culture medium. In some embodiments, about 250 µM 2-phospho-ascorbate and / or about 5 mg / ml albumin, and optionally about 2 mM L-glutamine, are added to the culture medium. In some embodiments, 250 µM 2- phospho-ascorbate and / or 5 mg / ml albumin, and optionally 2 mM L-glutamine, are added to the culture medium.

[0112] Inhibitors of ROCK include, as non-limiting examples, Y-27632, ripasudil, netarsudil, chroman 1, Y39983, Wf-536, SLx-2119, Azabenzimidazole-aminofurazans, DE-104 Olefins, Isoquinolines, Indazoles, pyridinealkene derivatives, H-1152P, XD-4000, HMN-1152, ROKα 4- (1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, rhostatin, BA-210, BA-207, BA-285, BA-1037, BA-215, Ki-23095, VAS-012, quinazoline,GSK-269962, and salts thereof, including Y-27632 dihydrochloride, H-1152 dihydrochloride, fasudil hydrochloride, SR-3677 dihydrochloride, glycyl-H-1152 dihydrochloride, SB-772077B dihydrochloride, TC-S-7001, RKI- 1447 dihydrochloride, and OXA-06 dihydrochloride. In some embodiments, the culture of PSCsPATENT ATTORNEY DOCKET NO.: N2041-03401 is contacted with a culture medium comprising a ROCK inhibitor, which can be any ROCK inhibitor featured in this disclosure, for at least one day. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the concentration of ROCK inhibitor is at a range between about 1 µM to about 15 µM. In some embodiments, the ROCK inhibitor is at a concentration that ranges from 1.0 to 2.0 µM, from 2.0 to 3.0 µM, from 3.0 to 4.0 µM, from 4.0 to 5.0 µM, from 5.0 to 6.0 µM, from 6.0 to 7.0 µM, from 7.0 to 8.0 µM, from 8.0 to 9.0 µM, from 9.0 to 10 µM, from 10 to 11 µM, from 11 to 12 µM, from 12 to 13 µM, from 13 to 14 µM, from 14 to 15 µM, or ranging between any two concentrations referred to above or herein. In some embodiments, the concentration of Y-27632 is 10 µM.

[0113] In some embodiments, the culture of PSCs is contacted with a culture medium comprising 10 µM Y-27632 for at least one day prior to contacting the culture of PSCs with a WNT signaling pathway activator and a BMP signaling pathway activator for about 2 to 5 days and then with an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for about 2 to 5 additional days, then subsequently partially exchanging the culture medium with a culture medium comprising a WNT signaling pathway inhibitor for one or more days. In some embodiments, the culture of PSCs is contacted with a culture medium comprising 10 µM Y-27632 for at least one day, then the culture medium is replaced with a culture medium contacting ROCK- inhibitor cultured cells with a WNT signaling pathway activator and a BMP signaling pathway activator for about 2 to 5 days and then with an FGF / ERK signaling pathway activator and / or a TGF-β signaling pathway activator for about 2 to 5 additional days, then subsequently partially exchanging the culture medium with a culture medium comprising a WNT signaling pathway inhibitor for one or more days. In some embodiments, culture medium contacting the ROCK- inhibitor cultured cells is replaced with a culture medium comprising about 0.5 µM CHIR99021, about 5 ng / ml BMP4, about 7.5 ng / ml FGF2, and about 7.5 ng / ml Activin A. In some embodiments, culture medium contacting the ROCK-inhibitor cultured cells is replaced with a culture medium comprising about 0.5 µM CHIR99021, about 5 ng / ml BMP4, about 5 ng / ml FGF2, and about 5 ng / ml Activin A. In some embodiments, the culture medium subsequently partially exchanged comprising a WNT signaling pathway inhibitor comprises 2 µM C-59.

[0114] In some embodiments, the culture of PSCs is an adherent layer of cells. In some embodiments, the layer of cells is grown in a two-dimensional culture system or on microcarriers.

[0115] In some embodiments, the PSCs are cultures on scaffolds composed of microcarriers, which are beads or particles. The beads may be microscopic or macroscopic and may further be dimensioned to permit penetration into tissues or compacted to form a particular geometry. In some embodiments, the framework for the cell cultures comprises particles that, in combinationPATENT ATTORNEY DOCKET NO.: N2041-03401 with the cells, form a three-dimensional tissue. The cells attach to the particles and to each other to form a three-dimensional tissue. Beads, or microcarriers are typically considered a two- dimensional system or scaffold.

[0116] As used herein, a “microcarrier” refers to a particle having a size of nanometers to micrometers, where the particles may be any shape or geometry, being irregular, non-spherical, spherical, or ellipsoid. The size of the microcarriers suitable for the purposes herein can be of any size suitable for the particular application.

[0117] In some embodiments, the size of microcarriers suitable for the three-dimensional tissues may be those administrable by injection. In some embodiments, the microcarriers have a particle size range of at least about 1 μm, at least about 10 μm, at least about 25 μm, at least about 50 μm, at least about 100 μm, at least about 200 μm, at least about 300 μm, at least about 400 μm, at least about 500 μm, at least about 600 μm, at least about 700 μm, at least about 800 μm, at least about 900 μm, at least about 1000 μm, or any range of sizes between any two of the sizes referred to above or herein.

[0118] In some embodiments, the microcarriers are made of biodegradable materials. In some embodiments, microcarriers comprising two or more layers of different biodegradable polymers may be used. In some embodiments, at least an outer first layer has biodegradable properties for forming the three-dimensional tissues in culture, while at least a biodegradable inner second layer, with properties different from the first layer, is made to erode when administered into a tissue or organ.

[0119] In some embodiments, the microcarriers are porous microcarriers. Porous microcarriers refer to microcarriers having interstices through which molecules may diffuse in or out of the microparticle. In other embodiments, the microcarriers are non-porous microcarriers. A nonporous microparticle refers to a microparticle in which molecules of a select size do not diffuse in or out of the microparticle.

[0120] Microcarriers for use in the compositions are biocompatible and have low or no toxicity to cells. The microcarriers may comprise various polymers, natural or synthetic, charged (i.e., anionic or cationic) or uncharged, biodegradable, or nonbiodegradable. The polymers may be homopolymers, random copolymers, block copolymers, graft copolymers, or branched polymers.

[0121] In some embodiments, the microcarriers comprise non-biodegradable microcarriers. Non- biodegradable microcapsules and microcarriers include, as non-limiting examples, those made of polysulfones, poly (acrylonitrile-co-vinyl chloride), ethylene-vinyl acetate, and hydroxyethyl methacrylate-methyl-methacrylate copolymers. These are useful to provide tissue bulking properties or in embodiments where the microcarriers are eliminated by the body.PATENT ATTORNEY DOCKET NO.: N2041-03401

[0122] In some embodiments, the microcarriers comprise degradable scaffolds. These include microcarriers made from naturally occurring polymers, non-limiting examples of which include, among others, fibrin, casein, serum albumin, collagen, gelatin, lecithin, chitosan, alginate, or poly- amino acids such as poly-lysine.

[0123] In some embodiments, the degradable microcarriers are made of synthetic polymers, non- limiting examples of which include, among others, polylactide (PLA), polyglycolide (PGA), poly (lactide-co-glycolide) (PLGA), poly (caprolactone), polydioxanone trimethylene carbonate, polyhybroxyalkonates (e.g., poly (hydroxybutyrate), poly (ethyl glutamate), poly DTH iminocarbony (bisphenol A iminocarbonate), poly (ortho ester), and polycyanoacrylates.

[0124] In some embodiments, the microcarriers comprise hydrogels, which are typically hydrophilic polymer networks filled with water. Hydrogels have the advantage of selectively triggering polymer swelling. Depending on the composition of the polymer network, swelling of the microparticle may be triggered by a variety of stimuli, including pH, ionic strength, thermal, electrical, ultrasound, and enzyme activities. Non-limiting examples of polymers useful in hydrogel compositions include, among others, those formed from polymers of poly (lactide-co- glycolide); poly (N-isopropylacrylamide); poly (methacrylic acid-g-polyethylene glycol); polyacrylic acid and poly (oxypropylene-co-oxyethylene) glycol; and natural compounds such as chrondroitan sulfate, chitosan, gelatin, fibrinogen, or mixtures of synthetic and natural polymers, for example, chitosan-poly (ethylene oxide). The polymers may be cross-linked reversibly or irreversibly to form gels adaptable for forming three-dimensional tissues.

[0125] In some embodiments, the microcarriers or beads for use in the present invention are composed wholly or partly of dextran.

[0126] In some embodiments, cardiomyocyte differentiation is performed in two- or three- dimensional culture.

[0127] In some embodiments, the cardiomyocyte differentiation is performed in suspension or adherent culture.

[0128] In some embodiments, the cardiomyocytes express maturation markers after about 4 weeks of differentiation. In some embodiments, the cardiomyocytes express maturation markers after about 5 weeks of differentiation. In some embodiments, the cardiomyocytes express maturation markers after about 6 weeks of differentiation. In some embodiments, the cardiomyocytes express maturation markers after more than about 6 weeks of differentiation.

[0129] Markers of cardiomyocyte maturation include, as non-limiting examples, CAS12, S100A4, CASQ1, ADRB1, ADRB2, ATP2A2, RYR2, PLN, JPH2, CAMK2D, ATP1A3, KCNJ2, CACNA2D1, KCNQ1, SCN5A, KCNE1, SLC8A1, KCNH2, SCN4B, KCNE5, MYH6, MYL2,PATENT ATTORNEY DOCKET NO.: N2041-03401 TNNI3, MYM2, MYH7, TPM1, MYL3, TNNC1, TNNT2, ACTC1, LPL, ACOX1, PPARA, PPARD, ACAT1, ACADVL, DGAT1, PPARGC1A, HADHB, AND ACSL1. In some embodiments, the maturation markers comprise MYL2, MYH7, and / or KCNQ1.

[0130] In some embodiments, cTnT positive cells are observed after 8 days of differentiation. In some embodiments, cTnT positive cells comprise at least 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of PSCs, or any range of cTnT positive cells falling between any two of the percentages above or herein, after at least 8 days of differentiation. In some embodiments, cTnT positive cells comprise at least 80% of the PSCs after 9 days of differentiation.

[0131] Stem cells are undifferentiated cells that have the ability to self-renew indefinitely and to remain in said undifferentiated state. As opposed to embryonic stem cells, which can only be isolated from the inner mass of a blastocyst, there are three known accessible sources of adult stem cells: the bone marrow, which requires the drilling of a bone; the adipose tissue, which is accessible by liposuction; and the blood, from which the cells can be extracted among other cells. The term “pluripotent stem cells,” as used herein, refers to cells that are capable of generating all the cell types of an organism, i.e., cells derived from any of the three germ layers. On the other hand, multipotent stem cells can differentiate into several cell types, but only those of a closely related family of cells, generally the cell types of the organ from which they originate. Most adult stem cells are multipotent, but small amounts of pluripotent adult stem cells can be retrieved from the umbilical cord or other tissues. The sources of cells used for cell therapy include stem cells such as embryonic stem cells (ESCs), adult stem cells, and induced pluripotent stem cells (iPSCs).

[0132] In some embodiments, the PSCs used in the methods described herein are human (hPSCs), and in some embodiments, the human PSCs are induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs).

[0133] Physical culture conditions include, as non-limiting examples, the culture environment of the cell (e.g., adherent versus suspension culture, or in two-dimensional versus in three- dimensional culture systems), the pH of the culture medium, the gas concentration in the incubator (e.g., CO2 concentration, O2 concentration), and the temperature.

[0134] There are two basic systems for growing cells in culture: monolayers on an artificial substrate (i.e., adherent culture) or free-floating in the culture medium (suspension culture). The majority of the cells derived from vertebrates, with the exception of hematopoietic cell lines and a few others, are anchorage-dependent and have to be cultured on a suitable substrate that is specifically treated to allow cell adhesion and spreading (i.e., tissue-culture-treated). However, many cell lines can also be adapted for suspension culture.PATENT ATTORNEY DOCKET NO.: N2041-03401

[0135] In some embodiments, the culture of PSCs is an adherent layer of cells. In some embodiments, the layer of cells is grown in a two-dimensional culture system or on microcarriers.

[0136] In addition to the treatment of the tissue-culture surface, cells can be required to be grown on coated surfaces to enhance or improve their adhesion and / or spreading (i.e., using a coating). “Coating” as an additional surface treatment stands for all additional modifications made to increase cell adhesion in addition to the standard plasma or corona treatment that is performed on all cell culture plastic by the manufacturer. Usually, coating is done with proteins or peptides. Various proteins can be used to coat tissue-culture-treated dishes, including poly-L-lysine, poly- D-lysine, poly-Ornithine, gelatin, collagen I, IV, fibronectin, laminin, vitronectin, osteopontin, fibronectin domains, MatrigelTM(a basement membrane extract containing several components of the extracellular matrix and growth factors), collagen gels, alginate gels, and lactate gels.

[0137] In some embodiments, the PSCs are cultured on a coated surface, including a laminin coating.

[0138] Physical culture conditions include the gas concentration in the incubator. Incubation of cell cultures is typically performed in a normal atmosphere with 15–22% oxygen and 5% CO2for expansion and seeding. In some embodiments, the PSCs are grown in a humidified atmosphere with about 5% CO2concentration and normoxic conditions (non-hypoxic O2concentration). While hypoxic culture conditions are thought to support stem cell performance in general, in the present methods, the PSCs are cultured under conditions that are not hypoxic. As used herein, “normoxic” conditions refer to culture conditions including atmospheric O2 concentration (e.g., about 15–25% O2concentration). As used herein, hypoxic conditions are characterized by a lower oxygen concentration as compared to the oxygen concentration of ambient air (approximately 15%–25% oxygen).

[0139] Chemical culture conditions include, as non-limiting examples, the agents or molecules that are added to the culture medium to achieve the desired effects sought after (i.e., differentiation of PSCs into cardiomyocytes). The terms “agent” and “molecule” are used interchangeably and include, as non-limiting examples, small molecules (including small molecules that do not have optimal cell permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers, and other polymers, for example proteins, peptides, hormones, carbohydrates, or polyamines.

[0140] Unless otherwise specified, pluripotent stem cells are maintained in a stem cell medium suitable for the culture and propagation of pluripotent stem cells. Non-limiting examples of such suitable media include, as a non-limiting example, StemMACS iPS-Brew XF. For cardiomyocytes differentiation, the pluripotent stem cells are switched to a “defined medium.” As used herein, thePATENT ATTORNEY DOCKET NO.: N2041-03401 term “basal medium” generally refers to a base medium without any additives added (for example, a basal medium refers to a medium as commercially available). Those generally include water, nutrients, salts, and amino acids, but no additives or supplements. A basal medium can be supplemented with general additives to obtain a “supplemented basal medium.” Non-limiting examples of supplements include, as non-limiting examples, insulin or ascorbate. A basal medium can also be completed with specific signaling molecules, such as those identified by a user as necessary to achieve a particular goal with the cell culture, such as, for example, driving the differentiation of a cell type of interest into a target cell type. A non-limiting example of a basal medium supplemented with a specific signaling molecule includes a ROCK inhibitor medium containing Y-27632. Such a complete basal medium can be referred to as a “final,” “complete,” or “cell-specific” medium.

[0141] Such basal or supplemented defined media used for cardiomyocytes generation can then be completed with small molecules of interest as needed.

[0142] In some embodiments, the culture medium is Knockout-DMEM or IMDM. In some embodiments, the culture medium is StemMACS iPS-Brew XF. In some embodiments, the culture medium is a preparation of Knockout-DMEM, IMDM, StemMACS iPS-Brew XF, or related base medium supplemented with growth factors, accessory factors, cytokines, activators, or inhibitors. In some embodiments, the culture medium is a basal medium, supplemented basal medium, defined medium, final medium, complete medium, or cell-specific medium.

[0143] Presented below are examples discussing methods of inducing the differentiation of PSCs into cardiomyocytes contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used. EXAMPLES

[0144] The invention is further illustrated by the following non-limiting examples: EXAMPLE 1 Design of Cardiomyocytes Differentiation Protocol

[0145] In a stepwise iterative developmental process, a new combination of factors promoting cardiac differentiation produces cardiomyocyte progenitors and cardiomyocytes from pluripotent stem cells (PSCs).

[0146] The cardiomyocyte differentiation protocol detailed here is structured into two main phases, designed to closely mimic the natural developmental progression from cardiac mesodermPATENT ATTORNEY DOCKET NO.: N2041-03401 to fully differentiated cardiomyocytes. Initially, the protocol employs a combination of mesoderm- inducing factors, specifically BMP and WNT, alongside FGF and TGF-β signaling pathway activators, to transition pluripotent stem cells into cardiac mesoderm. This phase is conducted in an insulin-rich medium, aiming for a duration of 1 to several days, optimally 2 days, to establish a conducive environment for mesodermal specification.

[0147] Following the mesodermal induction, the method transitions into a critical differentiation phase, marked by a strategic partial medium change to a formulation that includes a WNT pathway inhibitor. This switch is crucial for directing the nascent cardiac mesoderm towards terminal cardiomyocyte differentiation. The duration of this phase mirrors that of the initial induction, lasting from 1 to several days with an optimal period of 2 days, facilitating the maturation of the precursor cells into functional cardiomyocytes.

[0148] This protocol, founded on a deep understanding of cardiac development and cellular signaling, leverages the interplay of specific growth factors and signaling pathways to effectively guide pluripotent stem cells through a mimetic developmental trajectory. The introduction of a partial medium change at a pivotal transition point significantly enhances the efficiency and fidelity of the differentiation process, ensuring the generation of cardiomyocytes with improved functional characteristics. This stepwise, optimized approach not only demonstrates the potential for generating cardiac cells in vitro for research and therapeutic applications but also provides a valuable framework for understanding and manipulating the complex pathways involved in cardiac development. EXAMPLE 2 Materials and Methods of Cardiac Differentiation of Pluripotent Stem Cells

[0149] Table 1: Materials for Cell Culture Material Reference number / brief instructions iM t i 511 A bi 892011 al 2) re ix at ve ze w ayPATENT ATTORNEY DOCKET NO.: N2041-03401 In some embodiments the concentrations of the ROCK inhibitor and incubation times can be any ROCK i hi it i ti ti f t h i . ml C s. 20 m ix ee an he y ge ve in or 1 w g. P be . ve in at n. T be n. ve in or nd up F ng nyPATENT ATTORNEY DOCKET NO.: N2041-03401 A TGF-β signaling pathway R&D Systems 338-AC-500 / CF activator, e.g., Activin A Equilibrate vial to RT and briefly spin contents. Dissolve ti t t t 10 l i 01% HSA PBS Ali t in or nd up -β be n. ze C. to in in er T be . 2, # id id

[0150] Methods:

[0151] Maintenance of hiPSCs:

[0152] hiPSCs are split on Monday morning and Thursday afternoon at 200,000 cells per 6-well. Approximately 2.5 M cells can be expected from one well of a 6-well plate on a Monday (25,000 cells per cm²). Scale up to other for-mats like T75 if needed. Differentiation is initiated on Mondays (before noon) from fully confluent plates.PATENT ATTORNEY DOCKET NO.: N2041-03401

[0153] Monday: - Coat 6-wells for hiPSC maintenance with 3 µl iMatrix-511 per well in 2 ml XF medium with 10 µM Y-27632 for at least 1 hour at 37°C. - Prewarm Accutase and approximately 10 ml of XF medium. Transfer required volume of Accutase (1 ml per 6-well to be harvested) to separate tube. Add 1:1000 Y-27632 to both solutions and mix. - Maintenance wells of hiPSCs should be near-confluent and overall undifferentiated. Vigorously agitate plate with cells to collect all dead cells in supernatant. Completely soak off medium and wash with 4 ml PBS. Replace by 1 ml prewarmed Accutase containing Y-27632 and transfer to incubator for 10-15 min. Most cells should come off by gently agitating the plate. If this is not the case, prolong digestion for 2 more min and so forth, until cells lift off virtually by themselves. - Add 1 ml of XF medium containing Y-27632 per 6-well to a 15 ml tube. Flush cells off from each well by pipetting up / down + flushing 3-4 times. Transfer cells from all wells to 15 ml tube containing XF medium + Y. Centrifuge at 300 g for 2 min. Supernatant should be clear and cells should form a compact pellet. - Soak off supernatant and resuspend in 2 ml of XF+Y medium per harvested 6-well by pipetting up and down 3 times with a 1 ml pipette. Immediately and gently transfer 8-10 µl to a counting chamber and quantify cell titer. - Plate out 200,000 cells per well into each freshly coated 6-well. Transfer to incubator and agitate plate slowly describing and infinity symbol inside the incubator.

[0154] Tuesday: - Feed cells with 2.5 ml prewarmed XF medium per well.

[0155] Wednesday: - Feed cells with 3 ml prewarmed XF medium per well.

[0156] Thursday: - Confirm that cells are sub confluent and fully undifferentiated. In the afternoon, coat wells / flasks and split cells at 20,000 cells per cm² except that more wells may be required to start cardiac differentiation next Monday. Cells for maintenance may be kept in 6-well format, whereas cells for differentiation may be split to T75 flasks while proportionally scaling up volumes of solutions.

[0157] Friday: - In the afternoon, feed maintenance wells of hiPSCs with 6 ml XF medium per 6-well over the weekend. Scale up accordingly for other formats such as T75.PATENT ATTORNEY DOCKET NO.: N2041-03401

[0158] Small-scale cardiac 3D differentiation by differentiation upon embryoid body (EB) formation:

[0159] Monday: - Confirm near or fully confluent and undifferentiated state of hiPSCs forming a homogeneous smooth surface on the wells / flasks to be used for differentiation. In 6-well format, 1.5-3 M cells are needed per well. - Prepare required amount of differentiation medium + approximately 1 / 3 volume for washing and resuspension steps - about 3 ml per 6-well to be started. Scale up or down accordingly: o Knockout™ DMEM o 1 x ITS o PSG or just Gln or Glx o 250 µM 2-phospho-ascorbate (from 250 mM stock) o 0.05% (w / v) HSA (from 10% stock) o 10 µM Y-27632 (1:1000 from stock) o 5-10 ng / ml FGF2 (from 10 µg / ml stock) o 5-10 ng / ml Activin A (from 10 µg / ml stock) o 3-5 ng / ml BMP4 (from 1 or 10 µg / ml stock) o 0.5 µM CHIR99021 (from 1- or 3-mM stock) - Mix well and equilibrate to 37°C together with Accutase (1 ml per 6-well to be harvested) containing 10 µM Y-27632. Individual factors may also be added later, i.e., to individual samples / wells, or more than one master mix may be prepared for several experimental conditions to be started like, for instance, keeping the CHIR99021 concentration constant while varying the BMP dose. - Harvest iPSCs as for splitting, using 20-25 min of Accutase digestion after 2 x washing thoroughly with PBS. After this incubation time, the cells should come off the plates by themselves, essentially without need to strong agitation or pipetting. Pipet up / down three times per well / flask to be harvested for dissociating the cell aggregates into single cells, then transfer to centrifugation tube(s) containing an equal amount of differentiation medium. Centrifuge for 2 min at 300 g. - Confirm that supernatant is clear to then remove it. Resuspend cells in differentiation medium at 2 ml per harvested 6-well (9 cm²) by pipetting up / down 2-3 times. - Centrifuge and resuspend cells again as before. - Determine live cell titer using a NucleoCounter. Adjust cell titer and total volume as follows:PATENT ATTORNEY DOCKET NO.: N2041-03401 o Bring to 750,000-1,500,000 cells per ml. o 2 ml needed per differentiation 6-well (~9 cm²). - Distribute at 2 ml per 6-well into standard tissue culture 6-wells and incubate on Thermo Fisher Scientific or Heidolph wave shaker placed into cell culture incubator overnight.

[0160] Tuesday: - Confirm that the cells have formed EB-like aggregates with smooth surfaces and that the cultures contain relatively few single floating cells, otherwise indicating suboptimal starting conditions.

[0161] Wednesday: - Prepare basal differentiation medium with WNT inhibitor C-59 (~0.5 ml needed per differentiation 6-well): o Knockout™ DMEM o PSG or just Gln or Glx o 250 µM 2-phospho-ascorbate (from 250 mM stock) o 0.05% (w / v) HSA (from 10% stock) o 2 µM C-59 (from 2 mM stock) - Mix well and equilibrate to 37°C. - Remove differentiation plate from incubator. In cell culture hood, tilt plate by approximately 45°, leave for 0.5-1 min, then carefully, using a 1 ml pipette and without disturbing the settled aggregates at the bottom, soak off 25% of medium. - Add differentiation medium ad 2 ml (depending on how much has been withdrawn in the previous step) and place back onto wave shaker.

[0162] Friday: - Prepare basal differentiation medium as above (albeit without C-59 but IMDM with SB43 (Op178); 2 ml needed per differentiation 6-well), mix well, and equilibrate to 37°C. - Remove old medium from differentiation wells and add 2 ml of fresh basal differentiation medium to the cells. - Place back onto wave shaker and incubate over the weekend.

[0163] Following Monday and afterwards: - The cell aggregates should show spontaneous beating by day 7 and may be analyzed for the expression of cardiac markers or be kept in culture for extended periods of time using regular media changes. Overall cardiac differentiation efficiency may also be assessed after day 7, i.e., on day 8 or 9, although this should be kept consistent from experiment to experiment.

[0164] Small-scale cardiac 3D differentiation by differentiation after EB formation:PATENT ATTORNEY DOCKET NO.: N2041-03401 This procedure differs from the above one in that EBs are made in iPSC medium first. Then, differentiation is started on the next day using the already-formed EBs, which might bear advantages in terms of robustness.

[0165] Monday: - Confirm near or fully confluent and undifferentiated state of hiPSCs forming a homogeneous smooth surface on the wells / flasks to be used for differentiation. In 6-well format, 1.5-2 M cells are needed per well. - Prepare required amount of iPS Brew medium + approximately 1 / 3 volume for washing and resuspension steps - about 5 ml per 6-well to be started. Scale up or down accordingly: o iPS Brew medium o 10 µM Y-27632 - Mix well and equilibrate to 37°C together with Accutase (1 ml per 6-well to be harvested) containing 10 µM Y-27632. - Harvest iPSCs as for routine splitting, using 20-25 min of Accutase digestion after 2 x washing thoroughly with PBS. After this incubation time, the cells should come off the plates by themselves, essentially without need to strong agitation or pipetting. Pipet up / down three times per well / flask to be harvested for dissociating the cell aggregates into single cells, then transfer to centrifugation tube(s) containing an equal amount of iPSC medium. - Centrifuge for 2 min at 300 x g. - Confirm that supernatant is clear to then remove it. Resuspend cells in iPSC medium containing 10 µM Y-27632 at 2 ml per harvested 6-well (9 cm²) by pipetting up / down 2-3 times. - Centrifuge and resuspend cells again as before. - Determine live cell titer using a NucleoCounter. Adjust cell titer and total volume as follows: o Bring to 750,000-1,000,000 cells per ml. o 2 ml needed per differentiation 6-well (~9 cm²) - Distribute at 2 ml per 6-well into standard tissue culture 6-wells and incubate on Thermo Fisher Scientific or Heidolph wave shaker placed into cell culture incubator overnight.

[0166] Tuesday: - Confirm that the cells have formed EB-like aggregates with smooth surfaces and that the cultures contain relatively few single floating cells, otherwise indicating suboptimal starting conditions. - Prepare mesoderm induction medium (2 ml needed per well): o Knockout™ DMEMPATENT ATTORNEY DOCKET NO.: N2041-03401 o 1 x ITS o PSG or just Gln or Glx o 250 µM 2-phospho-ascorbate (from 250 mM stock) o 0.05% (w / v) HSA (from 10% stock) o 5 ng / ml FGF2 (from 10 µg / ml stock) o 5 ng / ml Activin A (from 10 µg / ml stock) o 3-5 ng / ml BMP4 (from 1 or 10 µg / ml stock) o 0.5 µM CHIR99021 (from 1- or 3-mM stock) - Mix well and equilibrate to 37°C. - Remove plate from incubator. In cell culture hood, tilt plate by approximately 45°, leave for 0.5-1 min, then carefully, using a 1 ml pipette and without disturbing the settled aggregates at the bottom, soak off medium almost completely. Add 2 ml of above differentiation medium and place back onto wave shaker.

[0167] Wednesday: - For 2 d mesoderm induction variant, do not disturb plate. - For 1 d mesoderm induction variant, prepare cardiac specification medium (basal differentiation medium with WNT inhibitor C-59; ~2 ml needed per differentiation 6-well): o Knockout™ DMEM o 1 x ITS o PSG or just Gln or Glx o 250 µM 2-phospho-ascorbate (from 250 mM stock) o 0.05% (w / v) HSA (from 10% stock) o 0.2-2 µM C-59 (from 2 mM stock) - Mix well and equilibrate to 37°C. - Remove differentiation plate from incubator. In cell culture hood, tilt plate by approximately 45°, leave for 0.5-1 min, then carefully, using a 1 ml pipette and without disturbing the settled aggregates at the bottom, soak off medium. - Add up to 2 ml of above cardiac specification medium (depending on how much has been withdrawn in previous step) and place back onto wave shaker until Friday.

[0168] Thursday: - For 2 d mesoderm induction variant, prepare and change to cardiac specification medium. - For 1 d mesoderm induction variant, do not disturb plate.

[0169] Friday:PATENT ATTORNEY DOCKET NO.: N2041-03401 - Prepare 2 ml basal differentiation medium per well as above, with or without C-59 depending on protocol variant: o For 2 d mesoderm induction variant, include C-59 o For 1 d mesoderm induction variant, omit C-59 - Mix well, and equilibrate to 37°C. - Remove old medium from differentiation wells and add 2 ml of fresh cardiac specification or basal differentiation medium to the cells. - Place back onto wave shaker and incubate over the weekend.

[0170] Following Monday and afterwards: - The cell aggregates should show spontaneous beating by day 7 and may be analyzed for the expression of cardiac markers or be kept in culture for extended periods of time using regular media changes. Overall cardiac differentiation efficiency may also be assessed after day 7, i.e., on day 8 or 9, although this should be kept consistent from experiment to experiment.

[0171] Small-scale cardiac 3D differentiation on microcarriers:

[0172] Monday: - Starting point are iPSCs expanded on microbeads according to SOP RD-12, e.g., cells seeded onto beads on the previous Thursday at 200 k cells per 6-well and 20 mg beads. - Harvest and count total number of cells in representative aliquot of iPSC-bead culture, e.g., 1 complete well from several replicate wells. Briefly, centrifuge beads for 0.5 min at 300 x g. aspirate supernatant, wash with 1 volume of PBS, pellet and wash again, then resuspend in 2 ml prewarmed Accutase containing Y. Transfer to a fresh well of a 6-well plate and incubate for 20- 25 min. Dissociate cells for ~5 times using a 1 ml pipette, confirm single-cell dissociation under the microscope, then pass through a 40 µM cell strainer plates onto a 50 ml tube. Wash strainer with several ml of medium (XF medium or below differentiation medium). Centrifuge at 300 x g for 2 min, resuspend cells in medium of choice containing Y. Work out volume of original iPSCs- bead suspension corresponding to a given number of cells on beads assuming 100% cell recovery from beads. - During Accutase digestion in the previous step, prepare required amount of differentiation medium + approximately 50% for washing and resuspension steps - about 3 ml per 6-well to be started. Scale up or down accordingly: o Knockout™ DMEM o 1 x ITS o PSG or just Gln or Glx o 250 µM 2-phospho-ascorbate (from 250 mM stock), optionalPATENT ATTORNEY DOCKET NO.: N2041-03401 o 0.05% (w / v) HSA (from 10% stock), optional o 5-10 ng / ml FGF2 (from 10 µg / ml stock) o 5-10 ng / ml Activin A (from 10 µg / ml stock) o 3-5 ng / ml BMP4 (from 1 or 10 µg / ml stock) o 0.5 µM CHIR99021 (from 1- or 3-mM stock) - Mix well and equilibrate to 37°C. - Transfer required volume of iPSC-beads into a 15- or 50-ml tube, then centrifuge at 300 g for 0.5 min. - Completely aspirate off supernatant, wash iPSC-beads in surplus differentiation medium, and pellet again. - Resuspend iPSC-beads in required volume of differentiation medium and distribute evenly into target wells. - Place plate(s) onto shaker inside cell culture incubator.

[0173] Wednesday: - Prepare basal differentiation medium with WNT inhibitor C-59 (~2 ml needed per differentiation 6-well): o Knockout™ DMEM o PSG or just Gln or Glx o 250 µM 2-phospho-ascorbate (from 250 mM stock) o 0.05% (w / v) HSA (from 10% stock) o 0.2-2 µM C-59 (from 2 mM stock) - Mix well and equilibrate on 37°C. - Remove differentiation plate from incubator. In cell culture hood, tilt plate by approximately 45°, leave for 0.5-1 min, then carefully, using a 1 ml pipette and without disturbing the beads at the bottom, soak off medium. There may be no need completely replace the old medium, i.e., a partial medium change may be sufficient. - Add up to 2 ml of above differentiation medium (depending on how much has been withdrawn in the previous step) and place back onto wave shaker.

[0174] Friday: - Prepare basal differentiation medium as above (albeit without C-59 – 2-4 ml needed per differentiation 6-well), mix well, and equilibrate to 37°C. - Remove old medium from differentiation wells and add 2-4 ml of fresh basal differentiation medium to the cells. - Place back onto wave shaker and incubate over the weekend.PATENT ATTORNEY DOCKET NO.: N2041-03401

[0175] Following Monday and afterwards: - The cell-beads should show spontaneous beating by day 7 and may be analyzed for the expression of cardiac markers or be kept in culture for extended periods of time using regular media changes. Overall cardiac differentiation efficiency may also be assessed after day 7, i.e., on day 8 or 9, although this should be kept consistent from experiment to experiment.

[0176] Cardiomyocytes maintenance after differentiation: - For 3D, following differentiation into cardiomyocytes, the cells may be further kept in plain defined conditions: o IMDM base medium o ITS o PSG (or just Gln or Glx) o 250 µM AscP o 0.05% HSA - 3D aggregates may also be dissociated into single cells in the presence of Y-27632 and be replated into the above medium (with Y-27632 on the first day) to re-form 3D aggregates. - For 2D, 3D CM aggregates may be dissociated into single cells in the presence of Y-27632 and be replated into the above medium (with Y-27632 on the first day) onto iMatrix-511 (1x) coated plates.

[0177] Analysis of cardiac differentiation efficiency by FACS: - On day 7-9 of differentiation, prepare the following solutions: o PEB-Buffer (stable for 4 weeks at 4°C): 100 ml PBS (- / -) with 0.5g BSA (end conc. 0.5%) and 200 µl 0.5M EDTA (end conc.2mM). o Fixation / Permeabilization Solution (prepare freshly): 1 Vol Fixation / Permeabilization Solution 1 + 3 Vol Fixation / Permeabilization Solution 2 - 0.5 ml needed per sample, keep chilled. o Permeabilization Buffer (prepare freshly): 1 Vol 10x Permeabilization Buffer + 9 Vol dest. H2O - 2.1 ml needed per sample. o Prewarm Accutase (containing 10 µM Y-27632) to 37°C. - Harvest cells: o Completely soak off medium and transfer EB’s in a 1.5 ml tube o Wash with 0,5-1 ml PBS. o Centrifuge 400 x g for 2 min., soak of supernatant o Replace by 0.5 ml prewarmed Accutase containing Y-27632 and transfer to incubator for 20 min on a rotator (MACSmix).PATENT ATTORNEY DOCKET NO.: N2041-03401 o After this pipette twice up and down, cells should be single cells. If this is not the case, prolong digestion for 2 more min. o Stop the reaction with 500 µl Medium containing serum and spin for 2 min at 400 x g. o When clumps inside, strain the cells through a 20-40 µm strainer. - Fix and stain cells: o Remove supernatant and wash cells with at least 1 ml PEB buffer and centrifuge again for 2 min at 400 x g. o Resuspend cells in 0.5 ml of cold, freshly prepared Fixation / Permeabilization Solution. o Mix well and incubate for 30 minutes in the refrigerator (2-8°C) on a rotator (MACSMix). o Centrifuge at 400 x g for 2 min. o Remove supernatant, add 0.5 ml of 1x Permeabilization buffer and centrifuge at 400 x g for 2 min. o Remove supernatant and resuspend in 100 µl 1x Permeabilization buffer +2 µl cTNT- FITC or isotype control. o Mix well and incubate for 30 minutes in the refrigerator (2-8°C)on a rotator (MACSMix). o Add 1 ml of 1x Permeabilization buffer and centrifuge at 400xg for 2 min. o Remove supernatant and resuspend cell pellet in 300 µl PEB-Buffer for analysis at the FACS machine. - Analyze cells on MACSQuant: o Switch on machine or start software by touching the screen. o After login, click on top right button > Acquisition mode, let machine warm up for ~30 min. o View > Hardware > Lens and Detection: Confirm that blue laser has stably reached ~37°C. o Run bead calibration: Fill one droplet of beads (stored at 2-8°C) into FACS tube and place into holder; Press barcode reader symbol at top right and scan barcode form stock vial containing calibration beads > Calibration run will start and terminate automatically. o Optionally flush after clicking droplet button at bottom right. o Open > Instrument Settings > Choose appropriate template (FSC lin at 380 / SSC lin at 634 / B1 hlog at 400 / Trigger FSC at 17.30).PATENT ATTORNEY DOCKET NO.: N2041-03401 o Open > Workspace > Choose appropriate template plotting SSC-A vs. FSC-A (P1) / SSC-A vs. SSC-H (P2, gate on P1) / Counts vs. FITC channel (gate on P2) / SSC-H vs. FITC channel (gate on P2). o Project > Enter experiment ID. o Settings > P2. o Events > 50,000. o Define sample and uptake volumes (e.g., 300 µl and 200 µl, respectively). o Define sample ID; Vortex and place first sample into holder, then click start button at bottom right. Repeat for remaining samples. o Cleaning: Fill ~2 ml cleaning solution into FACS tube and start cleaning program after pressing the droplet button at the bottom right. o Click on top right button > Switch instrument off.

[0178] Abbreviations and standard reagents: CHIR CHIR99021 % r, ts 6,EXAMPLE 3 Generation of Cardiomyocytes Differentiated from Human Pluripotent Stem Cells

[0179] The differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes replicates the phases of early in vivo cardiogenesis, directing pluripotent stem cells (PSCs) initially into mesoderm and then through cardiac specification to form cardiomyocytes. Over the years, a variety of strategies have been utilized to produce cardiomyocytes from human PSCs (hPSCs). It has been established that activating the WNT pathway with the small molecule CHIR99021, at an appropriate dose, can induce cardiac mesoderm (PMID: 22645348, 23257984). Similarly, protocols that leverage bone morphogenetic protein (BMP) have been employed (PMIDPATENT ATTORNEY DOCKET NO.: N2041-03401 18432194). These pathways are known to synergistically bolster the induction of cardiogenic fate. Notably, BMP4’s addition has been found to significantly enhance WNT signaling by modulating crucial target genes in the initial stages of differentiation. Consequently, when WNT and BMP agonists are combined, their effective dosage is reduced (BMP4 below 10 ng / ml and CHIR99021 below 2 µM). Additionally, FGF2 and Activin A serve as “accessory factors” in this process, boosting efficiency and consistency across experiments, even though their simultaneous activation is not deemed essential.

[0180] Cardiac specification, following mesoderm induction, is facilitated by the inhibition of the WNT pathway, achievable in vitro through pharmacological inhibitors like IWP2, IWR1, and C- 59 (PMID: 26748419, PMID: 21737789, PMID: 24930130). The criticality of this inhibition step immediately after WNT activation is underscored by research from Rao et al (Rao, Jyoti et al. “Stepwise Clearance of Repressive Roadblocks Drives Cardiac Induction in Human ESCs.” Cell stem cell vol.18,3 (2016): 341-53. doi:10.1016 / j.stem.2015.11.019).

[0181] Despite some protocols achieving differentiation efficiencies exceeding 90%, the consistency across independent experiments poses a challenge. Variations in outcomes, even under seemingly constant conditions, have highlighted an urgent need for methodological enhancements to improve robustness, crucial for therapeutic applications where inconsistencies can lead to significant delays.

[0182] The co-stimulation of BMP and WNT pathways, optionally combined with FGF / ERK and TGFβ / SMAD cascade activation, has proven more effective than sole WNT stimulation for cardiac mesoderm induction. This refined approach aims to mitigate sensitivity to cell titer fluctuations and boost reproducibility. Incorporating ascorbate and serum albumin into the differentiation medium has beneficial effects on reproducibility. A pivotal step towards achieving high reproducibility involved only partial medium changes when transitioning from WNT activation to inhibition, a practice diverging from the conventional full medium exchange aimed at reversing signaling pathway activities.

[0183] In a methodical developmental process, various parameters affecting cardiac differentiation in a 3D context were optimized. Co-stimulation of BMP and WNT pathways, alongside FGF and TGFβ signaling activators, initiates cardiac mesoderm in an insulin-containing medium for one to several days (ideally two), followed by a partial medium swap to include a WNT inhibitor for terminal cardiomyocyte differentiation for another one to several days (again, ideally two). This co-stimulation strategy, effectively applied in 3D settings, formed the foundation for comprehensive optimization in over 100 experiments focusing on efficiency and consistency. The final protocol, initiating differentiation upon rather than after 3D aggregatePATENT ATTORNEY DOCKET NO.: N2041-03401 formation, is detailed in FIGURE 1A. A comparison to a semi-optimized version (gray dots) shows that introducing a partial medium exchange at the transition to WNT inhibition (optimized protocol, blue dots) significantly boosts robustness, as illustrated in FIGURE 1B. Minor modifications, like adjustments to media change intervals and the addition of ascorbic acid, contributed to establishing a robust methodology on a smaller scale, as depicted in FIGURE 1C and the blue dots in FIGURE 1B.

[0184] FIGURE 2 demonstrates that a medium exchange of 25% or less optimizes consistency between experiments, emphasizing the critical nature of partial medium exchange for establishing a robust methodology and overcoming batch-to-batch variations.

[0185] Initially, BMP4 and CHIR99021 were identified as fundamental inducers of cardiogenic mesoderm. Titration experiments indicated that their combined application resulted in high differentiation efficiencies, as validated by cardiac troponin C staining, in contrast to the outcomes of sole WNT activation (FIGURE 3A). However, reproducing these promising results proved challenging. Further investigation identified cell titer as a crucial variable (FIGURE 3B), where even a modest reduction significantly impacted differentiation efficiencies, underscoring the need for consistent parameter control. A heat map representation of grid-type titration of BMP4 and CHIR99021 concentrations for optimizing cardiac mesoderm induction is illustrated in FIGURE 3C. WNT activation alone was inferior to co-stimulation of the BMP and WNT pathways.

[0186] The introduction of FGF2 and Activin A into the co-stimulation cocktail at optimized doses modestly improved cardiac differentiation efficiencies (FIGURE 4A). Crucially, the four- pathway stimulation strategy bolstered the protocol’s robustness by diminishing the impact of cell titer variations (FIGURE 4B).

[0187] FIGURE 5 compares various method variants, reaffirming that BMP and WNT pathway co-stimulation consistently yield superior and more uniform results, although WNT stimulation alone can also achieve high efficiency under certain conditions. This flexibility in signaling cue application timing is further evidenced in FIGURE 6, showcasing the protocol’s adaptability to different base media, including Knockout-DMEM and IMDM.

[0188] The essential role of WNT pathway inhibition is highlighted in FIGURE 7, where C-59 is shown to have a broad effective concentration range for cardiac specification (standard: 0.5 µM final).

[0189] The necessity of “unspecific” additives, such as L-ascorbate (or 2-phospho-L-ascorbate) and serum albumin, for high-efficiency cardiac differentiation is depicted in FIGURE 8, presenting a scenario where these compounds are indispensable.PATENT ATTORNEY DOCKET NO.: N2041-03401

[0190] Finally, iPSC-derived cardiomyocytes initially exhibiting an immature gene expression signature (FIGURE 9A) undergo a transition towards late myosin light and heavy chain-encoding genes (FIGURE 9B and 9C), acquiring a more defined sarcomeric structure, indicative of maturation towards a ventricular default fate.

[0191] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered accordingly.

Claims

PATENT ATTORNEY DOCKET NO.: N2041-03401 CLAIMS What is claimed is:

1. A method of inducing cardiomyocyte differentiation from pluripotent stem cells (PSCs) comprising: a) contacting PSCs in a culture medium for one or more days wherein the culture medium comprises a WNT signaling pathway activator and: (i) a bone morphogenetic protein (BMP) signaling pathway activator; (ii) optionally an FGF / ERK signaling pathway activator; and (iii) optionally a TGF-β signaling pathway activator; and b) partially exchanging the culture medium from a) with a culture medium comprising a WNT signaling pathway inhibitor and further culturing for one or more days, thereby inducing the PSCs to differentiate into cardiomyocytes.

2. The method of claim 1, wherein the FGF / ERK signaling pathway activator is FGF2.

3. The method of claim 2, wherein the concentration of FGF2 ranges from between about 2.5 ng / ml to 20 ng / ml.

4. The method of claim 1, wherein the TGF-β signaling pathway activator comprises Activin A.

5. The method of claim 4, wherein the concentration of Activin A ranges from between about 2.5 ng / ml to 20 ng / ml.

6. The method of claim 1, wherein the volume of the culture medium exchanged in b) is about 10%, 25%, 50%, or 75% of the culture medium in a).

7. The method of claim 6, wherein the volume of the culture medium exchanged in step b) is about 25% of the culture medium in a).

8. The method of claim 1, wherein the WNT signaling pathway activator comprises CHIR99021.

9. The method of claim 1, wherein the BMP comprises BMP4.

10. The method of claim 1, wherein the culture medium from a) and / or b) further comprises ascorbate and / or albumin, and optionally L-glutamine.

11. The method of claim 10, wherein the culture medium optionally comprises one or more salts of 2-phospho-L-ascorbate.PATENT ATTORNEY DOCKET NO.: N2041-03401 12. The method of claim 1, wherein the contacting in a) is for at least 2 days.

13. The method of claim 1, wherein the incubation in b) is for 2 to 5 days.

14. The method of claim 1, wherein the WNT signaling pathway inhibitor is selected from IWP2, IWR1, or C-59.

15. The method of claim 14, wherein the concentration of the WNT signaling pathway inhibitor is at least 0.1 µM.

16. The method of claim 1, wherein the cardiomyocytes express maturation markers after about 6 weeks of differentiation.

17. The method of claim 16, wherein the maturation markers comprise MYL2, MYH7, and / or KCNQ1.

18. The method of claim 1, wherein the cardiomyocyte differentiation is performed in a culture selected from: a two-dimensional culture, a three-dimensional culture, a suspension culture, and an adherent culture.

19. The method of claim 18, wherein the culture is a plate, a microcarrier bead or a mesh substrate.

20. The method of claim 1, wherein cardiac troponin T (cTnT) positive cells comprise at least 80% of the PSCs after 9 days of differentiation.

21. The method of claim 1, wherein the culture medium from a) and b) is Knockout-DMEM or IMDM.

22. The method of claim 1, wherein the PSCs are human PSCs (hPSCs).

23. The method of claim 22, wherein the hPSCs are human induced pluripotent stem cells (hiPSCs) or human embryonic stems cells (hESCs).

24. A method of inducing cardiomyocyte differentiation from pluripotent stem cells (PSCs) comprising: a) contacting PSCs with an iPSC culture medium for at least one day, wherein the iPSC culture medium comprises a Rho-associated kinase (ROCK) inhibitor to obtain ROCK- inhibitor cultured cells; b) contacting the ROCK-inhibitor cultured cells with a mesoderm induction medium for one or more days, wherein the mesoderm induction medium comprises:PATENT ATTORNEY DOCKET NO.: N2041-03401 (i) a WNT signaling pathway activator; (ii) a bone morphogenetic protein (BMP) signaling pathway activator; (iii) optionally an FGF / ERK signaling pathway activator; and (iv) optionally a TGF-β signaling pathway activator; c) partially exchanging the differentiation culture medium from b) with a culture medium comprising a WNT signaling pathway inhibitor and further culturing for one or more days, thereby inducing the PSCs to differentiate into cardiomyocytes.

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