Methods for producing committed cardiac progenitor cells

Abstract

Provided herein are methods for differentiating pluripotent stem cells into committed cardiac progenitor cells. Further provided herein are methods of using committed cardiac progenitor cells in the treatment of cardiac disease.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 243,606, filed September 13, 2021, the contents of which are incorporated herein by reference. [Background technology]

[0002] 1. Field The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to the differentiation of pluripotent stem cells into committed cardiac progenitor cells.

[0003] 2. Description of Related Technology Cardiac progenitor cells (CPCs) have the ability to differentiate into mature cardiomyocytes. These CPCs represent the final stage of commitment to cardiomyocytes. Therefore, these cells are attractive targets for regenerative medicine applications in drug development, such as for the treatment of myocardial infarction and congestive heart failure. Current methods for producing cardiomyocytes from pluripotent stem cells require long-term culture to make the cardiomyocytes stably contract. Therefore, there is a need to improve the method for producing committed cardiac progenitor cells from pluripotent stem cells in a more efficient way that requires less time in culture. Summary of the Invention [Means for solving the problem]

[0004] In certain embodiments, the disclosure provides an in vitro method for producing committed cardiac progenitor cells from human pluripotent stem cells (PSCs), comprising: (a) culturing PSCs in the presence of a Wnt agonist to initiate differentiation and a survival reagent to form cell aggregates; (b) further culturing the cell aggregates in the presence of a Wnt agonist for a period of time sufficient to produce a mesodermal cell population; and (c) differentiating the mesodermal cell population in the presence of a Wnt inhibitor to promote cardiac specification, thereby producing a population of committed cardiac progenitor cells.

[0005] In some embodiments, the PSCs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In certain embodiments, prior to step (a), the PSCs are cultured on a surface coated with an extracellular matrix. In some embodiments, the extracellular matrix is ​​vitronectin, collagen, laminin, Matrigel™, and / or fibronectin.

[0006] In certain embodiments, the survival reagent is a Rho-associated kinase (ROCK) inhibitor or a myosin II inhibitor. For example, the ROCK inhibitor is H1152 or Y-27632. In certain embodiments, the myosin II inhibitor is blebbistatin.

[0007] In some embodiments, the methods include culturing the cells in suspension culture. In certain embodiments, the suspension culture is carried out in one or more bioreactors, such as vertical-wheel bioreactors.

[0008] In certain embodiments, the Wnt agonist in step (a) is CHIR99021, SB216763, CHIR98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, or IM-12. In certain embodiments, the Wnt agonist is CHIR99021. In certain embodiments, CHIR99021 is present in the culture at a concentration of about 1 μM to 10 μM, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, particularly about 2 μM.

[0009] In some embodiments, step (a) is for 1 to 2 days, e.g., about 22, 23, 24, 25, or 26 hours, in particular about 24 hours. In certain embodiments, the culture of step (b) is free or substantially free of insulin.

[0010] In certain embodiments, the Wnt signaling agonist in step (b) is CHIR99021, SB216763, CHIR98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, or IM-12. In certain embodiments, the Wnt signaling agonist in step (b) is CHIR99021. In some embodiments, CHIR99021 is present in the culture at a concentration of 1 μM to 10 μM, such as at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, in particular at a concentration of about 4 μM to 5 μM, such as at a concentration of about 4.4 μM.

[0011] In some embodiments, the culture of step (b) further comprises an Activin / Nodal agonist and / or a BMP. In some embodiments, the Activin / Nodal agonist is Activin A or Nodal. In certain embodiments, step (b) is carried out for 1 to 5 days, such as about 1, 2, 3, 4, or 5 days, particularly about 1 or 2 days.

[0012] In some embodiments, the mesodermal cells express KDR, PDGFRα, CXCR4, and / or CD56. In particular embodiments, at least 5% (e.g., at least 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55%) of the mesodermal cells express CD56 prior to or during step (c). In some embodiments, at least 40% (e.g., at least 45, 50, 55, 60, 65, 70, or 75%) of the mesodermal cells express KDR and PDGFRα prior to or during step (c). In particular embodiments, after step (c) is initiated, the cells express KDR. In particular embodiments, prior to step (c), the mesodermal cells are positive for CXCR4 and CD56. In some embodiments, prior to step (c), at least 30% (e.g., at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, or 80%) of the positive mesodermal cell population are positive for CXCR4 and less than 60% (e.g., less than 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 45, 40, or 30%) of the mesodermal cell population are positive for CD56.

[0013] In some embodiments, prior to step (c), at least 20% (e.g., at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80%) of the cells of the mesoderm cell population are positive for CXCR4 and less than 60% (e.g., less than 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 45, 40, or 30%) of the mesoderm cell population are positive for CD56. In certain aspects, step (c) comprises adding a Wnt inhibitor when at least 20% (e.g., at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80%) of the positive mesodermal cell population are positive for CXCR4 and less than 60% (e.g., less than 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 45, 40, or 30%) of the mesodermal cell population are positive for CD56.

[0014] In certain embodiments, the Wnt inhibitor in step (c) is XAV939, IWR1, IWR2, IWR3, IWR4, ICG-001, IWR-1-endo, Wnt-C59, LGK-974, LF3, CP21R7, NCB-0846, PNU-74654, or KYA179K. In certain embodiments, the Wnt inhibitor is XAV939. In certain embodiments, XAV939 is present in the culture at a concentration of 5 μM to 10 μM, for example, at a concentration of 5, 6, 7, 8, 9, or 10 μM. In some embodiments, the culture of step (c) further comprises a TGFβ inhibitor, such as SB431542, LDN-193189, LY2157299, LY2109761, SB525334, SIS HCl, SB505124, GW788388, or LY364947. In certain embodiments, the TGFβ inhibitor is SB431542. In certain embodiments, SB431542 is present in the culture at a concentration of 1 μM to 5 μM, such as at a concentration of 1, 2, 3, 4, or 5 μM. In some embodiments, the culture of step (c) comprises insulin. In some embodiments, the culture of step (c) further comprises a BMP inhibitor or an AMPK inhibitor. In certain embodiments, the BMP inhibitor is dorsomorphin, LDN193189, DMH1, DMH2, or ML347. In some embodiments, step (c) is for 1 to 6 days, such as 1, 2, 3, 4, 5 or 6 days, for example 1 to 3 days, in particular about 2 days.

[0015] In certain embodiments, the method is serum-free. In some embodiments, the culturing is in a defined medium. In some embodiments, the method does not include performing drug resistance selection. In certain embodiments, the committed cardiac progenitor cells do not express a transgene.

[0016] In some embodiments, the method comprises administering at least 1×10 7 ~1x10 10 of committed cardiac progenitor cells.

[0017] In certain embodiments, less than 20% (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 10, or 5%) of the committed cardiac progenitor cells express EpCAM. In certain embodiments, less than 10% (e.g., less than 9, 8, 7, 6, or 5%) of the cells of the committed cardiac progenitor cells express EpCAM. In some embodiments, less than 20% (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 10, or 5%) of the committed cardiac progenitor cells are positive for KDR, CXCR4, and / or SAA. In some embodiments, at least 80% (e.g., 81, 82, 83, 84, 85, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the committed cardiac progenitor cells are positive for PDGFRα and CD56. In some embodiments, less than 20% (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 10, or 5%) of the committed cardiac progenitor cells are positive for EpCAM and SAA.

[0018] In certain embodiments, the methods are Good Manufacturing Practice (GMP) compliant. In some embodiments, the methods are, for example, characterized in that the committed cardiac progenitor cell population is at least 70% (e.g., 71, 72, 73, 74, 75, 75, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) positive for PDGFRa and less than 40% (e.g., 39, 38, 37, 36, 35, 34, 33, 32, 30, 29, 28, 31, 32, 33, 34, 35, 36, 37, 38, 39, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) positive for , 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10 or 5%) positive for EpCAM and less than 20% (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 10 or 5%) positive for SAA.

[0019] In further embodiments, the method further comprises maturing the committed cardiac progenitor cells to produce cardiomyocytes. In some embodiments, the committed cardiac progenitor cells are cultured in a monolayer. In particular embodiments, the committed cardiac progenitor cells are cultured on a surface coated with an extracellular matrix. In some embodiments, the extracellular matrix is ​​vitronectin, collagen, laminin, Matrigel™, and / or fibronectin. In particular embodiments, the extracellular matrix is ​​vitronectin.

[0020] In some embodiments, the cardiomyocytes express CTNT, MHC, MLC, CTNI, and / or sarcomeric alpha actinin. In certain embodiments, at least 80% (e.g., 81, 82, 83, 84, 85, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the cells are positive for sarcomeric alpha actinin.

[0021] In certain embodiments, the medium for maturation does not contain a Wnt inhibitor or a TGFβ inhibitor. In some embodiments, the culture for maturation is for 2 to 30 days, for example, 2 to 20 days, for example, 5 to 10 days.

[0022] In certain embodiments, the method comprises producing primed cardiac progenitor cells, comprising culturing the PSCs in suspension in the presence of a Wnt agonist to initiate differentiation, and when the population of cells comprises less than about 60% CD56 positive cells and at least about 20% CXCR4 positive cells, culturing the cells in the presence of a Wnt inhibitor to produce a population of primed cardiac progenitor cells that are at least 70% positive for PDGFRα, less than 40% positive for KDR, less than 20% positive for EpCAM, and less than 20% positive for SAA. The population of primed cardiac progenitor cells may be cryopreserved.

[0023] In some embodiments, the method further comprises differentiating the committed cardiac progenitor cells into a vascular endothelial cell population. In some embodiments, the differentiating comprises culturing the committed cardiac progenitor cells in the presence of fibroblast growth factor (FGF) and / or vascular endothelial growth factor (VEGF). In certain embodiments, the vascular endothelial cells are positive for CD33 and CD144. In some embodiments, at least 20% (e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70% or more) of the cells of the vascular endothelial cell population are positive for CD33 and CD144.

[0024] In some embodiments, the method further comprises differentiating the committed cardiac progenitor cell population into a smooth muscle cell population. In certain embodiments, the differentiating comprises culturing the committed cardiac progenitor cell population in the presence of FGF and / or VEGF. In certain embodiments, the smooth muscle cell population has at least 50% (e.g., 55%, 60%, 70%, 75%, 80% or more) cells positive for CD140b and CD90.

[0025] Further provided herein is a population of committed cardiac progenitor cells produced by the methods of this embodiment and aspects thereof. Also provided herein is a population of cardiomyocytes, vascular endothelial cells, or smooth muscle cells produced by the methods of this embodiment and aspects thereof.

[0026] Another embodiment provides a population of committed cardiac progenitor cells having at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, or 99%) expression of CD56, at least 80% (e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) expression of PDGFRα, and less than 10% (e.g., less than 9, 8, 7, 6, 5, 4, 3, 2, or 1%) expression of CXCR4, KDR, and EpCAM. In certain aspects, the committed cardiac progenitor cells are produced by the methods of this embodiment and aspects thereof. In certain aspects, the committed cardiac progenitor cell population is GMP compliant. In some aspects, the composition is a pharmaceutical composition.

[0027] A further embodiment provides a method for the treatment of a cardiac disorder in a subject, comprising administering an effective amount of committed cardiac progenitor cells of this embodiment or aspect thereof to a subject in need thereof.

[0028] In some embodiments, the committed cardiac progenitor cells are administered directly to the heart. In certain embodiments, administration is performed using an intramyocardial catheter. In some embodiments, the cells are administered in a suspension that includes human albumin (e.g., FLEXBUMIN®), e.g., at a concentration of 1% to 10%, e.g., at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%, particularly at a concentration of about 5%.

[0029] In some aspects, the administered committed cardiac progenitor cells exhibit engraftment, cell survival, and maturation into cardiomyocytes. In some aspects, the subject is a human. In certain aspects, the cardiac disorder is myocardial infarction, cardiomyopathy, congestive heart failure, ventricular septal defect, atrial septal defect, congenital heart defect, ventricular aneurysm, pediatric heart disease, ventricular aneurysm, or cardiac disease requiring ventricular reconstruction.

[0030] A further embodiment provides a method of generating cardiac progenitor cells, comprising providing pluripotent stem cells (PSCs), culturing the PSCs in suspension in the presence of a Wnt agonist to initiate cardiac differentiation, and adding a Wnt inhibitor when the cell population is comprised of less than about 60% CD56 positive cells and more than about 30% CXCR4 positive cells to promote robust cardiac specification, thereby producing a cardiac progenitor cell population.

[0031] In some embodiments, the cardiac progenitor cells are useful for treating diseases characterized by insufficient cardiac function. In certain embodiments, the cardiac progenitor cells can differentiate in vivo into cardiomyocyte, endothelial and vascular smooth muscle lineages. In some embodiments, a cardiac progenitor cell population committed by cardiac specification (CTC4) is produced. In certain embodiments, differentiation occurs in a bioreactor.

[0032] In certain embodiments, the method further comprises cryopreserving the cell population when the cell population is composed of cells that are more than 70% positive for PDGFRα, less than 40% positive for KRD, less than 20% positive for EPCAM, and less than 20% positive for sarcomeric α-actinin. In some embodiments, the CTC4 cells may be cryopreserved. In certain embodiments, the committed cardiac progenitor cells are administered directly to the subject's heart. In some embodiments, the differentiation in the presence of a Wnt inhibitor further comprises a TGFβ inhibitor. In certain embodiments, the differentiation in the presence of a Wnt inhibitor further comprises a BMP inhibitor. In some embodiments, the method is a serum-free medium. In certain embodiments, the method does not include drug resistance selection.

[0033] In some embodiments, the method further comprises maturing the committed cardiac progenitor cells to produce cardiomyocytes. In some embodiments, the maturation medium does not include a Wnt inhibitor or a TGFβ inhibitor. In certain embodiments, the cells can be further specified into endothelial cells or smooth muscle cells by addition of VEGF.

[0034] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description of the invention and the specific embodiments thereof, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0035] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0036] [Figure 1A-1B] Schematic diagram showing an exemplary protocol for differentiation of committed cardiac progenitor cells (CTC4) from induced pluripotent stem cells (iPSCs) into cardiomyocytes (Figure 1A). The process includes the initial stages of iPSC aggregate formation, mesoderm induction, and cardiac specification. Schematic of the processing days of the culture method (plating and suspension culture) and indication of the progression stages of cardiac differentiation (Figure 1B).

[0037] [Figure 2A-2B] Outline of processing days for culture methods (plate culture and suspension culture) and indicators of the progression stage of cardiac differentiation (Figure 2A). Schematic diagram showing the scale of iPSC and differentiation using multi-layer CELLSTACK® vessels and PBS3 VERTICAL-WHEEL™ bioreactor (Figure 2B). Cells can be cryopreserved at large scale (e.g., 3.0 x 106 cells / vial) using, for example, Aseptic Technologies' AT CLOSED-VIALS®.

[0038] [Figure 3A-3C]Schematic diagram depicting an exemplary protocol for differentiation of cardiomyocyte-committed cardiac progenitor cells (CTC4) from induced pluripotent stem cells (iPSCs) (Figure 3A). The process includes the initial stages of iPSC aggregate formation, mesoderm induction, and cardiac specification. Three different PBS3 bioreactors were sampled on days 2-5 of differentiation and analyzed for CXCR4 and CD56 by flow cytometry (Figure 3B). Committed cardiac progenitor cells were harvested from the three PBS3 bioreactors, pooled, and analyzed for CXCR4 and CD56 by flow cytometry (Figure 3C).

[0039] [Figure 4A-4B] Three different PBS3 bioreactors were sampled on differentiation days 2-5 and analyzed for EPCAM by flow cytometry (Figure 4A). Committed cardiac progenitor cells were harvested from the three PBS3 bioreactors, pooled, and analyzed for EPCAM by flow cytometry (Figure 4B).

[0040] [Diagram 5] PBS3 bioreactors were sampled on days 4-6 and analyzed for KDR and PDGFRα by flow cytometry. Committed cardiac progenitor cells harvested on day 6 of differentiation had significantly reduced expression of KDR.

[0041] [Figure 6A-6B] Gene expression from multiple batches of iPSCs and committed cardiac progenitor cells was analyzed by Fluidigm for the pluripotency genes NANOG, SOX2, and POU5F1 (Figure 6A). Gene expression from multiple batches of committed cardiac progenitor cells was analyzed by Fluidigm for the cardiac genes HAND2, GATA4, NKX2.5, PDGFRA, and TBX5 (Figure 6B).

[0042] [Figure 7A-7E]Schematic diagram depicting the protocol to confirm that CTC4 cells become cardiomyocytes after thawing and plating on vitronectin-coated vessels in RPMI+B27 medium (Figure 7A). Flow cytometric characterization of CTC4 cells after thawing showed that the majority of the cell population was CD56 positive, CXCR4 negative, EpCAM negative, KDR negative, PDGFRα positive, and SAA negative, indicating that these cells are committed to become cardiomyocytes but have not yet started expressing the cardiac marker sarcomeric alpha actinin (SAA) (Figure 7B). Immunocytochemical characterization of CTC4 cells after culturing them for 7 days on vitronectin-coated 96-well plates with RPMI+B27 medium (Figure 7C). The cardiac-specific transcription factor NKX2.5 was expressed along with the cardiac structural proteins sarcomeric alpha actinin (SAA), cardiac troponin I (CTNI), and cardiac troponin T (CTNT). Flow cytometry analysis of SAA after 7 days of culturing CTC4 cells in vitronectin-coated vessels with RPMI+B27 medium shows cardiomyocyte specification (Figure 7D). Contraction of CTC4 cell-derived cardiomyocytes after 7 days of culturing CTC4 cells in vitronectin-coated vessels with RPMI+B27 medium (Figure 7E).

[0043] [Figure 8A-8C]Schematic diagram depicting myocardial infarction model in NUDE rats (Figure 8A). Three days after surgical ligation of the left anterior descending artery (LAD), CTC4 cells suspended in 5% Flexbumin (1e7) were administered by intramyocardial injection multiple times (5 times) into the peri-infarct region of the left ventricle. Thirty days after CTC3 injection, hearts were processed and analyzed. Tissues were cut into 5 rings per heart, processed, and embedded in paraffin. Twenty serial sections were cut from each ring of each heart and slides were generated. Immunohistochemistry analysis for human ALU was performed on slides generated from sections 1, 5, 10, and 20 of each heart block to determine the distribution and engraftment success of human cells (Figure 8B). Once human cells were detected, serial sections were taken for further processing and characterization. We characterized the transplanted human cells in a multiplexed manner using fluorescent in situ hybridization for human Alu, followed by immunohistochemistry to detect Ki67, cardiac troponin T, or CX43 (Figure 8C).

[0044] [Figure 9A-9B] Schematic diagram showing the culture of iPSC-derived cardiac progenitor cells in RPMI+B27 medium containing growth factors FGF2 and / or VEGF for differentiation into vascular endothelial cells (CD31+CD144+) and smooth muscle cells (CD140b+CD90+) (Figure 9A). Flow cytometry of the expression of CD31 and CD144 for vascular endothelial cells, and CD90 and CD140b for smooth muscle cells (Figure 9B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Differentiation of pluripotent stem cells can be induced in a variety of ways, for example, by forming cell aggregates (these aggregates are called embryoid bodies (EBs)) in adherent colonies or in a low-adherent environment, for example. The morphogenetic signaling and activities of molecules and cells within EBs resemble many of the ways such cells perform during natural ontogeny in developing embryos. In certain embodiments, the present disclosure provides methods for producing committed cardiac progenitor cells from pluripotent stem cells (PSCs), such as induced pluripotent stem cells (iPSCs), in large quantities and in a short period of time. These committed cardiac progenitor cells are primed to become cardiomyocytes without additional growth factors or small molecule signals, yet retain the ability to differentiate into endothelium. In some embodiments, the differentiation process provided herein is optimized to establish stable and robust contraction rapidly after thawing and plating.

[0046] The differentiation process can include forming aggregates from PSCs, such as iPSCs, in the presence of an agent that promotes aggregate formation, such as a Wnt agonist and a ROCK inhibitor. The aggregates can then be induced to form mesodermal cells in the presence of a Wnt agonist, such as CHIR99021. In certain embodiments, the mesodermal induction medium does not contain insulin. The mesodermal induction medium can further include an activin agonist and / or a BMP. Mesodermal cells can be identified by positive expression of CXCR4, KDR, PDGFRα, and / or CD56, and the substantial absence of expression of pluripotency markers CKIT and / or EPCAM. The mesodermal induction step can be about 1-3 days. The mesodermal cells are then cardiac specified in the presence of a Wnt inhibitor, and optionally TGFβ and / or a BMP inhibitor, particularly in combination with insulin. Committed cardiac progenitor cells can be produced, such as about 1-3 days after the initiation of cardiac specification. In certain aspects, the aggregates at the mesodermal stage can be maintained in suspension culture to initiate cardiac specification, or the mesodermal cells can be individualized and plated as monolayer cultures prior to initiating cardiac specification. Committed cardiac progenitor cells can be produced in both culture systems. The committed cardiac progenitor cells can be further cultured to produce cardiomyocytes. Notably, the differentiation process can be performed in the absence of serum, without drug resistance or metabolic selection.

[0047] In this study, we developed a robust and scalable cGMP iPSC-derived cardiac differentiation protocol that can generate up to 1 × 10 cells per bioreactor. 8 ~3×10 9 yielding 1 x 10 committed cardiac progenitor cells (CTC4 cells) per cryopreservation vessel. 6 ~300×10 6These CTC4 cells can be cryopreserved on a cellular scale. These CTC4 cells are different from the earlier stage KDR+ cardiac progenitor cells previously described. For example, CTC4 cells have already rapidly decreased KDR expression and do not have the same differentiation potential as KDR+ cardiac progenitor cells at earlier developmental stages. Instead, CTC4 cells can be cryopreserved at a specific late developmental stage, but before the cells have committed to early cardiomyocytes.

[0048] In a further embodiment, the cryopreserved cardiac progenitor cells of the present invention can be thawed and cultured in a medium such as RPMI+B27 to further differentiate into highly pure cardiomyocytes. The cryopreserved cardiac progenitor cells can also become cardiomyocytes after injection into a subject's myocardium.

[0049] In a further aspect, the committed cardiac progenitor cells of the invention can be differentiated into vascular endothelial cells or smooth muscle cells, such as in medium containing FGF and / or VEGF.

[0050] Additionally, the present disclosure provides methods of treatment, including administering the CTC4 cells provided herein. The CTC4 cells can be delivered by direct injection, transendocardial, or intramyocardial catheter delivery. The dosage of iPSC-derived CTC4 cells is approximately 1×10 7 ~1×10 9 The CTC4 cells of the present disclosure can be produced from HLA-compatible iPSCs for compatibility with the subject of treatment. The current methods, including using all the materials and culture formats described, can be used for cGMP production. Thus, the present disclosure provides a stable, reproducible and suitable source of cells to advance drug development and cardiac regenerative medicine. The CTC4 cells can also be used to help identify and avoid toxicity issues of drug-mediated cardiogenesis.

[0051] I. Definition As used herein, "substantially free" with respect to a particular component is used herein to mean that none of the particular component is intentionally incorporated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from unintentional contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the particular component is undetectable by standard analytical methods.

[0052] As used herein, "a" or "an" may mean one or more. When used in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0053] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer only to alternatives or the alternatives are not mutually exclusive, however, the present disclosure supports a definition that refers only to alternatives and "and / or." As used herein, "another" may mean at least a second or more.

[0054] Throughout this application, the term "about" is used to indicate that a value includes the error of variation inherent in the device, method used to determine the value, or the variation that exists among study subjects. In some embodiments, the term generally means within the standard deviation of the stated value as determined by standard analytical techniques for determining the stated value. The term can also be used to refer to plus or minus 5% of the stated value, for example, the percentage of cells in a population of cells that are positive or negative for a particular marker.

[0055] The term "exogenous" when used with respect to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means. Or, when used with respect to a cell, the term refers to a cell that has been isolated and subsequently introduced into another cell or organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or may be one or more additional copies of a nucleic acid that is naturally present in the organism or cell. An exogenous cell may be from a different organism or may be from the same organism. Non-limiting examples of exogenous nucleic acids include those that are in a chromosomal location different from their location in the natural cell, or that are flanked by different nucleic acid sequences than those found in nature.

[0056] By "expression construct" or "expression cassette" is meant a nucleic acid molecule capable of directing transcription. An expression construct contains, at a minimum, one or more transcriptional regulatory elements (such as a promoter, enhancer, or functional equivalents thereof) that direct gene expression in one or more desired cell types, tissues, or organs. Additional elements may also be included, such as transcription termination signals.

[0057] A "vector" or "construct" (sometimes called a gene delivery system or gene transfer "vehicle") refers to a polymeric or molecular complex comprising a polynucleotide that is delivered to a host cell, either in vitro or in vivo.

[0058] A "plasmid" is a common type of vector; it is an extrachromosomal DNA molecule that can replicate independently of chromosomal DNA and is separate from the chromosomal DNA. In some cases, it may be circular or double-stranded.

[0059] The term "cell" is used herein in the broadest sense in the art and refers to a structural unit of tissue in a multicellular organism, surrounded by a membrane structure that separates it from the outside, capable of self-replication, and containing genetic information and a mechanism for its expression. As used herein, a cell may be a naturally occurring cell or an artificially modified cell (e.g., a fusion cell, a genetically engineered cell, etc.).

[0060] The term "stem cell" as used herein refers to a cell that is capable of differentiating into a diverse range of specialized cell types under appropriate conditions, but capable of self-renewal under other appropriate conditions, remaining essentially in an undifferentiated pluripotent state. The term "stem cell" also encompasses pluripotent cells, multipotent cells, precursor cells, and progenitor cells. Exemplary human stem cells can be derived from hematopoietic stem cells or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from fetal reproductive tissue. Exemplary pluripotent stem cells can also be produced from somatic cells by reprogramming somatic cells to a pluripotent state through the expression of certain transcription factors associated with pluripotency, and these cells are referred to as "induced pluripotent stem cells" or "iPSc" or "iPS cells".

[0061] "Embryonic stem (ES) cells" are undifferentiated pluripotent cells obtained from earlier stage embryos, such as the inner cell mass of the blastocyst stage, or produced by artificial means (e.g., nuclear transfer), that can give rise to all differentiated cell types of the embryo or adult, including germ cells (e.g., sperm and eggs).

[0062] "Induced pluripotent stem cells (iPSc or iPS cells)" are cells generated by reprogramming somatic cells by expressing or inducing the expression of a combination of factors (referred to herein as reprogramming factors). iPS cells can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct 3 / 4), Sox2, c-Myc, Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, at least four reprogramming factors, at least five reprogramming factors, at least six reprogramming factors, or at least seven reprogramming factors for reprogramming somatic cells into pluripotent stem cells.

[0063] "Pluripotent stem cells" refer to stem cells that have the potential to differentiate into any of the cells that form one or more tissues or organs, or preferably any of the three germ layers: endoderm (inner stomach lining, digestive tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), or ectoderm (epidermal tissue and nervous system).

[0064] The term "somatic cell", as used herein, refers to any cell other than a germ cell (e.g., egg, sperm, etc.) that does not directly transmit its DNA to the next generation. Typically, somatic cells do not have, or have limited, pluripotency. As used herein, somatic cells may be naturally occurring or genetically modified.

[0065] "Programming" is the process of changing the type of progeny a cell can produce. For example, a cell is programmed if it has been altered, either in culture or in vivo, so that it can form at least one new cell type progeny compared to the cell type it would have formed under the same conditions if not programmed. This means that after sufficient proliferation, a measurable proportion of progeny having the phenotypic characteristics of the new cell type is observed, whereas essentially no such progeny could have occurred before programming. Alternatively, it means that the proportion having the characteristics of the new cell type is measurably greater than before programming. This process includes differentiation, dedifferentiation, and transdifferentiation.

[0066] "Reprogramming" is the process of measurably enhancing the ability of a cell to form at least one new cell type progeny, either in culture or in vivo, after which it remains the same in the absence of reprogramming. More specifically, reprogramming is the process of conferring pluripotency to somatic cells, meaning that after sufficient proliferation, a measurable proportion of progeny have phenotypic characteristics of the new cell type, whereas essentially no such progeny could arise before reprogramming. Alternatively, it means that the proportion that have the characteristics of the new cell type are measurably greater than they were before programming.

[0067] "Differentiation" is the process by which a less specialized cell becomes a more specialized cell type. "Dedifferentiation" is the cellular process by which a partially or terminally differentiated cell reverts to an earlier developmental stage, such as pluripotency or multipotency. "Transdifferentiation" is the process by which one differentiated cell type is converted to another differentiated cell type. Typically, transdifferentiation by programming occurs without the cell passing through an intermediate pluripotency stage; that is, the cell is directly programmed from one differentiated cell type to another. Under certain conditions, the percentage of progeny with the characteristics of the new cell type may be at least about 1%, 5%, 25% or more, in order of increasing preference.

[0068] The term "forward programming" refers to the programming of a multipotent or pluripotent cell, as opposed to a differentiated somatic cell that does not have pluripotency, by providing the multipotent or pluripotent cell with one or more specific lineage-committing genes or gene products. For example, forward programming can refer to the process of programming ESCs or iPSCs into hematopoietic progenitor cells or other precursor cells, or hematopoietic cells or other differentiated somatic cells.

[0069] The term "subject" or "subject in need thereof" as used herein refers to a mammal, male or female, of any age, preferably a human, in need of cell or tissue transplantation. Typically, the subject is in need of cell or tissue transplantation (also referred to herein as a recipient) because of a disease, pathological or undesirable condition, condition, or syndrome, or a physical, morphological, or physiological abnormality that is amenable to treatment via cell or tissue transplantation.

[0070] "Survival reagent" refers to an agent that promotes and / or aids cell survival when added to cell culture medium. For example, Rho-associated kinase (ROCK) inhibitors or myosin II specific inhibitors can be used as survival reagents. In certain embodiments, these survival reagents promote cell congregation in culture.

[0071] "Rho-associated kinase inhibitor" is abbreviated as "ROCK inhibitor" and refers to any one of substances, such as small molecules, siRNA, miRNA, antisense RNA, etc., that inhibit or reduce the function of Rho-associated kinase or its signaling pathway in a cell. "ROCK signaling pathway" as used herein may include any signal processor involved in the ROCK-associated signaling pathway, such as the Rho-ROCK-myosin II signaling pathway, its upstream signaling pathway, or its downstream signaling pathway in a cell. Examples of ROCK inhibitors include, but are not limited to, Rho-specific inhibitors, ROCK-specific inhibitors, MRLC (myosin regulatory light chain)-specific inhibitors, or myosin II-specific inhibitors.

[0072] "Committed cardiac progenitor cells (CPCs), primed CPCs, or CTC4 cells" are used interchangeably herein and refer to cells that have not yet fully differentiated into cardiomyocytes, but have been engineered to differentiate toward a cardiac lineage. Thus, these CPCs are primed to become cardiomyocytes. Committed cardiac progenitor cells can be cryopreserved, and when plated or injected in vivo, over 90% differentiate into cardiomyocytes (e.g., SAA-positive cardiomyocytes) without the addition of additional growth factors or small molecule signals. Examples of committed cardiac progenitor cell markers include PDGFRα and CD56. In certain aspects, committed cardiac progenitor cells do not express CXCR4, KDR, CKIT, EPCAM, and / or sarcomeric alpha-actinin. These committed CPC or CTC4 cells are multipotent and can be further differentiated into other cell lineages, such as vascular endothelial cells or smooth muscle cells, for example, by culturing in the presence of growth factors.

[0073] "Cardiomyocytes" or "cardiac muscle cells" refers to muscle cells that constitute cardiac muscle. Examples of cardiac specific markers include α-sarcomeric actinin, troponin, myosin heavy chain, or L-type calcium current.

[0074] "Administering," as used herein, is intended to mean delivering in an affected or performed manner using any of a variety of methods and delivery systems known to those of skill in the art. For example, administration can be intravenous, oral, implant, transmucosal, transdermal, intramuscular, or subcutaneous. Local administration is specifically contemplated. For example, "administering" can be performed once, multiple times, and / or over one or more extended periods of time.

[0075] "Super donors" as used herein refer to individuals who are homozygous for certain MHC class I and MHC class II genes. These homozygous individuals serve as super donors whose cells (including tissues and other materials containing those cells) can be transplanted into individuals who are either homozygous or heterozygous for that haplotype. Super donors can be homozygous for each of the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ locus alleles.

[0076] II. Pluripotent stem cells In certain embodiments of the present disclosure, methods and compositions are disclosed for providing cardiac progenitor cells from pluripotent stem cells, which may be stem cells including, but not limited to, induced pluripotent stem cells and embryonic stem cells.

[0077] In certain aspects, pluripotent stem cells as used herein are human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that can be expanded in vitro for long periods of time while retaining the ability to differentiate into all cell types of the body, including the cardiac progenitor cells of the present disclosure. Thus, these cells can potentially provide an unlimited supply of patient-specific functional cardiac progenitor cells for both drug development and therapeutic use.

[0078] A. Embryonic stem cells In a particular embodiment, the pluripotent stem cells are sexual stem cells (ESCs). ES cells are derived from the inner cell mass of blastocysts and have a high differentiation potential in vitro. ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo and then culturing the inner cell mass on a feeder layer of non-growing cells. The re-plated cells can continue to grow to produce new colonies of ES cells, which can be removed, dissociated, plated, and grown again. This process of "passaging" undifferentiated ES cells can be repeated many times to produce cell lines containing undifferentiated ES cells (U.S. Patent Nos. 5,843,780; 6,200,806; 7,029,913). ES cells have the ability to proliferate while maintaining pluripotency. For example, ES cells are useful for studying cells and genes that control cell differentiation. The pluripotency of ES cells, in combination with genetic manipulation and selection, can be used for in vivo genetic analysis studies via the generation of transgenic, chimeric, and knockout mice.

[0079] Methods for producing mouse ES cells are well known. In one method, preimplantation blastocysts of 129 mouse strain are treated with mouse antisera to remove the trophectoderm, and the inner cell mass is cultured on a feeder layer of chemically inactivated mouse embryonic fibroblasts in medium containing fetal bovine serum. The resulting undifferentiated ES cell colonies are subcultured on a feeder layer of mouse embryonic fibroblasts in the presence of fetal bovine serum to produce ES cell populations. In some methods, mouse ES cells can be grown without a feeder layer by adding the cytokine leukemia inhibitory factor (LIF) to serum-containing culture medium (Smith, 2000). In another method, mouse ES cells can be grown in serum-free medium in the presence of bone morphogenetic protein and LIF (Ying et al., 2003).

[0080] Human ES cells can be produced or derived from zygote or blastocyst stage mammalian embryos produced by sperm and egg cell fusion, nuclear transfer, pathogenesis, or by chromatin reprogramming and subsequent incorporation of the reprogrammed chromatin into the cell membrane to produce embryonic cells, by methods previously described (Thomson and Marshall, 1998; Reubinoff et al., 2000). In one method, human blastocysts are exposed to anti-human serum, trophectoderm cells are lysed, and the inner cell mass is removed and cultured on a feeder layer of mouse embryonic fibroblasts. Furthermore, cell clumps derived from the inner cell mass are chemically or mechanically dissociated and replated, and colonies with undifferentiated morphology are selected by micropipette, dissociated, and replated. In some methods, human ES cells can be grown without serum by culturing them on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor (Amit et al., 2000). Alternatively, human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix such as MATRIGEL™ or laminin in the presence of “conditioned” medium containing basic fibroblast growth factor ( Xu et al., 2001 ).

[0081] ES cells can also be obtained from mouse and human established cell lines, as well as from other organisms, including rhesus monkeys and marmosets, by methods previously described (Thomson and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000; U.S. Pat. No. 5,843,780). For example, human established ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14, and ACT30. As a further example, mouse established ES cell lines include the CGR8 cell line established from the inner cell mass of 129 mouse embryos, and cultures of CGR8 cells can be grown in the presence of LIF without a feeder layer.

[0082] ES stem cells can be detected by protein markers including transcription factor Oct4, alkaline phosphatase (AP), stage-specific embryonic antigen SSEA-1, stage-specific embryonic antigen SSEA-3, stage-specific embryonic antigen SSEA-4, transcription factor NANOG, cancer regression antigen 1-60 (TRA-1-60), cancer regression antigen 1-81 (TRA-1-81), SOX2, or REX1.

[0083] B. Induced pluripotent stem cells In another aspect, pluripotent stem cells as used herein are induced pluripotent stem (iPS) cells, commonly abbreviated as iPS cells or iPSCs. Induction of pluripotency was achieved in 2006 with mouse cells (Yamanaka et al. 2006) and in 2007 with human cells (Yu et al. 2007; Takahashi et al. 2007) by reprogramming somatic cells via the introduction of pluripotency-associated transcription factors. The use of iPSCs avoids most of the ethical and practical issues associated with the large-scale clinical use of ES cells, and patients receiving iPSC-derived autologous transplants will not require lifelong immunosuppressive treatments to prevent graft rejection.

[0084] Any cell can be used as the starting point for iPSCs, except for germ cells. For example, the cell type can be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, hepatocytes, or gastric cells. T cells can also be used as a source of somatic cells for reprogramming (U.S. Patent No. 8,741,648; U.S. Patent Publication No. 2015 / 0191697). There is no limit to the degree of cell differentiation or the age of the animal from which the cells are taken, and undifferentiated progenitor cells (including somatic cells) and even terminally differentiated mature cells can be used as a source of somatic cells in the methods disclosed herein. iPS cells can be grown under conditions known to differentiate human ES cells into specific cell types and express human ES cell markers, including SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

[0085] Somatic cells can be reprogrammed to produce iPSC cells by using methods known to those skilled in the art. Those skilled in the art can easily produce iPS cells, for example, see published US Patent Application Nos. 2009 / 0246875, 2010 / 0210014, 2012 / 0276636, US Patent Nos. 8,058,065, 8,129,187; PCT Publication No. WO2007 / 069666 A1, US Patent Nos. 8,268,620; 8,546,140; 9,175,268; 8,741,648; US Patent Application No. 2011 / 0104125 and US Patent No. 8,691,574, which are incorporated herein by reference. In general, nuclear reprogramming factors are used to produce pluripotent stem cells from somatic cells. In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized, in other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are utilized, or Oct3 / 4, Sox2, Nanog, and Lin28 are utilized.

[0086] The mouse and human cDNA sequences for these nuclear reprogramming agents are available by reference to the NCBI accession numbers described in WO2007 / 069666 and U.S. Patent No. 8,183,038, which are incorporated herein by reference. Methods for introducing one or more reprogramming agents, or nucleic acids encoding these reprogramming agents, are known in the art and are also disclosed in, for example, U.S. Patent Nos. 8,268,620, 8,691,574, 8,741,648, 8,546,140, ​​published U.S. Patent No. 8,900,871, and 8,071,369, all of which are incorporated herein by reference.

[0087] Once induced, iPSCs can be cultured in a medium sufficient to maintain pluripotency. iPSCs can be used with various media and techniques developed for culturing pluripotent stem cells, more specifically embryonic stem cells, as described in U.S. Pat. No. 7,442,548 and U.S. Pat. Publication No. 2003 / 0211603. For mouse cells, leukemia inhibitory factor (LIF) is added to the normal medium as a differentiation inhibitor. For human cells, it is desirable to add basic fibroblast growth factor (bFGF) instead of LIF. Other methods for culturing and maintaining iPSCs can be used with the methods disclosed herein, as known to those skilled in the art.

[0088] In certain embodiments, undefined conditions can be used, for example, pluripotent cells can be cultured on fibroblast feeder cells or medium exposed to fibroblast feeder cells to maintain the stem cells in an undifferentiated state. In some embodiments, cells are cultured in the presence of mouse embryonic fibroblasts as feeder cells that have been treated with radiation or antibiotics to terminate cell division. Alternatively, pluripotent cells can be cultured and maintained in an essentially undifferentiated state using defined feeder-independent culture systems, such as TESR™ medium (Ludwig et al., 2006a; Ludwig et al., 2006b) or E8™ / Essential 8™ medium (Chen et al., 2011).

[0089] Plasmids are designed with many goals in mind, such as achieving a controlled high copy number, avoiding potential sources of plasmid instability in bacteria, and providing a means for plasmid selection compatible with use in mammalian cells, including human cells. For plasmids for use in human cells, particular attention is paid to a twofold requirement. First, they must be suitable for maintenance and fermentation in E. coli so that large amounts of DNA can be produced and purified. Second, they must be safe and suitable for use in human patients and animals. The first requirement requires high copy number plasmids that can be relatively easily selected and stably maintained during bacterial fermentation. The second requirement requires attention to elements such as selectable markers and other coding sequences. In some embodiments, the plasmid encoding the marker is composed of (1) a high copy number origin of replication, (2) a selectable marker, such as but not limited to, the neo gene for antibiotic selection with kanamycin, (3) a transcription termination sequence including a tyrosinase enhancer, (4) a multiple cloning site for integrating various nucleic acid cassettes, and (5) a nucleic acid sequence encoding the marker operably linked to a tyrosinase promoter. In certain aspects, the plasmid does not include a tyrosinase enhancer or promoter. A number of plasmid vectors are known in the art for inducing nucleic acids encoding certain proteins. These include, but are not limited to, the vectors disclosed in U.S. Pat. No. 6,103,470, U.S. Pat. No. 7,598,364, U.S. Pat. No. 7,989,425, U.S. Pat. No. 6,416,998, and U.S. Patent Application Serial No. 12 / 478,154, which are incorporated herein by reference.

[0090] The episomal gene transfer system can be a plasmid, an Epstein-Barr virus (EBV)-based episomal vector (U.S. Pat. No. 8,546,140), a yeast-based vector, an adenovirus-based vector, a simian virus 40 (SV40)-based episomal vector, a bovine papilloma virus (BPV)-based vector, or a lentivirus vector. The viral gene transfer system can be an RNA-based or DNA-based viral vector (PCT / JP2009 / 062911, PCT / JP2011 / 069588).

[0091] C. Embryonic stem cells derived from somatic cell nuclear transfer Pluripotent stem cells for producing hematopoietic progenitor cells can also be prepared by somatic cell nuclear transfer, in which a donor nucleus is transferred into a spindle-free oocyte. Stem cells produced by nuclear transfer are genetically identical to the donor nucleus. In one method, donor fibroblast nuclei from rhesus skin fibroblasts are introduced into the cytoplasm of spindle-free mature metaphase II rhesus oocytes by electrofusion (Byrne et al., 2007). The fused oocytes are activated by exposure to ionomycin and then incubated until the blastocyst stage. The inner cell mass of selected blastocysts is then cultured to produce embryonic stem cell lines. Embryonic stem cell lines display normal ES cell morphology, express various ES cell markers, and differentiate into multiple cell types both in vitro and in vivo.

[0092] D. MHC haplotype matching Major histocompatibility antigens are the main cause of immune rejection of allogeneic organ transplants. There are three major haplotypes in MHC class I (A, B, and C) and three major haplotypes in MHC class II (DR, DP, and DQ). The HLA locus is highly polymorphic and distributed over 4 Mb on chromosome 6. The haplotypic potential of HLA genes within this region is clinically important because this region is associated with autoimmune and infectious diseases and HLA haplotype compatibility between donor and recipient can affect the clinical outcome of transplantation. HLA corresponding to MHC class I presents peptides from within the cells, whereas HLA corresponding to MHC class II presents antigens from outside the cells to T lymphocytes. Incompatibility of MHC haplotypes between the graft and the host induces an immune response against the graft, leading to its rejection. Therefore, patients can be treated with immunosuppressants to prevent rejection. HLA-compatible stem cell lines may overcome the risk of immune rejection.

[0093] Due to the importance of HLA in transplantation, HLA loci are usually typed by serology and PCR to determine preferred donor-recipient pairs. Serological testing for HLA class I and class II antigens can be performed on purified T or B lymphocytes using the complement-mediated lymphocytotoxicity test. This procedure is primarily used to match HLA-A and HLA-B loci. Molecular-based tissue typing is often more accurate than serological testing. Low-resolution molecular techniques, such as SSOP (sequence-specific oligonucleotide probe) methods, which analyze PCR products against a series of oligonucleotide probes, can be used to identify HLA antigens and are currently the most common methods used for class II-HLA typing. High-resolution techniques, such as SSP (sequence-specific primer) methods, which utilize allele-specific primers for PCR amplification, can identify specific MHC alleles.

[0094] If donor cells are HLA homozygous, i.e., have identical alleles for each antigen-presenting protein, the MHC compatibility between donor and recipient increases significantly. Most individuals are heterozygous for MHC class I and class II genes, but certain individuals are homozygous for these genes. These homozygous individuals can act as super donors, and grafts generated from their cells can be transplanted into all individuals who are either homozygous or heterozygous for that haplotype. Furthermore, if homozygous donor cells have a haplotype that is frequently found in the population, these cells can be utilized in transplantation therapy for multiple individuals.

[0095] Thus, in some embodiments, the iPSCs of the method can be produced from somatic cells of the subject to be treated or another subject with the same or substantially the same HLA type as the patient. In one case, the donor's major HLA (e.g., the three major loci HLA-A, HLA-B, and HLA-DR) is identical to the recipient's major HLA. In some cases, the somatic cell donor can be a super donor. Thus, iPSCs from a super donor that is MHC homozygous can be used to generate a committed cardiac progenitor cell population. Thus, the committed cardiac progenitor cells from the super donor can be transplanted into a subject that is either homozygous or heterozygous for its haplotype. For example, the committed cardiac progenitor cells can be homozygous for two HLA alleles, such as HLA-A and HLA-B. Thus, the committed cardiac progenitor cells produced from the super donor can be used in the methods disclosed herein to produce committed cardiac progenitor cells that may be "compatible" with a large number of potential recipients.

[0096] Thus, certain embodiments of the present disclosure provide a repository (e.g., library) of HLA homozygous committed cardiac progenitor cells. The HLA haplotypes represented in the primary library can reflect the most common HLA haplotypes found in the human population, e.g., common HLA haplotypes in Caucasians, common HLA haplotypes found in individuals of African descent, common HLA haplotypes in Asians, common HLA haplotypes in Latinos, common HLA haplotypes in Native Americans, etc. For example, a single abundant haplotype may be present in a very large proportion of a population, such that a single HLA homozygous cell line can serve as a histocompatible donor for a very large proportion of patients. The library includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 20-25, 25-30, or more than 30 different types of HLA homozygous cells. The primary library can include a first HLA homozygous cell that is homozygous for a first HLA haplotype and a second HLA homozygous cell that is homozygous for at least a second HLA haplotype. The primary library can include a single cell type or two or more different cell types. The primary library can be cataloged, for example, by a searchable computer database, and information regarding HLA haplotypes, and optionally additional information such as cell surface markers, karyotype information, etc., can be stored and retrieved.

[0097] The HLA homozygous committed cardiac progenitor cells described herein may be used in a wide range of clinical applications, including cell and / or tissue transplantation. The HLA homozygous committed cardiac progenitor cells are HLA-compatible with the recipient and therefore can be introduced into the recipient without the need for immunosuppressive therapy, or at least with reduced need for immunosuppressive therapy. Standard immunosuppressive drug regimens can cost thousands of dollars / month and have undesirable side effects, including infections and cancers that are often life-threatening and expensive to treat. Thus, the present HLA homozygous committed cardiac progenitor cells overcome several obstacles that currently limit the use of human cells for clinical applications.

[0098] III. Differentiation into committed cardiac progenitor cells (CTC4) Embodiments of the present disclosure relate to the differentiation of PSCs, particularly iPSCs, into cardiac progenitor cells (committed or primed to further differentiate into cardiomyocytes). The schematic in Figure 1A shows an exemplary 6-day differentiation process. The process begins with iPSCs grown in Essential 8 medium on vitronectin-coated vessels prior to initiating large-scale differentiation, such as in a bioreactor.

[0099] In some embodiments, the methods relate to the modulation of Wnt signaling in a complete suspension bioreactor process. During cardiac development, Wnt signaling in the mesoderm can be rapidly modulated to further cardiac specification. To date, the timing at which Wnt signaling must be reduced has not been well described. In this study, the expression of two cell surface markers, CXCR4 and CD56, was followed. Cardiac differentiation can be followed by daily analysis of these two markers, and decisions can be made to steadily differentiate into the heart based on the expression profile. The early mesoderm stage is represented by a population of CXCR4+CD56- cells, followed by CXCR4+CD56+ double positive cells, and then by the loss of CXCR4 expression, resulting in CXCR4-CD56+ committed cardiac progenitor cells.

[0100] As shown in Figure 3B, Wnt signaling can be inhibited for the steady cardiac specification of cells to become cardiomyocytes.Preferably, the culture on day 3 is at least 30% positive for CXCR4 and less than 60% positive for CD56.If the culture becomes more than 60% positive for CD56 before Wnt signaling is inhibited, the steady specification to cardiac cells may not occur, and thus the efficiency of becoming cardiomyocytes may be low.In addition, if the culture has already become more than 20% CXCR4-CD56+, showing a decrease in the expression of CXCR4, it is also too late to inhibit Wnt signaling for steady cardiac specification.

[0101] A. Aggregate formation Pluripotent stem cells are first differentiated with a Wnt agonist (e.g., CHIR99021) and induced to form aggregates to differentiate into CTC4 cells. In the aggregates, differentiation is initiated and cells begin to recapitulate embryonic development to a limited extent. They cannot form trophectoderm tissues (including placenta), but can develop into virtually all other types of cells present in the organism. The present disclosure can further promote cardiac differentiation after aggregate formation.

[0102] Pluripotent cells can form embryoid bodies or aggregates as part of the differentiation process. The formation of "embryoid bodies" (EBs), clusters of proliferating cells, to induce differentiation is commonly associated with the in vitro aggregation of human pluripotent stem cells into EBs, allowing the human pluripotent stem cells to differentiate spontaneously and randomly into multiple tissue types representing endodermal, ectodermal, and mesodermal origins.

[0103] In certain embodiments, pluripotent stem cells are cultured in the presence of a ROCK inhibitor and a chemical agonist of the Wnt pathway, such as a GSK3 inhibitor (e.g., CHIR99021), to activate the Wnt pathway. Wnt pathway agonists can include CAS 853220-52-7 (2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine), SB216763, CHIR98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, or IM-12. The medium can include a Wnt agonist, such as CHIR99021, at a concentration of about 1-10 μM, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. In certain embodiments, the medium includes a Wnt agonist, such as CHIR99021, at a concentration of about 4.4 μM. In certain embodiments, the method includes culturing the cells in the presence of about 2 μM of a Wnt agonist during aggregate formation, e.g., from day 0 to day 1, and then culturing the cells in the presence of about 4.4 μM for mesoderm induction, e.g., from day 1 to day 3.

[0104] ROCK inhibitors can be used for the culture and passage of pluripotent stem cells and / or the differentiation of stem cells. Thus, ROCK inhibitors can be present in the medium of any cell culture (e.g., adherent culture or suspension culture) when pluripotent stem cells are growing, dissociating, forming aggregates or differentiating. Rho-specific inhibitors, such as botulinum C3 enzyme, and / or myosin II-specific inhibitors can also be used as ROCK inhibitors in certain embodiments of the present disclosure. In certain embodiments, myosin II inhibitors, such as blebbistatin, can be used to induce aggregate formation.

[0105] An exemplary ROCK-specific inhibitor is Y-27632, which selectively targets ROCK1 (and also inhibits ROCK2) and also inhibits TNF-α and IL-1β. Y-27632 is cell permeable and inhibits ROCK1 / ROCK2 by competing with ATP (IC 50 =800 nM). Other ROCK inhibitors include, for example, H1152, Y-30141, Wf-536, HA-1077, hydroxyl HA-1077, GSK269962A, and SB-772077-B. In certain embodiments, the ROCK-specific inhibitor used in the method is H1152. In some embodiments, H1152 is present in the culture at a concentration of 50-200 μM, for example, at a concentration of about 100 μM.

[0106] Other non-limiting examples of ROCK inhibitors include antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (e.g., siRNAs), competitive peptides, antagonist peptides, inhibitory antibodies, ScFV antibody fragments, dominant negative mutants, and expression vectors thereof. In addition, other small molecule compounds are known as ROCK inhibitors, and such compounds or derivatives thereof can also be used in the embodiments (see, for example, U.S. Patent Publication Nos. 20050209261, 20050192304, 20040014755, 20040002508, 20040002507, 20030125344, and 20030087919, and International Patent Publication Nos. 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, and 2004 / 039796, which are incorporated herein by reference). One or more ROCK inhibitors can also be used in combination in the present methods.

[0107] According to some embodiments, PSCs can be treated with a ROCK inhibitor in culture medium. Thus, the culture medium used in the disclosed method may already contain a ROCK inhibitor, or the disclosed method may include a step of adding a ROCK inhibitor to the culture medium. The concentration of the ROCK inhibitor in the culture medium is not particularly limited, as long as it can achieve the desired effect, such as improved survival rate of stem cells. Such ROCK inhibitors, for example, Y-27632, HA-1077, or H-1152, can be used at an effective concentration of at least or about 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 500 to about 1000 μM, or any range of concentrations derivable therein. These values ​​may indicate the amount of ROCK inhibitor individually or in combination with one or more ROCK inhibitors.

[0108] For example, when Y-27632 is used as a ROCK inhibitor, it can be used at a concentration of about 0.01 to about 1000 μM, more specifically at a concentration of about 0.1 to about 100 μM, even more specifically at a concentration of about 1.0 to about 30 μM, most specifically at a concentration of about 2.0 to 20 μM, or at any concentration range that can be derived therefrom. When fasudil (HA1077) is used as a ROCK inhibitor, it can be used at a concentration about twice the concentration of the above-mentioned Y-27632. When H1152 is used as a ROCK inhibitor, it can be used at about 1 / 50 of the concentration of the above-mentioned Y-27632.

[0109] The aggregate formation step is carried out for a time sufficient to induce the production of aggregates. For example, pluripotent stem cells, such as induced pluripotent stem cells, can be contacted with a ROCK inhibitor for about 10, 15, 20, 25, 30 minutes to several hours (e.g., at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, 36 hours, 48 ​​hours, or any range derivable therein). In certain embodiments, a period of 1 to 3 days, such as about 1 day, is sufficient to induce the cells to form aggregates.

[0110] The density of the stem cells to be treated with the ROCK inhibitor is not particularly limited as long as it is a density that can obtain the desired effect, such as improving the survival rate of the stem cells. The cell density is, for example, about 1.0 × 10 1 ~1.0×10 7 cells / ml, more specifically, approximately 1.0 × 10 2 ~1.0×10 7 cells / ml, and more specifically, about 1.0×10 3 ~1.0×10 7 cells / ml, most specifically, approximately 3.0 × 10 4 ~2.0×10 6 cells / ml.

[0111] In certain embodiments, PSCs are cultured in the presence of a ROCK inhibitor to improve survival, cloning efficiency or passage efficiency at low density (dissociated single cells or small aggregates). In certain embodiments, PSCs are cultured in the absence of feeder cells, feeder cell extracts and / or serum. Before subcloning or passage, for example, at least 1 hour before subcloning or passage, PSCs can be cultured in the presence of a ROCK inhibitor. Alternatively or additionally, PSCs are maintained in the presence of a ROCK inhibitor during or after subcloning or passage.

[0112] Pluripotent stem cells can be seeded in aggregate formation promotion medium using any method known in the art of cell culture. For example, pluripotent stem cells can be seeded in aggregate formation promotion medium as single colonies or clonal groups, or pluripotent stem cells can be seeded essentially as individual cells. In some embodiments, pluripotent stem cells are dissociated into essentially individual cells using mechanical or enzymatic methods known in the art. As a non-limiting example, pluripotent stem cells can be exposed to proteolytic enzymes that destroy the bonds between cells and culture surfaces and between cells themselves. Enzymes that can be used to individualize pluripotent stem cells and induce aggregate formation and differentiation can include, but are not limited to, trypsin, various commercially available preparations of trypsin, such as TrypLE, or mixtures of enzymes, such as Accutase®.

[0113] A variety of matrix components can be used to culture pluripotent cells, including collagen (e.g., collagen IV), laminin, vitronectin, Matrigel™, gelatin, polylysine, thrombospondin (e.g., TSP-1, TSP-2, TSP-3, TSP-4, and / or TSP-5), fibronectin, and / or ProNectin-F™. Combinations of these matrix components can provide the added benefit of promoting cell proliferation and cell viability. In certain embodiments, cells can be cultured using one, two, three, four, five, six, or more of the above matrix components. In some aspects, pluripotent cells are cultured on a vitronectin-coated surface.

[0114] In certain embodiments, the pluripotent cells can be added or seeded into culture medium as essentially individual (or dispersed) cells to form a culture on the culture surface. The culture medium into which the cells are seeded can include Essential 8 (E8) medium, a survival factor such as a ROCK inhibitor, and a Wnt pathway agonist. In these embodiments, the culture surface can be composed of essentially any material that is compatible with standard sterile cell culture methods in the art, such as a non-adherent surface. The culture surface can further include a matrix component (e.g., vitronectin) as described herein. In certain embodiments, the matrix component can be injected into the culture surface before contacting the culture surface with the cells and medium.

[0115] B. Mesoderm induction Then, the aggregate of pluripotent stem cells, such as the aggregate of iPS cells, is cultured in a medium to promote mesoderm induction. The aggregate can be contacted with a Wnt agonist, and optionally with an Activin / Nodal agonist and / or a BMP. In certain embodiments, the medium does not contain a ROCK inhibitor or insulin. The medium can contain a higher concentration of one or more Wnt agonists compared to the aggregate formation step. The Wnt agonist can be the same as the Wnt agonist in the aggregate formation step, or a different Wnt agonist. Agonists of the Wnt pathway can include CHIR99021, IWP-1, IWP-2, IWP-3, IWP-4, CAS 853220-52-7 (2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine), SB216763, CHIR98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, or IM-12. The Wnt agonist can be CHIR99021 and can be present at a concentration of about 1-10 μM, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. In a particular embodiment, the Wnt agonist is CHIR99021 and is present at a concentration of about 4-5 μM, such as about 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 μM, particularly about 4.4 μM.

[0116] Activin agonists are compounds that activate the Activin / Nodal signaling pathway, for example by binding to TGFβ or activin receptors. Examples of activin agonists include activin A, activin B, activin AB, TGFβ1, growth differentiation factor (GDF)-3, BML-284, and Nodal. For example, activin agonists or BMPs can be used at a concentration of 0.1 ng / mL to 12 ng / mL.

[0117] The basal medium for mesoderm induction can be any medium known in the art for culturing stem cells. Exemplary media include E8 medium, TeSR medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham medium, RPMI 1640 medium, and Fischer's medium. In certain embodiments, the basal medium is RPMI medium supplemented with B27. In certain embodiments, the medium is insulin-free or substantially insulin-free.

[0118] The mesoderm induction step can be for a period of time sufficient to induce mesoderm markers such as CXCR4, KDR, PDGFRα, and / or CD56, and to lose expression of CKIT and / or EPCAM. For example, the aggregates can be cultured in the presence of a Wnt agonist, an Activin / Nodal agonist, and / or a BMP for about 1-5 days, e.g., about 1, 2, 3, 4, or 5 days. In certain embodiments, the aggregates are cultured for about 2-3 days for mesoderm induction.

[0119] In certain embodiments, CXCR4 begins to be expressed at an early mesoderm stage, such as day 2. Stronger expression of CXCR4 is then detected along with the onset of CD56 expression.

[0120] C. Cardiac Specification The mesodermal cells are then directed to cardiac specification in the presence of a Wnt inhibitor and, optionally, a TGFβ inhibitor. The culture may further include insulin, an activin inhibitor, and / or a BMP inhibitor. Cardiac specification may be promoted by the addition of insulin. Once the cells are less than 60% positive for CD56 and at least 20% positive for CXCR4, a Wnt inhibitor may be added. After Wnt inhibition, such as on day 4, the cells are at an early cardiac mesoderm stage, characterized by loss of expression of CXCR4, the majority of the cells expressing CD56, and the emergence of a KDR+PDGRFα+ population. On day 5, a population of cardiac progenitor cells that are KDR+PDGFRα+ is seen with continued expression of CD56 and loss of CXCR4 expression. Then, on day 6, a primed or committed cardiac precursor population appears, characterized by loss of KDR expression.

[0121] In certain aspects, the aggregates at the mesodermal stage can be maintained in suspension culture and initiated into cardiac specification, or the mesodermal cells can be individualized and plated as monolayer cultures prior to initiating cardiac specification. CTC4 cells can be produced by both culture methods. CTC4 cells can be further cultured to produce cardiomyocytes. Notably, the differentiation process can be serum-free without the use of drug resistance or metabolic selection.

[0122] The Wnt inhibitor can be XAV939, ICG-001, IWR-1-endo, Wnt-C59, LGK-974, LF3, CP21R7, NCB-0846, PNU-74654, IWR-1, IWR-2, IWR-3, IWR-4, or KYA179K. The Wnt inhibitor, such as XAV939, can be present at a concentration of about 1 to 25 mM, for example at a concentration of about 5 mM, 10 mM, or 15 mM, particularly at a concentration of about 10 mM.

[0123] The TGFβ inhibitor can be SB431542, LDN-193189, LY2157299, LY2109761, SB525334, SIS HCl, SB505124, GW788388, or LY364947. The TGFβ inhibitor, such as SB431542, can be present at a concentration of about 1 to 25 mM, such as about 5 mM, 10 mM, or 15 mM, particularly about 10 mM. 193189, LY2157299, LY2109761, SB525334, SIS HCl, SB505124, GW788388, or LY364947. The TGFβ inhibitor, such as SB431542, may be present at a concentration of about 1-5 μM, such as about 1, 2, or 3 μM, particularly about 2 μM.

[0124] BMP inhibitors include 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin), 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 4-[6-[4-(1-methyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline hydrochloride (LDN193189), and 4-[6-[4-(1-methyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline hydrochloride (LDN193189). The BMP inhibitor may be 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-2), and 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (ML 347). The BMP inhibitor, such as dorsomorphin, may be present at a concentration of about 0.1 μM to 5 μM, for example at a concentration of about 1, 2, or 3 μM, and particularly at a concentration of about 2 μM.

[0125] CTC4 cells can be produced from mesoderm about 1-4 days after the initiation of cardiac specification. Cardiac specification can occur for about 1-4 days, for example, about 2 or 3 days. CTC4 cells can then be cryopreserved or differentiated into cardiomyocytes in an appropriate medium, such as RPMI medium with B27 supplement. In certain embodiments, the committed cardiac progenitor cell population can be isolated or cryopreserved once the cell population is at least 70% positive for PDGFRα, less than 40% positive for KDR, less than 20% positive for EPCAM, and less than 20% positive for SAA.

[0126] The committed cardiac progenitor cell aggregates can be dissociated and cryopreserved. Dissociation of the aggregates can be performed using any known procedure. These procedures include treatment with chelating agents (e.g., EDTA, etc.), enzymes (e.g., trypsin, collagenase, etc.), and mechanical dissociation (e.g., pipetting, etc.). The cells can be cultured on a matrix as described above, for example, on a vitronectin-coated surface.

[0127] D. Differentiation of CTC4 into cardiomyocytes As described in Figure 7A, CTC4 cells can be further matured or differentiated into cardiomyocytes. In particular, CTC4 cells can be matured into subpopulations of cardiomyocytes, such as atrial cells, ventricular cells, and pacemaker cells, by differentiation conditions known in the art. When plated into vessels of various sizes, CTC4 cells can be differentiated into highly pure and contracting monolayers of cardiomyocytes (Figures 7C-E).

[0128] To promote the cardiomyocyte phenotype, cells can be cultured with factors and combinations of factors that enhance the proliferation or survival of cells of the cardiomyocyte type or inhibit the growth of other cell types. This effect can be due to a direct effect on the cells themselves or due to an effect on another cell type, resulting in enhanced cardiomyocyte formation. For example, factors that induce the formation of cells equivalent to endoderm or ectoderm, or that cause these cells to manufacture elements that promote their own cardiac differentiation, are all included within the scope of cardiotropic or differentiation factors for differentiation into cardiomyocytes.

[0129] For example, induction medium for cardiac differentiation may include, but is not limited to, precardiac explants, precardiac mesoderm conditioned medium, mesoderm secreted growth factors such as HGF. In certain aspects, the differentiation factor may be a growth factor involved in cell development. Differentiation factors include, but are not limited to, regulators of one or more signal transduction pathways of bone morphogenetic proteins, Activin A / Nodal, vascular endothelial growth factor (VEGF), Dickkopf homolog 1 (DKK1), basic fibroblast growth factor (bFGF), insulin growth factor (IGF), and / or epidermal growth factor (EGF).

[0130] CTC4 cells can be cultured in medium to promote maturation into cardiomyocytes. An exemplary maturation medium can include RPMI with B27 supplement. In some embodiments, the term "maturation medium" refers to the medium used to further differentiate cells to produce a population of cells that are more than 70% positive for PDGFRα, less than 40% positive for KRD, less than 20% positive for EPCAM, and less than 20% positive for sarcomeric alpha actinin. For example, cells can be differentiated into cardiomyocytes or endothelial cells.

[0131] In one method, CTC4 cells are matured into cardiomyocytes in a medium supplemented with the above-mentioned Wnt inhibitor and TGFβ inhibitor.For example, the medium can be Williams E medium containing cell maintenance cocktail B (i.e., penicillin / streptomycin, insulin, transferrin, selenium acid, BSA, linoleic acid, GlutaMAX, and HEPES), Wnt inhibitor (e.g., XAV939) and TGFβ inhibitor (e.g., SB431542).Alternatively, instead of or in addition to TGFβ inhibitor, CTC4 cells can be contacted with activin inhibitor and / or BMP inhibitor.Cells can be cultured as a monolayer, such as on an extracellular matrix coating (e.g., vitronectin).

[0132] E. Cell culture conditions The culture conditions according to the present invention are appropriately determined depending on the medium and stem cells used. The medium according to the present disclosure can be prepared using a medium used for culturing animal cells as a basal medium. The basal medium can be any of E8 medium, TeSR medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham medium, RPMI 1640 medium, and Fischer's medium, and any combination thereof, but is not limited thereto as long as it is a medium that can be used for culturing animal cells.

[0133] In certain aspects, the medium according to the present disclosure is a serum-free medium. Serum-free medium refers to a medium that does not contain raw or unpurified serum, and thus may include a medium that contains purified blood-derived components or animal tissue-derived components (such as growth factors). The medium according to the present disclosure may or may not contain any substitute for serum. The serum substitute may include materials that suitably contain albumin (e.g., lipid-rich albumin or albumin substitutes, such as recombinant albumin, vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. The serum substitute may be prepared, for example, by the methods disclosed in WO 98 / 30679. Alternatively, any commercially available material may be used for greater convenience. Commercially available materials include Knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco) and Glutamax (Gibco).

[0134] The medium of the present disclosure may also contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and inorganic salts. The concentration of 2-mercaptoethanol may be, for example, about 0.05 to 1.0 mM, particularly about 0.1 to 0.5 mM, but is not particularly limited thereto as long as it is suitable for culturing stem cells.

[0135] Culture vessels used to culture stem cells may include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multiwell plates, microslides, chamber slides, tubes, trays, CellSTACK® chambers, culture bags, roller bottles, and bioreactors, such as PBS500 and / or PBS3, but are not limited thereto as long as stem cells can be cultured in them. Stem cells can be cultured in volumes of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, 2000 ml, or any range derivable therein, depending on the needs of the culture. In certain embodiments, the culture vessel may be a bioreactor, which may refer to any device or system that supports a biologically active environment. A bioreactor may be at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 L, 1, 2, 4, 6, 8, 10, 15 m 3 , or any range of volumes derivable therein.

[0136] The culture vessel can be cell-adhesive or non-adhesive, and can be selected according to the purpose. The cell-adhesive culture vessel can be coated with any substrate for cell adhesion, such as extracellular matrix (ECM), to improve the adhesion of the vessel surface to cells. The substrate for cell adhesion can be any material for attaching stem cells or feeder cells (if used). Substrates for cell adhesion include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin and mixtures thereof, such as Matrigel™, and dissolved cell membrane preparations (Klimanskaya et al., 2005).

[0137] Other culture conditions can be appropriately determined. For example, the culture temperature can be about 30 to 40°C, for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, 39°C, but is not particularly limited thereto. The CO2 concentration can be about 1 to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen partial pressure can be at least or about 1, 5, 8, 10, 20%, or any range derivable therein.

[0138] The disclosed method can also be used for suspension culture of stem cells, including suspension culture on carriers (Fernandes et al., 2007) or gel / biopolymer encapsulation (US Patent 20070116680). The term suspension culture of stem cells means that stem cells are cultured in a medium under non-adherent conditions to a culture vessel or feeder cells (if used). The suspension culture of stem cells includes dissociated culture of stem cells and aggregate suspension culture of stem cells. The term dissociated culture of stem cells means culturing stem cells in suspension, and dissociated culture of stem cells includes culturing a single stem cell or culturing a small cell aggregate consisting of multiple stem cells (e.g., about 2 to 400 cells). By continuing the dissociated culture, the cultured dissociated cells form larger stem cell aggregates, after which aggregate suspension culture can be performed. Aggregated suspension cultures include embryoid body culture methods (see Keller et al., 1995) and SFEB methods (Watanabe et al., 2005; WO 2005 / 123902). The methods of the present disclosure can significantly improve the survival rate and / or differentiation efficiency of stem cells in suspension cultures.

[0139] Bioreactors can be classified according to general categories, including static bioreactors, stirred flask bioreactors, rotating wall vessel bioreactors, hollow fiber bioreactors, and direct perfusion bioreactors. Within the bioreactor, cells can be free or immobilized, or seeded on a porous three-dimensional scaffold (hydrogel). In certain embodiments, the bioreactor uses a suspension bioreactor due to the efficient mixing and low shear stress that results in a uniform particle suspension.

[0140] F. GMP manufacturing process The methods disclosed and used herein use all GMP-compliant materials and can scale multiple (e.g., 3 L) bioreactor manufacturing batches to produce the purity and cell numbers required for cardiac cell therapy development. As shown in Figure 2B, scaling iPSC expansion in multi-layer culture vessels produces enough iPSCs to seed multiple 3 L bioreactors. The CTC4 cryopreservation step during manufacturing can produce 300 x 10 CTC4 cells. 6 It can be frozen at the scale of individual cells / container (Figure 2C), reducing the need for thawing and handling of containers during preclinical development in large animal models and future clinical studies.

[0141] G. Characterization of committed cardiac progenitor cells (CTC4 cells) Cells obtained according to the present method can be characterized according to a number of phenotypic criteria. CTC4 cells express known cardiac genes while pluripotency genes are downregulated, as shown in Figure 6A-B, and are characterized by a unique combination of cell surface markers: CD56+ PDGFRA+ KDR- CXCR4- EPCAM- (Figure 7B). Cardiomyocytes and progenitor cells derived from pluripotent stem cell lines often have morphological characteristics of cardiomyocytes from other sources. They can be spindle, round, triangular, or polygonal, and they may exhibit characteristic striations in sarcomere structures detectable by immunostaining. They can form flat sheets of cells, or aggregates that remain attached to a substrate or float in suspension, and when examined by electron microscopy, they may exhibit typical sarcomeres and atrial granules.

[0142] Pluripotent stem cell derived cardiomyocytes and their precursors usually have at least one cardiomyocyte specific marker, including: cardiac troponin I (cTnI), a subunit of the troponin complex that provides a calcium-sensitive molecular switch that regulates striated muscle contraction; cardiac troponin T (cTnT); or Nkx2.5, a cardiac transcription factor that is expressed in the cardiac mesoderm during early mouse embryogenesis and persists in the developing heart. The cells also usually express at least one (often at least three, five, or more) of the following markers: atrial natriuretic factor (ANF), myosin heavy chain (MHC), especially the cardiac specific β chain, MLC, titin, tropomyosin, α-sarcomeric actinin, and desmin. ANF is a hormone that is expressed in the developing heart and fetal cardiomyocytes, but is downregulated in adults. It is considered to be a good marker for cardiomyocytes because it is expressed in a very specific manner in cardiac cells, but not in skeletal muscle cells. Additional markers include MEF-2A, MEF-2B, MEF-2C, MEF-2D (transcription factors expressed in cardiac mesoderm and persist in the developing heart), N-cadherin (mediates adhesion between cardiac cells), connexin 43 (forms gap junctions between cardiomyocytes), β1-adrenergic receptor (β1-AR), creatine kinase MB (CK-MB) and myoglobin (which are elevated in serum after myocardial infarction), α-cardiac actin, early growth response-I, cyclin D2, and GATA-4 (transcription factor highly expressed in cardiac mesoderm and persists in the developing heart), which regulates many cardiac genes and is involved in cardiogenesis.

[0143] Tissue-specific markers can be detected using any suitable immunological technique, such as flow immunocytometry or affinity adsorption for cell surface markers, immunocytochemical analysis (e.g., of fixed cells or tissue sections) for intracellular or cell surface markers, Western blot analysis of cell extracts, and enzyme-linked immunoassays for products secreted into cell extracts or medium. Antibodies that distinguish cardiac markers such as cTnI and cTnT from other isoforms are commercially available from suppliers such as Sigma and Spectral Diagnostics. Expression of an antigen by a cell is said to be antibody-detectable if a significant detectable amount of antibody binds to the antigen in a standard immunocytochemical or flow cytometric analysis (optionally after fixation of the cells and optionally using a labeled secondary antibody).

[0144] Expression of tissue-specific gene products can also be detected at the mRNA level by the following methods: using publicly available sequence data (GenBank), Northern blot analysis, dot blot hybridization analysis, or reverse transcriptase-initiated polymerase chain reaction (RT-PCR) using sequence-specific primers in standard amplification methods. Expression of tissue-specific markers is considered positive when protein or mRNA is detected at a level at least or about 2, 3, 4, 5, 6, 7, 8, or 9 times, more specifically more than 10, 20, 30, 40, or 50 times, above the level of control cells, such as undifferentiated pluripotent stem cells or other unrelated cell types.

[0145] Once markers have been identified on the surface of cells of the desired phenotype, they can be subjected to immunoselection and the cell population can be further enriched by techniques such as immunopanning or antibody-mediated fluorescence activated cell sorting.

[0146] Under the right circumstances, pluripotent stem cell-derived cardiomyocytes often exhibit spontaneous and periodic contractile activity. This means that when cultured in a suitable tissue culture environment with the right Ca2+ concentration and electrolyte balance, the cells can be observed to contract across one axis of the cell and then release the contraction without the need to add any additional components to the culture medium. The contractions are periodic, meaning that they repeat in a regular or irregular manner in a normal buffer solution, at a frequency of about 6 to 200 contractions / min, often at a frequency of about 20 to about 90 contractions / min. Individual cells can exhibit spontaneous and periodic contractile activity by themselves, or in concert with neighboring cells in a tissue, cell aggregate, or cultured cell mass.

[0147] Contractile activity of cells can be characterized according to the effect of culture conditions on the nature and frequency of contractions. Compounds that reduce available Ca2+ concentration or otherwise interfere with transmembrane transport of Ca2+ often affect contractile activity. For example, the L-type calcium channel blocker diltiazem inhibits contractile activity in a dose-dependent manner. On the other hand, adrenergic receptor agonists such as isoprenaline and phenylephrine have positive chronotropic effects. Further characterization of the functional properties of cells may include characterizing Na+, K+, and Ca2+ channels. Electrophysiology, such as action potentials, can be studied by patch clamp techniques on cardiomyocytes. See Igelmund et al., 1999; Wobus et al., 1995; and Doevendans et al., 2000.

[0148] Although functional attributes provide a way to characterize cells and their precursors in vitro, they may not be necessary for some of the use aspects mentioned in this disclosure. For example, a mixture of enriched cells with some of the markers listed above, even if they do not have all of the functional or electrophysiological properties, may be of great therapeutic benefit if they can be transplanted into defective cardiac tissue and acquire the necessary functional properties in vivo to compensate for cardiac function.

[0149] When derived from an established pluripotent stem cell line, the cell populations and isolated cells of the present disclosure can be characterized as having the same genome as their derived line. This means that the chromosomal DNA between the pluripotent stem cells and the cardiac cells is more than 90% identical, which can also be assumed when cardiac cells are obtained from an undifferentiated line through the normal mitotic process. The characteristic that the cells of the cardiomyocyte lineage are derived from a parent cell population is important in several ways. In particular, the undifferentiated cell population can be used to produce additional cell populations with a shared genome. The additional cell population can be either a further batch of cardiac cells or another cell type useful for therapy, such as a population that can pre-tolerize the patient to a histocompatible type of cardiac allograft (US 2002 / 0086005; WO 03 / 050251).

[0150] IV.How to use CTC4 cells or cells derived therefrom, such as cardiomyocytes, vascular endothelial cells, or smooth muscle cells, obtained by the methods and compositions of certain embodiments can be used in a variety of applications, including, but not limited to, in vivo organ transplantation or cell transplantation, in vitro screening of cytotoxic compounds, carcinogens, mutagens, growth / regulatory factors, pharmaceutical compounds, etc., elucidating the mechanisms of cardiac disease and disorders, studying the mechanisms by which drugs and / or growth factors act, diagnosing and monitoring cancer in patients, gene therapy, and manufacturing biologically active pharmaceuticals.

[0151] The CTC4 cells of the present disclosure or cells derived therefrom, such as cardiomyocytes, vascular endothelial cells, or smooth muscle cells, can be used commercially to screen for factors (e.g., solvents, small molecule drugs, peptides, oligonucleotides, etc.) or environmental conditions (e.g., culture conditions or manipulations) that affect the properties of such cells and their various progeny.

[0152] In some embodiments, CTC4 cells or cells derived therefrom, such as cardiomyocytes, vascular endothelial cells, or smooth muscle cells, can be used to screen for factors that promote maturation into late cardiomyocyte precursors or terminally differentiated cells, or promote the growth and maintenance of such cells in long-term culture. For example, candidate maturation or growth factors are added to cells in different wells, and any resulting phenotypic changes are then examined, determined according to desired criteria for further culture and use of the cells.

[0153] Another screening application of the present disclosure relates to testing for the effect of pharmaceutical compounds on the maintenance or repair of myocardial tissue. Screening may be performed because the compound is designed to have a pharmacological effect on the cells, or because a compound designed to have an effect elsewhere may have unintended side effects on cells of this tissue type. Screening may be performed using any of the progenitor or terminally differentiated cells of the present disclosure.

[0154] The reader is generally referred to the standard textbook In vitro Methods in Pharmaceutical Research, Academic Press, 1997, and U.S. Patent No. 5,030,015. Evaluation of the activity of a candidate pharmaceutical compound generally involves combining the candidate compound, alone or in combination with other pharmaceutical agents, with the differentiated cells of the present disclosure. The researcher measures any changes in morphology, marker phenotype, or functional activity of the cells that are caused by the compound (compared to untreated cells or cells treated with an inactive compound) and then correlates the effect of the compound with the observed changes.

[0155] Cytotoxicity can be determined primarily by effects on cell viability, survival, morphology, and expression of specific markers and receptors. The effect of pharmaceutical agents on chromosomal DNA can be determined by measuring DNA synthesis or repair. 3 Incorporation of [H]-thymidine or BrdU, especially at unscheduled times in the cell cycle or above levels required for cell replication, is consistent with the action of a pharmaceutical drug. Unwanted effects may also include abnormal rates of sister chromatid exchange as determined by metaphase spreads. The reader is referred to Vickers (in vitro Methods in Pharmaceutical Research, Academic Press, 1997, pp 375-410) for further details.

[0156] The effect on cell function can be assessed using any standard analytical method for observing cardiomyocyte phenotype or activity, such as marker expression, receptor binding, contractile activity, or electrophysiology, either in cell culture or in vivo. Drug candidates can also be analyzed for their effect on contractile activity, such as whether they increase or decrease the extent or frequency of contraction. If an effect is observed, the concentration of the compound can be titrated to determine whether it is effective at 50% (ED 50 ) can be determined.

[0157] The present disclosure further provides a method for screening drugs that have an effect on human cardiovascular progenitor cells, cardiovascular colonies, cardiomyocytes, endothelial cells, and vascular smooth muscle cells. The method includes contacting one of the cell populations described herein above with a candidate drug and determining whether the drug has an effect on the cell population. The drug tested may be natural or synthetic, a compound or mixture, a small molecule or polymer (including polypeptides, polysaccharides, polynucleotides, etc.), an antibody or antibody fragment, a compound from a library of natural or synthetic compounds, a compound obtained from rational drug design, a condition such as a cell culture condition, or any drug whose effect on the cell population can be evaluated using analytical methods known in the art. The effect on the cell population can be measured using any standard phenotypic or activity assay, such as marker expression, receptor binding, contractile activity, electrophysiology, cell viability, survival, morphology, or DNA synthesis or DNA repair assays. Standard proliferation and differentiation assays are described in U.S. Pat. No. 6,110,739. Such agents are useful in controlling cell proliferation, differentiation, viability, and tissue maintenance, regeneration and repair in vivo and in vitro.

[0158] A. Pharmaceutical Compositions The present disclosure further provides compositions comprising committed cardiac progenitor cells or cells derived therefrom, such as cardiomyocytes, vascular endothelial cells, or smooth muscle cells. The compositions may include pharma- ceutically acceptable carriers and diluents. The compositions may further include ingredients that promote engraftment. Compositions comprising these cells are useful for cell and tissue replacement and repair, and for the generation of cardiomyocytes in vitro and in vivo. Compositions comprising CTC4 cells are useful for the expansion of progenitor cell populations. The compositions may be formulated as pharmaceuticals or delivery devices for treating cardiac conditions.

[0159] The CTC4 cells of the present disclosure or cells derived therefrom (e.g., cardiomyocytes, vascular endothelial cells, or smooth muscle cells, etc.) can be provided in the form of a pharmaceutical composition and can include an isotonic excipient prepared under sufficiently sterile conditions for administration to humans. In certain embodiments, it may be desirable to disperse the cells using proteases or by gentle mechanical manipulation into a suspension of single cells or smaller clusters. To reduce the risk of cell death during engraftment, the cells can be heat shocked or cultured with about 0.5 U / mL erythropoietin about 24 hours prior to administration.

[0160] For general principles of pharmaceutical formulation, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, 1996; and Hematopoetic Stem Cell Therapy, 2000. The choice of cell excipient and any components associated with the composition may be varied depending on the route and device of administration. The composition may also contain or be combined with one or more other components that promote engraftment or functional recruitment of cardiomyocytes. Suitable components include matrix proteins that support or promote adhesion of cardiomyocytes or complementary cell types, particularly endothelial cells.

[0161] The present disclosure also includes a reagent system, which includes a set or combination of cells that may exist at any point in manufacture, distribution, or use. A set of cells includes any combination of two or more cell groups described in the present disclosure, including, but not limited to, a combination of a type of differentiated cell (cardiomyocyte, cardiomyocyte precursor, etc.) with undifferentiated pluripotent stem cells or other differentiated cell types that often share the same genome. Each cell type in the set may be packaged together, in separate containers in the same facility, in different locations, at the same time or at different times, and under the control of the same owner or different owners who share a business relationship.

[0162] The pharmaceutical compositions of the present disclosure may optionally be packaged in a suitable container along with instructions for the reconstitution of CTC4 cells or cells derived therefrom (e.g., cardiomyocytes, vascular endothelial cells, or smooth muscle cells) for a desired purpose, such as to ameliorate a disease state or abnormality of the myocardium.

[0163] B. Therapeutic use The cells provided in certain aspects of the present disclosure can be used to treat any subject in need thereof.Human conditions that may be suitable for such treatment include cardiac disorders such as myocardial infarction, cardiomyopathy, congestive heart failure, ventricular septal defect, atrial septal defect, congenital heart defect, ventricular aneurysm, pediatric heart disease, ventricular aneurysm, or heart disease requiring ventricular reconstruction.

[0164] In human treatment, the dosage is generally about 10 8 ~10 12 cells, typically about 2 × 10 8 ~1×10 9 The amount of cells administered is adjusted to accommodate the subject's weight, the nature and severity of the disease, and the replicative capacity of the administered cells. Ultimate responsibility for determining the treatment method and appropriate dosage rests with the attending clinician.

[0165] Certain embodiments also provide for the use of CTC4 cells to enhance myocardial tissue maintenance or repair in response to any recognized need, such as congenital errors of metabolic function, the effects of a pathology, or the effects of severe trauma.

[0166] To determine the suitability of the cell composition for therapeutic administration, the cells can first be tested in an appropriate animal model. At one level, the cells are evaluated for their ability to survive and maintain their phenotype in vivo. The cell composition is administered to an immunodeficient animal (e.g., NUDE rats, or animals rendered immunodeficient chemically or by irradiation). After an engraftment period, tissues are harvested to assess whether pluripotent stem cell-derived cells are still present. CTC4 cells were shown to engraft and survive at least 30 days after injection (Figure 8B). CTC4 cells can also continue to differentiate into cardiomyocytes, as shown in Figure 8C by co-staining hAlu+ cells with the gap junction protein connexin 43 (CX43) and the structural protein cardiac troponin T (CTNT).

[0167] Other methods for tracking cells in vivo include administering cells that express a detectable label (such as green fluorescent protein or β-galactosidase) or by administering cells that are pre-labeled (e.g., BrdU or 3 Methods for detecting human phenotypes include administering cells labeled with human-specific antibodies (e.g., H]thymidine) or by subsequent detection of constitutive cell markers (e.g., using human-specific antibodies). The presence and phenotype of the administered cells can be assessed by immunohistochemistry or ELISA using human-specific antibodies, or by RT-PCR analysis using primers and hybridization conditions that specifically amplify human polynucleotides according to published sequence data.

[0168] Compatibility can also be determined by assessing the degree of cardiac recovery resulting from treatment with pluripotent stem cell-derived cardiomyocytes. Many animal models are available for such analysis. For example, the heart can be cryoinjured by placing a pre-chilled aluminum rod in contact with the surface of the left ventricular anterior wall (Murry et al., 1996; Reinecke et al., 1999; U.S. Patent No. 6,099,832; Reinecke et al., 2004). In larger animals, cryoinjury can be produced by placing a 30-50 mm copper disk probe cooled in liquid nitrogen on the left ventricular anterior wall for approximately 20 minutes (Chiu et al., 1995). Infarction can be induced by ligating the left main coronary artery (Li et al., 1997). The injury site is treated with the cell preparation of the present disclosure, and the cardiac tissue is examined by histology for the presence of cells in the injured area. Cardiac function can be monitored by measuring parameters such as left ventricular end-diastolic pressure, developed pressure, rate of pressure rise, and rate of pressure decay.

[0169] After due consideration, the differentiated cells of the present disclosure can be used for tissue reconstruction or regeneration in human patients or other subjects in need of such treatment. The cells are administered in such a way that they can be transplanted or transferred to the intended tissue site and reconstruct or regenerate the functionally deficient area. Specialized devices are available that are suitable for administering cells capable of reconstructing cardiac function directly into the ventricle, pericardium, or inside the myocardium at the desired location.

[0170] Patients receiving allografts of pluripotent stem cell-derived CTC4 cells can be treated to reduce immune rejection of the transplanted cells, if necessary. Methods under consideration include administration of traditional immunosuppressants such as cyclosporine A (Dunn et al, Drugs 61:1957, 2001) or induction of immune tolerance using matched cell populations of pluripotent stem cell-derived cells (WO 02 / 44343; US Patent 6,280,718; WO 03 / 050251). Another approach is to modify the CTC4 cell population, for example by treating with allopurinol, to reduce the amount of uric acid produced by the cells upon transplantation into the subject. Alternatively or in combination, the patient is primed by administering allopurinol or an enzyme that metabolizes uric acid, such as uric acid oxidase (PCT / US04 / 42917).

[0171] Suitable patients for regenerative medicine according to the present method include those with various types of acute and chronic heart disease, such as coronary heart disease, cardiomyopathy, endocarditis, congenital cardiovascular defects, and congestive heart failure. The efficacy of treatment can be monitored by clinically accepted criteria, such as reduction in the area occupied by scar tissue or revascularization of scar tissue, and reduction in the frequency and severity of angina, or improvement in developed cardiac pressure, systolic pressure, end diastolic pressure, patient mobility, and quality of life.

[0172] In another embodiment, the present disclosure provides a method of cell replacement and a method of tissue replacement useful for treating diseases characterized by insufficient cardiac function, including, for example, congenital heart disease, coronary heart disease, cardiomyopathies, endocarditis, and congestive heart failure. Both differentiated cells and cardiovascular progenitor cells are useful for replacement therapy, since the progenitor cell population can differentiate in vivo into cardiomyocyte, endothelial, and vascular smooth muscle lineages. The cells are also useful for generating cardiovascular tissue in vitro. Methods for engineering cardiac tissue are known in the art and are reviewed, for example, in "Stem Cell Therapy and Tissue Engineering for Cardiovascular Repair" by Birla, Springer, 2006. Thus, in one embodiment, the present disclosure provides a method of cardiomyocyte replacement therapy, comprising administering to a subject in need of such treatment, a composition comprising cardiomyocytes isolated from an enriched population of human cardiovascular progenitor cells obtained according to the present disclosure. In another embodiment, the present disclosure provides a method of treating a disease characterized by insufficient cardiac function, comprising administering a composition comprising human cardiovascular progenitor cells to a subject in need of such treatment. In a preferred embodiment, the subject is a human. The composition may be administered by a route of administration that results in delivery or migration to cardiac tissue, including, for example, injection or implantation, and under conditions that result in the reduction of at least one side effect or symptom or disease.

[0173] The therapeutic methods of the present disclosure are not intended to limit the administration of CTC4 cells to a mammal to a particular mode of administration, dosage, or frequency of administration, and the present disclosure contemplates all methods of administration, including intramuscular, intravenous, intraarticular, intralesional, subcutaneous, or any other route sufficient to provide an appropriate dosage to prevent or treat a disease. The CTC4 cells may be administered to a mammal in a single dose or multiple doses. When administered multiple doses, the doses may be spaced apart, for example, one week, one month, one year, or ten years, from one dose to the next. One or more growth factors, hormones, interleukins, cytokines, small molecules, or other cells may be administered before, during, or after administration of the cells to further bias them toward a particular cell type.

[0174] [Example] V. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should recognize that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to work well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should recognize in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0175] [Example 1] iPSC-derived cardiac progenitor cells iPSCs were thawed and expanded in feeder-free monolayer culture on vitronectin-coated plates (2.5 μg / mL) in Essential 8 medium (E8) for 3 days, with daily medium changes.

[0176] On day 0 of differentiation in suspension, iPSCs were harvested with TrypLE, washed with E8, and resuspended in aggregate formation medium containing E8, 1 uM H1152 (Rho kinase inhibitor), and 2 uM CHIR99021 (Wnt agonist) to initiate aggregate formation and mesoderm induction. Cell densities were adjusted to 1 × 10 6 The culture was adjusted to cells / mL and seeded into the bioreactor (PBS500 or PBS3).

[0177] On day 1, aggregates were transferred to fresh media by allowing aggregates to settle and replacing 80% of the media with RPMI + B27 (no insulin) and 5 μM CHIR99021. Day 2 aggregates were fed by first allowing aggregates to settle and replacing 80% of the media with RPMI, B27 (no insulin) and 4.4 μM CHIR99021.

[0178] For cardiac specification, day 3 aggregates were first spun down and 80% of the medium was replaced with RPMI, B27 (containing insulin) and 10 uM XAV939 (Wnt inhibitor). In some instances, additional small molecules were added to efficiently induce cardiac specification for certain iPSCs. For example, these small molecules include 2 uM SB431542 (TGFβ / Activin inhibitor) and / or 1-2 uM Dorsomorphin (BMP inhibitor).

[0179] Cultures were fed as on previous days while cells continued to specify towards the cardiomyocyte lineage. On days 4 and 5, after initial settling of aggregates, 80% of the medium was replaced with RPMI and B27 (containing insulin). The entire process was performed serum-free and without drug resistance selection.

[0180] On day 6, cells were committed but not yet differentiated into cardiomyocytes. The aggregates were harvested and washed with D-PBS (- / -), then dissociated with TrypLE and cryopreserved as single cell suspensions in CryoStor CS10 in a regulated freezer.

[0181] Cardiac progenitor cells were analyzed for cardiac mesoderm markers (i.e., KDR, CKIT, and PDFGRα) and cardiomyocyte markers (i.e., SAA and SMA). The present differentiation method resulted in cardiomyocytes with more than 95% SAA. Thus, the present method efficiently produced committed cardiac progenitor cells and cardiomyocytes.

[0182] [Example 2] Identifying the differentiation stage at which Wnt inhibition is applied The point during cardiac differentiation when Wnt inhibition is required has not been well documented. The cell surface markers CXCR4 and CD56 can be used to monitor the status of the cultures and help determine when Wnt inhibition should be administered.

[0183] Day 1-6 aggregate samples were taken from both PBS500 or PBS3 bioreactor cultures, dissociated, and stained for CXCR4 and CD56. Cell populations shifted toward different expression profiles over the days. Initially, they showed expression of CXCR4, followed by CXCR4. pos CD56 pos The cells showed both CXCR4 and CXCR5 expression and finally lost CXCR4 expression by days 4 to 6.

[0184] If the cultures overexpressed CD56 on day 3 or had already begun to lose CXCR4 expression, it was too late to inhibit Wnt signaling and commit the cultures to a cardiomyocyte fate. However, we found that efficient cardiac specification occurred when the CXCR4-positive cell population was just beginning to express CD56. Strong expression of CD56 was one indication that either too much CHIR99021 had been used or the cell density was too low.

[0185] Another indicator of potentially unsuccessful cardiac differentiation was whether there was overexpression of CXCR4 on day 2. This was a clear indicator that too much CHIR99021 had been used by day 2.

[0186] [Example 3] Scales of Differentiation This cardiac differentiation was initially carried out using PBS500 vessels, but at this scale, 1 × 10 8 ~1×10 9 The scale was too small to produce cell therapy doses of cells (Figure 2B). Volume, PBS wheel speed, pH, and dissolved oxygen were explored using the PBS500 format and adapted to optimize clinically relevant differentiation scales using multiple PBS3 bioreactors.

[0187] [Example 4] Cryopreservation scale Standard 1.5-2.0 ml cryovials can be used to cryopreserve small-scale iPSC-derived product samples. However, in order to cryopreserve a sufficient amount of committed cardiac progenitor cells in a single vial that could be used as a clinically administered dose, we tested several different sizes of cryovials from Aseptic Technologies. After testing several AT vial sizes, we determined that the AT6 vial would provide a clinically relevant dose in a single vial (Figure 2C). 6 Freezing up the cells was tested and as a result the cells passed all quality release assays.

[0188] [Example 4] Committed cardiac progenitor cells express specific markers A time course study was performed testing multiple markers each day during the differentiation process. There was a clear induction of CXCR4 and PDGFRα from day 1 to day 3, indicating cardiac mesoderm specification (Figure 3B). Dynamic expression of KDR was also detected, with highest expression seen on day 4, followed by a rapid decline in expression between days 5 and 6 (Figure 5A). CD56 was also induced during day 3 and maintained throughout the differentiation process. Expression of EpCAM was monitored and decreased daily during the process, with less than 10% positive cells by day 6 (Figure 4). Cardiomyocyte structural proteins were also minimally expressed by day 6.

[0189] [Example 5] Committed cardiac progenitor cells become cardiomyocytes Day 6 CTC4 cells were thawed and plated to test their cardiomyocyte differentiation potential. They were seeded at various densities on vitronectin-coated vessels in RPMI and B27 (with insulin) and cultured for approximately 7 days (Figure 7A). Medium was replaced with a complete volume exchange every other day. Monolayers began to contract 2-6 days after plating. Contracting cells were harvested and analyzed by flow cytometry for cardiomyocyte-specific markers. Cells analyzed were >90% sarcomeric α-actinin positive (Figure 7D).

[0190] Day 6 CTC4 cells were also plated on vitronectin-coated 96-well plates in RPMI and B27 (with insulin) and cultured for 7 days. Cells were stained by immunocytochemistry for various cardiomyocyte markers and stained positive for cardiac troponin T, cardiac troponin I, and sarcomeric α-actinin. Cells also stained for the cardiac-specific transcription factor NKX2.5 (Figure 7C).

[0191] [Example 6] Engraftment of committed cardiac progenitor cells (CTC4) in a myocardial infarction model The NUDE rat myocardial infarction model was used to test for engraftment and differentiation of CTC4 cells (Figure 8A-C). Three days after infarction, CTC4 cells were thawed, counted, and resuspended in 5% Flexbumin. Cells were administered by direct injection at multiple injection sites. One month after injection, hearts were harvested and human cells were stained for human Alu using immunohistochemistry or in situ hybridization detection methods. Once human cells were found at specific sites within the rat myocardium, serial sections were further processed and stained by immunohistochemistry for cardiac troponin T (cardiomyocytes), Ki67 (proliferation), and CX43 (gap junction) markers. Human cells were detected one month after injection, and robust expression of cardiac troponin T and CX43 indicates that CTC4 cells continued to differentiate in vivo and became electrically connected cardiomyocytes. Additionally, a small number of cells were also stained for Ki67, indicating the possibility of a slight expansion of the human transplant site.

[0192] [Example 7] Differentiation of committed cardiac progenitor cells into vascular endothelial or smooth muscle cells A study was conducted to show that committed cardiac progenitor cells have the capacity to further differentiate into other cell lineages, such as endothelial cells (CD31+CD144+) and smooth muscle cells (CD140b+CD90+) (Figure 9). iPSC-derived committed cardiac progenitor cells were cultured in RPMI+B27 medium containing specific growth factors. Committed cardiac progenitor cells produced vascular endothelial cells or smooth muscle cells when cultured in medium containing FGF and / or VEGF for about 7 days. * * *

[0193] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications may be applied to the methods in the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be obtained. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0194] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Amit et al., Dev. Bio., 227:271-278, 2000. Byrne et al., Nature, 450(7169):497-502, 2007. Doevendans et al., J. Mol. Cell Cardiol., 32:839, 2000. Dunn et al., Drugs 61:1957, 2001. Fernandes, et al., J. Biotechnology, 132(2):227-236, 2007. Igelmund et al., Pflugers Arch., 437:669, 1999. International Patent Publication No. PCT / JP2009 / 062911 International Patent Publication No. PCT / JP2011 / 069588 International Patent Publication No. WO 02 / 44343 International Patent Publication No. WO 03 / 050251 International Patent Publication No. WO 03 / 050251 International Patent Publication No. WO 2007 / 069666 International Patent Publication No. WO 2007 / 069666 International Patent Publication No. WO2005 / 123902 International Patent Publication No. WO98 / 30679 Keller et al., Curr. Opin. Cell Biol., 7:862-869, 1995. Klimanskaya et al., Lancet., 365:P1636-1641, 2005. Ludwig et al., Nat. Biotechnol., 24:185-187, 2006b. Ludwig et al., Nat. Methods, 3:637-646, 2006a. Takahashi et al., Cell, 131, 861-872, 2007. Thomson and Marshall, Curr. Top. Dev. Biol., 38:133-165, 1998. Thomson and Odorico, Trends Biotechnol., 18(2):53-57, 2000. Thomson et al. Proc. Natl. Acad. Scie. USA, 92:7844-7848, 1995. U.S Application No. 12 / 478,154 U.S. Patent 8,546,140 U.S. Patent No. 5,843,780 U.S. Patent No. 5,843,780 U.S. Patent No. 6,103,470 U.S. Patent No. 6,110,739 U.S. Patent No. 6,200,806 U.S. Patent No. 6,280,718 U.S. Patent No. 6,416,998 U.S. Patent No. 7,029,913 U.S. Patent No. 7,442,548 U.S. Patent No. 7,598,364 U.S. Patent No. 7,989,425 U.S. Patent No. 8,058,065 U.S. Patent No. 8,071,369 U.S. Patent No. 8,129,187 U.S. Patent No. 8,183,038 U.S. Patent No. 8,268,620 U.S. Patent No. 8,268,620 U.S. Patent No. 8,546,140 U.S. Patent No. 8,546,140 U.S. Patent No. 8,691,574 U.S. Patent No. 8,691,574 U.S. Patent No. 8,741,648 U.S. Patent No. 8,741,648 U.S. Patent No. 8,741,648 U.S. Patent No. 8,900,871 U.S. Patent No. 9,175,268 U.S. Patent Publication No. 2003 / 0211603 U.S. Patent Publication No. 2009 / 0246875 U.S. Patent Publication No. 2010 / 0210014 U.S. Patent Publication No. 2011 / 0104125 U.S. Patent Publication No. 2012 / 0276636 U.S. Patent Publication No. 2015 / 0191697 U.S. Patent Publication No. US 2002 / 0086005 United States Patent 20070116680 Watanabe et al., Nature Neurosci., 8:288-296, 2005. Wobus et al., Ann. N.Y. Acad. Sci., 27:752, 1995. Xu et al., Nat. Biotechnol., 19:971-974, 2001. Yu et al., Science, 318: 1917-1920, 2007.

Claims

1. 1. An in vitro method for producing committed cardiac progenitor cells derived from human pluripotent stem cells (PSCs), comprising: (a) culturing PSCs in the presence of a Wnt agonist to initiate differentiation and a survival agent to form cell aggregates; (b) further culturing the cell aggregates in the presence of a Wnt agonist for a period of time sufficient to produce a mesodermal cell population; and (c) differentiating said mesodermal cells in the presence of a Wnt inhibitor to promote cardiac specification, thereby producing a population of committed cardiac progenitor cells. A method comprising:

2. the PSCs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), and / or the PSCs are cultured on a surface coated with an extracellular matrix prior to step (a), and optionally the extracellular matrix comprises vitronectin, collagen, laminin, MATRIGEL®, and / or fibronectin; The method of claim 1.

3. 2. The method of claim 1, wherein the survival reagent is a Rho-associated kinase (ROCK) inhibitor or a myosin II inhibitor, optionally wherein the ROCK inhibitor is H1152 or Y-27632, or the myosin II inhibitor is blebbistatin.

4. 10. The method of claim 1, wherein the method comprises culturing cells in suspension culture, and optionally the suspension culture is carried out in one or more bioreactors.

5. (i) the Wnt agonist in step (a) is CHIR99021, SB216763, CHIR98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, or IM-12; (ii) the Wnt inhibitor in step (c) is XAV939, IWR1, IWR2, IWR3, IWR4, ICG-001, IWR-1-endo, Wnt-C59, LGK-974, LF3, CP21R7, NCB-0846, PNU-74654, or KYA179K; and / or the culturing in step (c) further comprises a TGFβ inhibitor; (iii) the culturing in step (c) further comprises a TGFβ inhibitor, and the TGFβ inhibitor is SB431542, LDN-193189, LY2157299, LY2109761, SB525334, SIS HCl, SB505124, GW788388, or LY364947; or (iv) the culture in step (c) further comprises a BMP inhibitor, and the BMP inhibitor is dorsomorphin, LDN193189, DMH1, DMH2, or ML347; The method of claim 1.

6. 10. The method of claim 1, wherein step (a) is carried out for 1 to 2 days, step (b) is carried out for 1 to 5 days, or step (c) is carried out for 1 to 6 days.

7. the culture of step (b) is insulin-free or substantially insulin-free; the Wnt signaling agonist in step (b) is CHIR99021, SB216763, CHIR98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, or IM-12; the culturing in step (b) further comprises an Activin / Nodal agonist, wherein the Activin / Nodal agonist is Activin A or Nodal; and / or The culture in step (b) further comprises an Activin / Nodal agonist and / or BMP. The method of claim 1.

8. the mesodermal cells express KDR, PDGFRα, CXCR4, and / or CD56; at least 5% of said mesodermal cells express CD56 before or during step (c); at least 40% of said mesodermal cells express KDR and PDGFRα before or during step (c); the mesodermal cell population is positive for CXCR4 and CD56 prior to step (c); and / or prior to step (c), at least 20% of said mesodermal cell population are positive for CXCR4 and less than 60% of said mesodermal cell population are positive for CD56; The method of claim 1.

9. step (c) comprises adding a Wnt inhibitor when at least 20% of said mesodermal cell population are positive for CXCR4 and less than 60% of said mesodermal cell population are positive for CD56; or prior to step (c), at least 30% of said mesodermal cell population are positive for CXCR4 and less than 60% of said mesodermal cell population are positive for CD56; The method of claim 1.

10. The method of claim 1, wherein the culturing in step (c) contains insulin and / or further contains a BMP inhibitor or an AMPK inhibitor.

11. wherein the method is serum-free; the method is compliant with Good Manufacturing Practice (GMP), and / or The culturing is carried out in a defined medium. The method of claim 1.

12. The method comprises: 7 ~1×10 10 Produce committed cardiac progenitor cells The method does not involve performing drug resistance selection, and / or the committed cardiac progenitor cells do not express the transgene; The method of claim 1.

13. less than 20% or less than 10% of the committed cardiac progenitor cells express EpCAM; less than 20% of the committed cardiac progenitor cells are positive for KDR, CXCR4, and / or SAA; less than 20% of the committed cardiac progenitor cells are positive for EpCAM and SAA; and / or At least 80% of the committed cardiac progenitor cells are positive for PDGFRα and CD56. The method of claim 1.

14. 10. The method of claim 1, further comprising cryopreserving the committed cardiac progenitor cell population that is at least 70% positive for PDGFRα, less than 40% positive for KDR, less than 20% positive for EpCAM, and less than 20% positive for SAA.

15. 15. The method of any one of claims 1 to 14, further comprising maturing the committed cardiac progenitor cells to produce cardiomyocytes, wherein the culture for maturation does not contain a Wnt inhibitor or a TGFβ inhibitor, and the culture for pre-maturation is for 2 to 30 days.

16. and further comprising differentiating the committed cardiac progenitor cells into vascular endothelial cells. differentiation comprises culturing the committed cardiac progenitor cell population in the presence of fibroblast growth factor (FGF) and / or vascular endothelial growth factor (VEGF); the vascular endothelial cells are positive for CD33 and CD144; Optionally, at least 20% of the vascular endothelial cell population is positive for CD33 and CD144.

15. The method of any one of claims 1 to 14.

17. further comprising differentiating the committed cardiac progenitor cells into smooth muscle cells; differentiation comprises culturing the committed cardiac progenitor cell population in the presence of FGF and / or VEGF; Optionally, at least 50% of the smooth muscle cell population are positive for CD140b and CD90.

15. The method according to any one of claims 1 to 14.

18. A population of committed cardiac progenitor cells produced by the method of claim 1.

19. A composition comprising a population of committed cardiac progenitor cells having at least 90% expression of CD56, at least 80% expression of PDGFRα, substantially no expression of CTNT, and less than 10% expression of CXCR4, KDR and EpCAM.

20. 20. A method for treating a cardiac disorder in a subject, comprising administering an effective amount of the committed cardiac progenitor cells of claim 18 or 19 to a subject in need thereof.

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