Method for producing cardiac fibroblasts

Abstract

Provided herein are methods for generating quiescent cardiac fibroblasts from pluripotent stem cells. Further provided herein are microtissue compositions comprising cardiac fibroblasts along with endothelial cells and cardiomyocytes.

Description

[Technical Field]

[0001] Priority claims This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 412,212, filed September 30, 2022, the entire disclosure of which is incorporated herein by reference. [Background technology]

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

[0003] 2. Description of Related Technology It is well known that most pluripotent stem cell-derived cardiomyocytes (PSC-CMs) have an immature phenotype, more closely related to fetal cardiomyocytes than to mature cardiomyocytes in terms of cellular structure, calcium handling, metabolism, contractile function, and gene expression. This maturation bottleneck hinders the use of PSC-CMs for in vitro modeling of disease, drug discovery, or cell therapy (Guo and Pu et al., 2020).

[0004] Recent studies have demonstrated that cardiac supportive cell types play important roles in cardiomyocyte development, maturation, and cardiovascular disease. Multicellular microtissue models combining PSC-CMs with primary human cardiac fibroblasts and primary human endothelial cells in scaffold-free microtissues have demonstrated more relevant "mature" responses in compound testing, including inotropic responses to isoproterenol (Ravenscroft, 2016). Similar models have also been used for high-throughput screening of structural cardiotoxins (Archer, 2018).

[0005] Most researchers wishing to form multicellular cardiac microtissues can use one or more cell types derived from induced pluripotent stem cells. Replacing primary human cells with an isogenic PSC-derived supporter cell type offers many advantages that improve the relevance and reproducibility of cardiac multicellular microtissue models (Giacomelli, 2022). However, the application of cardiac microtissue assay platforms is limited by the need to differentiate all three cell types from iPSCs and synchronize the cell type cultures before combining them in microtissues. Therefore, there is an unmet need for a cardiac microtissue assay platform constructed from purified, cryopreserved PSC-derived cell types (i.e., cardiomyocytes, endothelial cells, and cardiac fibroblasts) and co-culture media. Summary of the Invention [Means for solving the problem]

[0006] In certain embodiments, the present disclosure provides an in vitro method for generating cardiac progenitor cells derived from human pluripotent stem cells (PSCs), the method comprising: (a) culturing PSC aggregates in a medium containing a Wnt agonist, an activin agonist, and BMP4 for mesoderm induction; (b) further culturing the PSC aggregates in a medium containing an activin agonist and BMP4, but essentially free of a Wnt agonist, to generate a population of NCAM-positive and CXCR4-low mesodermal progenitor cells; and (c) culturing the mesodermal progenitor cells in a medium containing a Wnt inhibitor to generate a population of cardiac progenitor cells. In some aspects, step (c) is further defined as generating a mixed population of cardiac progenitor cells and cTNT+ cardiomyocytes. In certain aspects, the method further comprises generating PSC-derived cardiac fibroblasts, comprising differentiating the cardiac progenitor cells in a medium containing basic FGF (bFGF) to generate a population of cardiac fibroblasts. In some embodiments, differentiation into cardiac fibroblasts is carried out in the absence of a TGFβ inhibitor.

[0007] Further embodiments provide an in vitro method for generating human pluripotent stem cell (PSC)-derived cardiac fibroblasts, the method comprising: (a) culturing PSC aggregates in a medium comprising a Wnt agonist, an activin agonist, and BMP4 for mesoderm induction; (b) further culturing the PSC aggregates in a medium comprising an activin agonist and BMP4, and essentially no Wnt agonist, to generate a population of NCAM-positive and CXCR4-low mesodermal progenitor cells; (c) culturing the mesodermal progenitor cells in a medium comprising a Wnt inhibitor to generate a population of cardiac progenitor cells; and (d) differentiating the cardiac progenitor cells to generate a population of cardiac fibroblasts in a medium comprising basic FGF (bFGF).

[0008] In some embodiments, the mesodermal progenitor cells are NCAM positive and CXCR4 (NCAM+ / CXCR4 low In certain embodiments, less than 10% (e.g., less than 5%) of the mesodermal progenitor cells are CXCR4 positive.

[0009] In certain embodiments, the cardiac progenitor cells include epicardial progenitor cells, endothelial fibroblast progenitor cells, second heart field progenitor cells, and / or neural crest progenitor cells. In some embodiments, about 30-70% (e.g., 30-50%, 40-60%, 50-70%, about 30%, about 40%, about 50%, about 60%, or about 70%) of the cardiac progenitor cells are second heart field progenitor cells, such as GATA4 and / or HAND2-positive cells. In certain embodiments, about 10-50% (e.g., 10-30%, 20-40%, 30-50%, about 10%, 20%, 30%, 40%, or 50%) of the cardiac progenitor cells are endothelial fibroblast progenitor cells, such as TEK-positive cells. In some embodiments, approximately 5-40% (e.g., 5-15%, 10-20%, 15-30%, 20-40%, approximately 5%, 10%, 15%, 20%, 30%, or 40%) of the cardiac progenitor cells are epicardial progenitor cells, such as cells positive for WT1, SNAI1, TBX19, and / or TBX20. In some embodiments, the cardiac progenitor cells are oligopotent. In some embodiments, the cardiac fibroblasts express vimentin (VIM), COL1A1, COL1A2, and / or secreted protein acidic and rich in cysteine ​​(SPARC). In some embodiments, the cardiac fibroblasts express discoidin domain receptor 2 (DDR2) and periostin (POSTN). In some embodiments, the cardiac fibroblasts are at least 40% (e.g., at least 40%, 45%, 50%, 55%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%) GATA-4 positive. In certain embodiments, the cardiac fibroblasts are at least 50%, e.g., at least 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% CD90 positive.

[0010] In some embodiments, the PSCs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In certain embodiments, the iPSCs are derived from a healthy subject or a subject with a genetic disease genotype. In certain embodiments, the iPSCs are engineered to contain a disease-associated mutation for a cardiovascular disease. In some embodiments, the iPSCs are derived from a subject with dilated cardiomyopathy and contain the LMNA-L35P mutation.

[0011] 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 some embodiments, CHIR99021 is present in step (a) at a concentration of about 5 μM to 10 μM, e.g., 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. In some embodiments, CHIR99021 is present in step (a) at a concentration of about 1 μM to about 20 μM, eg, 1-5 μM, 5-10 μM, 10-15 μM, or 15-20 μM.

[0012] In certain embodiments, the medium of steps (a)-(c) is free of or essentially free of insulin. In certain embodiments, the activin agonist is activin A. In some embodiments, the medium of step (a) further comprises albumin. In certain embodiments, the medium of any of steps (a)-(c) further comprises albumin.

[0013] In some embodiments, the PSC aggregates of step (a) are obtained by culturing PSCs in the presence of a Wnt agonist and a pro-life agent. In some embodiments, the Wnt agonist 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 some embodiments, CHIR99021 is present in step (a) at a concentration of about 0.5 to 5 μM, e.g., about 1 μM to 3 μM, e.g., 1 μM, 2 μM, or 3 μM. In certain embodiments, the pro-life agent is a Rho-associated kinase (ROCK) inhibitor or a myosin II inhibitor. In some embodiments, the ROCK inhibitor is H1152 or Y-27632. In certain embodiments, the myosin II inhibitor is H1152.

[0014] In certain embodiments, the mesodermal progenitor cells are dissociated into essentially single cells prior to step (c). In some embodiments, the medium in step (c) does not contain or is essentially free of BMP4, a Wnt agonist, and retinoic acid. 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 some embodiments, the Wnt inhibitor is XAV939. In certain embodiments, XAV939 is present in the medium at a concentration of 0.5 μM to 10 μM, e.g., 1 μM to 5 μM, e.g., 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM.

[0015] In some embodiments, the population of cardiac progenitor cells in step (c) comprises at least 30% (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more) WT1+ progenitor cells. In certain embodiments, the population of cardiac progenitor cells in step (c) comprises less than 80% (e.g., less than 85%, less than 90%, less than 95%, less than 98%, less than 99%, or less than 100%, e.g., less than 75%, less than 70%, less than 65%, less than 60%, less than 50%, or less than 40%) WT1+ progenitor cells.

[0016] In certain aspects, the method does not include a purification step between step (c) and differentiating the cells into cardiac fibroblasts. In some aspects, the purification step is further defined as cell sorting.

[0017] In certain embodiments, the TGFβ inhibitor is SB431542, LDN-193189, LY2157299, LY2109761, SB525334, SIS HCl, SB505124, GW788388, or LY364947. In certain embodiments, the TGFβ inhibitor is SB431542. In some embodiments, SB431542 is present in the medium at a concentration of 1 μM to 10 μM, such as 1 to 5 μM, 5 to 10 μM, 1 to 3 μM, 3 to 7 μM, or 7 to 10 μM.

[0018] In certain embodiments, the medium in step (a) is free of or essentially free of Wnt agonists and Wnt inhibitors. In some embodiments, the medium in step (b) is free of or essentially free of TGFβ inhibitors. In certain embodiments, the medium in step (b) further comprises serum.

[0019] In certain embodiments, bFGF is present in the medium at a concentration of 50-200 ng / mL. In some embodiments, the medium of step (b) is serum-free or essentially serum-free. In certain embodiments, bFGF is present in the medium at a concentration of 10-100 ng / mL. In further embodiments, the medium further comprises VEGF, EGF, and / or IGF.

[0020] In some embodiments, the population of cardiac fibroblasts comprises at least 75% (e.g., 80%, 85%, 90%, 95%, 99%, or 100%) TE-7, CD29, and / or CD90 positive cells. In certain embodiments, the population of cardiac fibroblasts comprises at least 85% TE-7 and / or CD29 / CD90 positive cells.

[0021] In certain embodiments, the cardiac fibroblasts are quiescent cardiac fibroblasts. In some embodiments, the population of cardiac fibroblasts comprises less than 15%, less than 10%, or less than 5% (e.g., 4%, 3%, 2%, or 1% or less) of alpha-smooth muscle actin (αSMA)-positive cells. In some embodiments, the population of cardiac fibroblasts comprises at least 85% TE-7, CD90, and CD29-positive cells, and less than 5% αSMA-positive cells.

[0022] In a further aspect, the method further comprises cryopreserving the population of cardiac fibroblasts.

[0023] In some embodiments, the method further comprises culturing the population of cardiac fibroblasts in the presence of TGFβ to produce an activated population of cardiac fibroblasts. In some embodiments, TGFβ is present at a concentration of 10-100 ng / mL, e.g., 10-25, 25-50, 50-75, or 75-100 ng / mL. In some embodiments, the activated population of fibroblasts exhibits increased fibronectin secretion and / or increased αSMA expression compared to the population of cardiac fibroblasts prior to culturing in the presence of TGFβ.

[0024] In certain embodiments, the culturing is performed in a defined medium. In certain embodiments, the method complies with good manufacturing practice (GMP).

[0025] In certain aspects, the present disclosure provides an in vitro method for generating human pluripotent stem cell (PSC)-derived epicardial progenitor cells, the method comprising: (a) culturing PSC aggregates in a medium containing a Wnt agonist, an activin agonist, and BMP4 for mesoderm induction; (b) further culturing the PSC aggregates in a medium containing an activin agonist and BMP4 but essentially free of a Wnt agonist to generate a population of NCAM+ / CXCR4+ (e.g., cells with NCAM-positive and low CXCR4 expression) mesodermal progenitor cells; and (c) culturing the mesodermal progenitor cells in a medium containing a Wnt inhibitor to generate a population of WT1+ epicardial progenitor cells. In some aspects, step (c) is further defined as generating a mixed population of WT1+ epicardial progenitor cells and cTNT+ cardiomyocytes. In certain embodiments, the method further comprises generating PSC-derived cardiac fibroblasts, comprising (a) culturing WT1+ epicardial progenitor cells in a medium comprising a TGFβ inhibitor; and (b) differentiating the WT1+ epicardial progenitor cells to generate a population of cardiac fibroblasts in a medium comprising basic FGF (bFGF). In certain embodiments, the method further comprises generating PSC-derived cardiac fibroblasts, comprising differentiating the WT1+ epicardial progenitor cells to generate a population of cardiac fibroblasts in a medium comprising basic FGF (bFGF). In some embodiments, differentiation into cardiac fibroblasts is performed in the absence of a TGFβ inhibitor.

[0026] Further embodiments provide an in vitro method for generating human pluripotent stem cell (PSC)-derived cardiac fibroblasts, the method comprising: (a) culturing PSC aggregates in a medium comprising a Wnt agonist, an activin agonist, and BMP4 for mesoderm induction; (b) further culturing the PSC aggregates in a medium comprising an activin agonist and BMP4, and essentially no Wnt agonist, to generate NCAM+ / CXCR4+ (e.g., NCAM+CXCR4 low positive(c) culturing the mesodermal progenitor cells in a medium containing a Wnt inhibitor to produce a population of WT+ epicardial progenitor cells, and (d) culturing the WT1+ epicardial progenitor cells in a medium containing a TGFβ inhibitor; and (e) differentiating the WT1+ epicardial progenitor cells to produce a population of cardiac fibroblasts in a medium containing basic FGF (bFGF).

[0027] In some embodiments, the mesodermal progenitor cells are NCAM positive and CXCR4 (NCAM+ / CXCR4 low ) In certain embodiments, less than 10% (e.g., less than 5%) of the mesodermal progenitor cells are CXCR4 positive.

[0028] In some embodiments, the PSCs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In certain embodiments, the iPSCs are derived from a healthy subject or a subject with a genetic disease genotype. In certain embodiments, the iPSCs are engineered to contain a disease-associated mutation for a cardiovascular disease. In some embodiments, the iPSCs are derived from a subject with dilated cardiomyopathy and contain the LMNA-L35P mutation.

[0029] 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 some embodiments, CHIR99021 is present in step (a) at a concentration of about 5 μM to 10 μM, e.g., 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. In some embodiments, CHIR99021 is present in step (a) at a concentration of about 1 μM to about 20 μM, eg, 1-5 μM, 5-10 μM, 10-15 μM, or 15-20 μM.

[0030] In certain embodiments, the medium of steps (a)-(c) is free of or essentially free of insulin. In certain embodiments, the activin agonist is activin A. In some embodiments, the medium of step (a) further comprises albumin. In certain embodiments, the medium of any of steps (a)-(c) further comprises albumin.

[0031] In some embodiments, the PSC aggregates of step (a) are obtained by culturing PSCs in the presence of a Wnt agonist and a pro-life agent. In some embodiments, the Wnt agonist 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 some embodiments, CHIR99021 is present in step (a) at a concentration of about 0.5 to 5 μM, e.g., about 1 μM to 3 μM, e.g., 1 μM, 2 μM, or 3 μM. In certain embodiments, the pro-life agent is a Rho-associated kinase (ROCK) inhibitor or a myosin II inhibitor. In some embodiments, the ROCK inhibitor is H1152 or Y-27632. In certain embodiments, the myosin II inhibitor is H1152.

[0032] In certain embodiments, the mesodermal progenitor cells are dissociated into essentially single cells prior to step (c). In some embodiments, the medium in step (c) does not contain or is essentially free of BMP4, a Wnt agonist, and retinoic acid. 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 some embodiments, the Wnt inhibitor is XAV939. In certain embodiments, XAV939 is present in the medium at a concentration of 0.5 μM to 10 μM, e.g., 1 μM to 5 μM, e.g., 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM.

[0033] In some embodiments, the population of WT1+ epicardial progenitor cells in step (c) comprises at least 30% (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more) positive WT1+ epicardial progenitor cells. In certain embodiments, the population of WT1+ epicardial progenitor cells in step (c) comprises less than 80% (e.g., less than 85%, less than 90%, less than 95%, less than 98%, less than 99%, or less than 100%, e.g., less than 75%, less than 70%, less than 65%, less than 60%, less than 50%, or less than 40%) positive WT1+ epicardial progenitor cells.

[0034] In certain aspects, the method does not include a purification step between step (c) and differentiating the cells into cardiac fibroblasts. In some aspects, the purification step is further defined as cell sorting.

[0035] In certain embodiments, the TGFβ inhibitor is SB431542, LDN-193189, LY2157299, LY2109761, SB525334, SIS HCl, SB505124, GW788388, or LY364947. In certain embodiments, the TGFβ inhibitor is SB431542. In some embodiments, SB431542 is present in the medium at a concentration of 1 μM to 10 μM, such as 1 to 5 μM, 5 to 10 μM, 1 to 3 μM, 3 to 7 μM, or 7 to 10 μM.

[0036] In certain embodiments, the medium in step (a) is free of or essentially free of Wnt agonists and Wnt inhibitors. In some embodiments, the medium in step (b) is free of or essentially free of TGFβ inhibitors. In certain embodiments, the medium in step (b) further comprises serum.

[0037] In certain embodiments, bFGF is present in the medium at a concentration of 50-200 ng / mL. In some embodiments, the medium of step (b) is serum-free or essentially serum-free. In certain embodiments, bFGF is present in the medium at a concentration of 10-100 ng / mL. In further embodiments, the medium further comprises VEGF, EGF, and / or IGF.

[0038] In some embodiments, the population of cardiac fibroblasts comprises at least 75% (e.g., 80%, 85%, 90%, 95%, 99%, or 100%) TE-7, CD29, and / or CD90 positive cells. In certain embodiments, the population of cardiac fibroblasts comprises at least 85% TE-7, CD29, and / or CD90 positive cells.

[0039] In certain embodiments, the cardiac fibroblasts are quiescent cardiac fibroblasts. In some embodiments, the population of cardiac fibroblasts comprises less than 15%, less than 10%, or less than 5% (e.g., 4%, 3%, 2%, or 1% or less) of alpha-smooth muscle actin (αSMA)-positive cells. In some embodiments, the population of cardiac fibroblasts comprises at least 85% TE-7, CD90, and CD29-positive cells, and less than 5% αSMA-positive cells.

[0040] In a further aspect, the method further comprises cryopreserving the population of cardiac fibroblasts.

[0041] In some embodiments, the method further comprises culturing the population of cardiac fibroblasts in the presence of TGFβ to produce an activated population of cardiac fibroblasts. In some embodiments, TGFβ is present at a concentration of 10-100 ng / mL, e.g., 10-25, 25-50, 50-75, or 75-100 ng / mL. In some embodiments, the activated population of fibroblasts exhibits increased fibronectin secretion and / or increased αSMA expression compared to the population of cardiac fibroblasts prior to culturing in the presence of TGFβ.

[0042] In certain embodiments, the culturing is performed in a defined medium. In certain embodiments, the method complies with good manufacturing practice (GMP).

[0043] Further embodiments provide populations of epicardial progenitor cells or cardiac fibroblasts produced by the methods of this embodiment and its aspects.

[0044] Another embodiment provides a composition comprising a population of PSC-derived cardiac fibroblasts that express TE-7 and CD29 at at least 75% (e.g., 80%, 85%, 90%, 95%, 99%, or 100%) and αSMA at less than 15%, 10%, or 5% (e.g., less than 4%, 3%, 2%, or 1%). In some aspects, the population of cardiac fibroblasts comprises at least 85% TE-7, CD90, and / or CD29 positive cells. In particular aspects, the population of cardiac fibroblasts comprises at least 85% TE-7, CD90, and / or CD29 positive cells and less than 5% αSMA positive cells. In some aspects, cardiac fibroblasts are produced by the methods of this embodiment and its aspects. In some aspects, the population of cardiac fibroblasts is GMP compliant. In particular aspects, the composition is a pharmaceutical composition.

[0045] Further embodiments provide methods for treating cardiac disease in a subject, comprising administering an effective amount of cardiac fibroblasts of this embodiment or aspect thereof to a subject in need thereof. In some aspects, the cardiac fibroblasts are administered directly to the heart. In particular aspects, administration is by using an intramyocardial catheter. In some aspects, the cells are administered in a suspension comprising human albumin. In particular aspects, the human albumin is present at a concentration of 1% to 20%, e.g., 1% to 10%, e.g., 1 to 5% or 5 to 10%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some aspects, the human albumin is present at a concentration of 5%. In particular aspects, the subject is human. In certain aspects, the cardiac disease is fibrosis, myocardial infarction, cardiomyopathy, congestive heart failure, ventricular septal defect, atrial septal defect, congenital heart defect, ventricular aneurysm, cardiac disease of pediatric origin, ventricular aneurysm, or cardiac disease requiring ventricular reconstruction.

[0046] Another embodiment provides a co-culture comprising cardiac fibroblasts, endothelial cells, and cardiomyocytes of this embodiment or any aspect thereof. In some aspects, the co-culture comprises about 40% (e.g., 45%, 50%, 55%, 60%, 65%, 70%, or 75%) cardiomyocytes, about 5% (e.g., 10%, 15%, 20%, 25%, or 30%) endothelial cells, and about 10% (e.g., 15%, 20%, 25%, or 30%) cardiac fibroblasts. In particular aspects, the co-culture comprises about 75% cardiomyocytes, about 30% endothelial cells, and about 30% cardiac fibroblasts. In some aspects, the co-culture comprises about 60% cardiomyocytes, about 20% endothelial cells, and about 20% cardiac fibroblasts. In particular aspects, the co-culture comprises about 75% cardiomyocytes, about 15% endothelial cells, and about 15% cardiac fibroblasts. In some aspects, the co-culture is further defined as a microtissue. In some aspects, the microtissue is in a microwell plate. In certain aspects, the microtissue does not include a scaffold. In some aspects, the cardiomyocytes, endothelial cells, and cardiac fibroblasts are isogenic. In certain aspects, the co-culture exhibits an inotropic response to the beta-adrenergic agonist isoproterenol.

[0047] Another embodiment provides a method of screening a test compound comprising introducing the test compound into a cardiac fibroblast population of this embodiment or aspect thereof. In some aspects, the method further comprises measuring cardiac fibroblast viability, cardiotoxicity, and / or cardiomyocyte function.

[0048] Further provided herein is a kit comprising the cardiac fibroblast population of this embodiment or aspect thereof. In some aspects, the kit further comprises endothelial cells and / or cardiomyocytes. Also provided herein is the use of the kit of this embodiment for disease modeling or disease screening.

[0049] 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 and specific examples, while indicating preferred embodiments of the present 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. [Brief explanation of the drawings]

[0050] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present 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.

[0051] [Figure 1] Schematic diagram representing the cardiac fibroblast protocol.

[0052] [Figure 2A-2B](Figure 2A) Schematic diagram showing cardiac progenitor cell induction using the ABC method (activin A, BMP4, and CHIR) versus CHIR alone (GSK3 inhibitor CHIR99021). (Figure 2B) Activin A and BMP4 in the mesoderm reduce the percentage of CXCR4+ progenitors compared to CHIR alone. CHIR alone was supplemented with 7 μM Chir99021. In the ABC condition, 9 ng / mL activin A, 5 ng / mL BMP4, and 7 μM CHIR were added on day 1 of differentiation, and only activin and BMP were added on day 2.

[0053] [Figures 3A-3E] (Figure 3A) Schematic of pathways for fibroblast progenitor cell induction: single CHIR, ABC, and dual CHIR. (Figure 3B) Dose-dependence of single CHIR epicardial cells. Increasing CHIR does not increase the percentage of WT1+ epicardial (Wilms tumor suppressor protein % (WT1%))-positive cells. (Figure 3C) Dose-dependence of single CHIR epicardial cells. Epicardial induction by single CHIR or the ABC step produces a mixed population of epicardial cells and cardiomyocytes. The percentage of WT-1+ epicardial cells ranges from 30% to 90% WT-1, and these cells do not proliferate highly. (Figure 3D) Epicardial morphology of single CHIR-treated cardiac progenitor cells. Single CHIR epicardial cells have a flat cobblestone morphology and prominent round, large nuclei (right). Addition of activin and BMP to CHIR results in cells retaining the flat cobblestone morphology, but the distinct cell-cell boundaries are less prominent (center). Notably, the increased proliferation of epicardial progenitor cells after the second CHIR treatment is reflected by a reduction in cell size (right) and a more elongated morphology. (Figure 3E) Quantitative real-time PCR analysis of action potentials in cardiac fibroblasts generated by the dual-CHIR and single-CHIR methods. Cardiac fibroblasts were differentiated by the two methods. Cardiac fibroblasts from each method were treated with 20 ng / mL TGFβ for 4 days. TGFβ activation increased the expression of POSTN, DDR2, COL1A1, COL1A2, GJA1, and ACTA2 in single-CHIR cardiac fibroblasts and also in double-CHIR epicardium-derived cardiac fibroblasts. Notably, the basal level of ACTA2 expression in the single-CHIR protocol was lower than that in the dual-CHIR protocol.

[0054] [Figures 4A-4H](Figure 4A) Schematic diagram of the cardiac fibroblast induction method without TGFβ inhibitors (SB43152 is shown with a dark bar). (Figure 4B) Percentage of CD29-positive cells at various concentrations of basic FGF during cardiac fibroblast differentiation in serum. Basic FGF can be used for cardiac fibroblast differentiation in serum. Integrin β-1 (ITGB1), also known as CD29, is a cell surface receptor encoded by the ITGB1 gene in humans. Increased CD29 expression was observed in cardiac fibroblasts differentiated using this method. The integrin β-1D isoform of CD29 is specifically expressed in cardiac and skeletal muscle. (Figure 4C) Purity of cardiac fibroblasts generated from ABC progenitors in serum-containing medium. Seven independent iPSC lines were differentiated into cardiac fibroblasts using ABC during mesoderm induction and cardiac fibroblast specification in serum-containing medium with a high concentration of basic FGF. All cardiac fibroblasts were highly fibroblast-specific (TE-7) and quiescent (aSMA) with <5% alpha-smooth muscle actin purity. (Figure 4D) Purity of cardiac fibroblasts from ABC progenitor cells induced to cardiac fibroblasts under serum-free conditions. Cardiac fibroblasts were induced from epicardial cells by culturing for five passages in serum-free medium without TGFβ inhibitors. The TE-7 and a-SMA purity of the resulting cardiac fibroblasts are shown. Cardiac fibroblasts induced in serum-free medium were highly TE-7 pure (>85%) and quiescent (aSMA <5%). A volcano plot comparison of gene expression of cardiac fibroblasts generated from both methods is shown on the right. (Figure 4E) iPSC-activated cardiac fibroblasts. Fibronectin secretion is shown as μg / mL per 106 cells. iPSC-derived cardiac fibroblasts were seeded at 104 cells / cm2 on VTN-coated plates. On day 2, TGFβ was added at the indicated concentrations to activate cardiac fibroblasts, and the medium was changed on day 4. On day 5, the medium supernatant was collected and analyzed for fibronectin secretion by human FN ELISA Kit Cat #BMS2028. On days 3 and 5, cardiac fibroblasts were harvested and counted in ViCellXR. Fibronectin secretion is shown as μg / mL per 106 cells.After 5 days, cells were harvested for flow cytometry. Alpha-smooth muscle actin populations emerge in the presence of TGFβ (right). (Figure 4F) Gene expression of iPSC cardiac fibroblasts by quantitative real-time PCR (qRT-PCR). iPSC cardiac fibroblasts express more GATA4, TBX20, NkX2-5, TBX18, and TCF21 than primary dermal fibroblasts. iPSC cardiac fibroblasts express collagen and connexin-43 at levels similar to primary cardiac fibroblasts. Two lots of iPSC-CFs were compared with primary human cardiac fibroblasts, primary human dermal fibroblasts, and primary human smooth muscle cells as controls. Notably, expression of activation-related genes aSMA (ACTA2), POSTN, and GJA1 was lower in iPSC-CFs compared to primary hCFs. (Figure 4G) Heatmap of bulk RNA-seq data comparing iPSC-CF (iCFB) lots with published datasets from adult primary cardiac fibroblasts (aCFs), fetal primary cardiac fibroblasts (fCFs), and iPSC-derived cardiomyocytes for characteristic genes. (Figure 4H) Population distribution of various gene expression in iPSC-derived cardiac fibroblasts by single-cell RNA-sequencing. Nearly all iPSC-CFs express characteristic fibroblast markers (VIM, FN1, COL1A1, SPARC). Within the pure TE-7 population, there are subpopulations of CFs that express various lineage transcription factors, including TBX3, TWIST1, GATA4, TIE-2 (TEK), SNA1I, and WT1. Only 2% of cells express the canonical epicardial marker Tcf21.

[0055] [Figures 5A-5E](Figure 5A) Schematic of the cardiac tri-culture microtissue assay. Cryopreserved iPSC-derived cell types are combined at specific ratios and formed into microtissues in microwell plates. The microtissues are supplied with co-culture medium to support all three cell types. 14 days after construction, the microtissues respond to exposure to inotropic compounds. (Figure 5B) Time-lapse 10x phase-contrast images of microtissues containing a total of 5,000 cells collected in an Incucyte S3 plate in an S-bio plate. (Figures 5C-5D) At DIV14, the diameters of the tri-culture microtissues were quantified and compared between microtissues containing different cell numbers. (Figure 5E) A tri-culture cardiac microtissue at DIV14 consisting of a total of 10,000 cells, including 15% iPSC-derived cardiac fibroblasts labeled with the viability dye Acridine orange to label viable cells in green.

[0056] [Figures 6A-6B] (Figure 6A) Isogenic triple-coculture cardiac microtissues exhibit inotropic responses to positive inotropes and the beta-adrenergic agonist isoproterenol across iPSC-derived cardiac fibroblast clones, multiple cardiac fibroblasts, and donor strains. This response occurred in triple-coculture microtissues from all three donor strains, two independently generated cardiac fibroblast lots, and microtissues containing cardiac fibroblasts from two independently derived clones. (Figure 6B) Inotropic responses increased with increasing proportions of cardiac fibroblasts in triple-coculture microtissues. At DIV14, responses to dobutamine (DOB), digoxin (DIG), isoproterenol (ISO), and epinephrine (EPI) were assessed in cardiac triple-coculture microtissues consisting of 10,000 total cells, 20% iPSC-derived endothelial cells, iPSC-derived cardiomyocytes, and varying concentrations of iPSC-derived cardiac fibroblasts (CF). Increasing CF content in cardiac 3D microtissues resulted in increased calcium amplitude inotropic responses to dobutamine, digoxin, and epinephrine.

[0057] [Figure 7] Screening of compound libraries using cardiac tri-species co-culture microtissues derived from isogenic cell types. Cardiac tri-species co-culture microtissues are highly suitable for high-throughput screening for cardiovascular drug discovery. Small numbers of cells can be used in multi-well plates (e.g., 96, 384, 1536) in a number of high-throughput assay platforms.

[0058] [Figure 8A-8B] (Figure 8A) Isogenic cardiac microtissues prepared from pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells harboring a mutation in the lamin (LMNA) gene reveal that non-cardiomyocytes exhibit disease phenotypic characteristics and contribute to the cardiomyocyte phenotype in vitro. Three-species coculture microtissues were generated from healthy control (NHC) and isogenic LMNA-L35P iPSC-derived cell types as previously described. 14 days after microtissue formation, the microtissues were exposed to isoproterenol. Healthy donor iPSC-derived microtissues responded to 100 nM isoproterenol with an increase in calcium transient amplitude. Disease model microtissues did not respond to the increase in isoproterenol. Immunofluorescence of LMNAL35P iPSC-derived cardiomyocytes reveals altered nuclear lamin, as predicted by the genotype. (Figure 8B) In vitro models, such as cardiac three-species coculture microtissues, can also be used to understand the impact of environmental stressors on inherited cardiovascular disease. A significant effect of hypoxia was observed on the beating rate of isogenic LMNA l35P microtissues. Triple coculture microtissues were generated from healthy control (NHC); isogenic LMNA-corrected, and isogenic LMNA-L35P iPSC-derived cell types. After cells were beating, they were placed in hypoxic (5% O2) or normoxic (20% O2) conditions. (Left) Healthy control microtissues and isogenic LMNA mutation-corrected microtissues exhibited similar beating rates in normoxic and hypoxic conditions, whereas LMNA mutant triple coculture MTs exhibited significantly slower beating rates in hypoxic conditions. (Right) Example of a DIV14 Ca2+ transient. DETAILED DESCRIPTION OF THE INVENTION

[0059] Description of exemplary embodiments In certain aspects, the present disclosure provides methods for the production of WT1 cells, such as epicardial cells, by adding serum-containing or serum-free medium in the presence of basic FGF. + A method for producing a highly purified population of cryopreserved quiescent cardiac fibroblasts from a population of cardiac progenitor cells is provided ( FIG. 1 ). Further provided herein is a method for producing a mixed population of WT1+ cardiac progenitor cells, such as cardiomyocytes and epicardial cells. In some embodiments, cardiac progenitor cells are oligopotent and can differentiate into multiple cardiac cell types.

[0060] In some embodiments, the starting population of iPSCs can be cultured under either hypoxic or normoxic conditions before differentiation into cardiac fibroblasts. To differentiate iPSCs into cardiac progenitor cells, the starting iPSCs can be formed into aggregates with a low concentration of a GSK3 inhibitor in E8 medium containing a ROCK inhibitor. To initiate differentiation, on day 1, the iPSCs can be treated with high levels of a GSK3 inhibitor in the presence of activin A and bone morphogenetic protein 4 (BMP4) growth factor for one day. Activin A and BMP4 treatment is continued for another day, for a total of two days.

[0061] In this study, we observed a decrease in the expression of the cardiac progenitor marker CXCR4 when activin and BMP4 (ABC, Figure 2A) were added to the mesoderm induction medium compared to GSK3 inhibition alone (Figure 2B). On day 3, cardiac progenitor cells were plated and treated with low levels of Wnt inhibitors. On day 5, no additional Wnt modulation was performed (Figure 3A), and a mixture of epicardial cells and cardiomyocytes was generated (Figure 3B). TGFβ inhibitors were then added to support epicardial cells. Notably, without secondary GSK3 inhibition, we found that epicardial progenitor proliferation was reduced (Figure 3C). By day 10, a mixed population of epicardial progenitor cells (WT1+) and cardiomyocytes (cTNT+) was generated. During long-term culture of epicardial progenitor cells from day 10 to day 25, the cardiomyocyte population decreased, and this cardiomyocyte population was subsequently eliminated after cardiac fibroblast induction and passaging.

[0062] In some embodiments, this method comprises the assemblen broid approach, which uses three separate differentiations to produce three separate pure cell populations, and then combines them.The assemblen broid approach allows the tightly controlled and reproducible control of cell composition.Alternatively, the organoid approach can be used, in which different cell types are co-differentiated, and for example, produce a mixed population of cardiomyocytes and epicardial cells.

[0063] Two methods for cardiac fibroblast induction were developed (Figure 4A). Cardiac fibroblast induction was initiated by removing TGFβ inhibitors. For serum-containing cardiac fibroblast induction, day 25 epicardial cells were transferred to DMEM low-glucose medium containing ascorbic acid and basic FGF, 10% serum. Cardiac fibroblasts were passaged when they reached 80% confluence and a TE-7 purity of greater than 75% within P3–P5. Increased purity of cardiac fibroblasts by TE-7 and CD-29 was observed with increasing basic FGF (Figure 4B). These cardiac fibroblasts were quiescent (e.g., <5% αSMA) and expressed genes and proteins characteristic of cardiac fibroblasts (Figure 4C).

[0064] For cardiac therapeutic applications, an alternative serum-free cardiac fibroblast induction protocol has also been developed. In some embodiments, epicardial cells were induced to develop into cardiac fibroblasts as early as day 17 by culturing in serum-free medium containing VEGF, EGF, FGF, and / or IGF. In some embodiments, the medium does not contain VEGF. Within five passages, a pure population of quiescent cardiac fibroblasts with a similar gene expression profile (e.g., 85% TE-7 and less than 5% aSMA) was achieved (Figure 4D). Cardiac fibroblasts from serum-containing and serum-free cultures were activated by treatment with TGFβ, and increased fibronectin secretion and α-smooth muscle actin expression were observed (Figure 4E). Cardiac fibroblasts were cryopreserved between P5 and P15 and used for microtissue assays.

[0065] Accordingly, further provided herein are isogenic microtissue compositions constructed from cryopreserved purified cell populations derived from PSCs, including cardiomyocytes, cardiac fibroblasts, and endothelial cells that exhibit a "mature" inotropic response.

[0066] Isogenic tri-species co-culture microtissues can be created from cryopreserved cardiomyocytes, endothelial cells, and cardiac fibroblasts derived from apparently healthy isogenic iPSCs (Figure 5A). Tri-species co-culture isogenic microtissues can be formed with a total of 500–20,000 cells (Figure 5B). Half-medium changes can be performed every other day, and the cultures continued in co-culture medium for 14–21 days (Table 8). Microtissues contract spontaneously within 2–4 days. Microtissues formed with a total of 5,000 cells can grow in size from an average diameter of 300–450 µm over 14 days. Microtissues can be assayed for their response to isoproterenol after 14 days of co-culture.

[0067] In this study, unlike microtissues from PSC-derived cardiomyocytes alone, isogenic triple coculture microtissues showed increases in both beating rate and amplitude due to calcium transients in the presence of isoproterenol (inotropic response, Figure 6A). The magnitude of the inotropic response increased with increasing proportion of cardiac fibroblasts incorporated into the microtissues (Figure 6B).

[0068] Further provided herein is a screening kit containing the present PSC-derived cardiac fibroblasts and microtissues for cardiotoxicity screening, which can contain all three cell types derived from iPSCs of apparently healthy donors used to screen compound libraries (Figure 7A).

[0069] Also provided herein are kits for isogenic disease modeling or screening for rare diseases comprising the present PSC-derived cardiac fibroblasts and microtissues, which may include all three cell types derived from iPSCs carrying a genetic disease genotype or derived from iPSCs engineered with a disease-associated mutation (Figure 7B).

[0070] Further embodiments provide methods for applying cardiac three-species co-culture assays involving three stem cell-derived cell types, namely cardiomyocytes, endothelial cells, and fibroblasts, to in vitro disease modeling, drug discovery, and toxicity testing.

[0071] Additionally, the present disclosure provides therapeutic methods that involve administering the cardiac fibroblasts or cardiac fibroblast-derived materials (i.e., matrix, vesicles, and / or secreted factors) provided herein. The cells can be delivered by direct injection or as part of a cardiomyocyte and cardiac fibroblast patch via transcardial, intramyocardial catheter delivery. The cardiac fibroblasts of the present disclosure can be produced from HLA-matched iPSCs for compatibility with the subject being treated. Current methods, including the use of all described materials and culture formats, are amenable to cGMP manufacturing. Thus, the present disclosure provides a robust, reproducible, and suitable cell source that will advance drug discovery and cardiac regenerative medicine.

[0072] II. Definition As used herein, "essentially free" with respect to a particular component is used herein to mean that none of the particular components are intentionally formulated into the composition and / or that the particular component is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any unintentional contamination of the composition is well below 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the specified component is undetectable by standard analytical methods.

[0073] As used herein, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0074] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer to alternatives only or where the alternatives are mutually exclusive, but the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" may mean at least a second or more.

[0075] Throughout this specification, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method employed to determine the value, or the variation that exists among study subjects.

[0076] The term "exogenous," when used with reference to a protein, gene, nucleic acid, or polynucleotide within 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 reference to a cell, the term refers to a cell that has been isolated and then introduced into another cell or organism by artificial or natural means. An exogenous nucleic acid can be from a different organism or cell, or it can be one or more additional copies of a nucleic acid that naturally occurs in an organism or cell. An exogenous cell can be from a different organism or it can be from the same organism. As a non-limiting example, an exogenous nucleic acid is a nucleic acid that is in a different chromosomal location than in a native cell, or that is adjacent to a different nucleic acid sequence than that found in nature.

[0077] "Expression construct" or "expression cassette" refers to a nucleic acid molecule capable of directing transcription. An expression construct contains one or more transcriptional control elements (e.g., promoters, enhancers, or functional equivalents thereof) that direct gene expression in at least one or more desired cell types, tissues, or organs. Additional elements, such as transcription termination signals, may also be included.

[0078] A "vector" or "construct" (sometimes called a gene delivery system or gene transfer "vehicle") refers to a macromolecule or molecular complex comprising a polynucleotide that is delivered to a host cell either in vitro or in vivo.

[0079] A "plasmid," a common type of vector, is an extrachromosomal DNA molecule that can replicate independently of chromosomal DNA and is sometimes circular and double-stranded.

[0080] The term "cell" is used herein in its broadest sense in the art and refers to a structural unit of tissue in a multicellular organism, surrounded by a membrane structure that isolates it from the outside, capable of self-replication, and containing genetic information and a mechanism for expressing it. As used herein, a cell may be a naturally occurring cell or an artificially modified cell (e.g., a fused cell, a genetically modified cell, etc.).

[0081] The term "stem cell" as used herein refers to a cell that can differentiate into a diverse range of specialized cell types under appropriate conditions and can self-renew and maintain an essentially undifferentiated, pluripotent state under other appropriate conditions. 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 generated from somatic cells by reprogramming them to a pluripotent state through the expression of specific transcription factors associated with pluripotency; these cells are referred to as "induced pluripotent stem cells" or "iPSc or iPS cells."

[0082] "Embryonic stem (ES) cells" are undifferentiated pluripotent cells obtained from an earlier stage embryo, such as the inner cell mass of the blastocyst stage, or created by artificial means (e.g., nuclear transfer), that can give rise to any differentiated cell type in the embryo, including germ cells (e.g., sperm or eggs), or in the adult.

[0083] "Induced pluripotent stem cells (iPSc or iPS cells)" are cells produced by reprogramming somatic cells by expressing or inducing the expression of a combination of factors (hereinafter referred to as reprogramming factors). iPS cells can be produced 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 (also called Oct3 / 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 to reprogram somatic cells into pluripotent stem cells.

[0084] "Pluripotent stem cells" refer to stem cells that have the potential to differentiate into all of the cells that make up one or more tissues or organs, or preferably, stem cells that have the potential to differentiate into 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).

[0085] As used herein, the term "somatic cell" refers to any cell other than a germ cell, such as an egg or sperm, that does not directly transfer its DNA to the next generation. Typically, somatic cells have limited or no pluripotency. As used herein, somatic cells may be naturally occurring or genetically modified.

[0086] "Programming" is the process of changing the type of progeny a cell can produce. For example, a cell is programmed if it has been modified so that it can form at least one new cell type progeny, either in culture or in vivo, compared to the cell that would have formed under the same conditions without programming. This means that after sufficient proliferation, when essentially no such progeny could have formed before programming, a measurable proportion of progeny is observed that have phenotypic characteristics of the new cell type; or, alternatively, the proportion that has the characteristics of the new cell type is measurably greater than before programming. This process includes differentiation, dedifferentiation, and transdifferentiation.

[0087] "Reprogramming" is a process that endows a cell with a measurable increase in the ability to form at least one new cell-type progeny, either in culture or in vivo, which it will subsequently possess under the same conditions without reprogramming. More specifically, reprogramming is the process that confers pluripotency to somatic cells. This means that after sufficient proliferation, if essentially no such progeny could be formed before reprogramming, a measurable proportion of progeny will be observed that have phenotypic characteristics of the new cell type; otherwise, the proportion that has characteristics of the new cell type will be measurably greater than before reprogramming.

[0088] "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 into another differentiated cell type. Typically, transdifferentiation by programming occurs without the cell passing through an intermediate pluripotent stage, i.e., the cell is directly programmed from one differentiated cell type to another. Under certain conditions, the proportion of progeny with characteristics of the new cell type can be at least about 1%, 5%, or 25% or more, in order of increasing preference.

[0089] The term "forward programming" refers to the programming of a multipotent or pluripotent cell, as opposed to a differentiated somatic cell that does not possess pluripotency, by providing the 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.

[0090] As used herein, the term "subject" or "subject in need thereof" 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 because of a disorder, or pathological or undesirable symptom, condition, syndrome, or physical, morphological, or physiological abnormality amenable to treatment with cell or tissue transplantation (also referred to herein as a recipient).

[0091] "Survival agents" refer to agents that promote and / or support cell survival when added to cell culture media. For example, Rho-associated kinase (ROCK) inhibitors or myosin II-specific inhibitors can be used as survival agents. In certain embodiments, these survival agents promote cell aggregation in culture.

[0092] "Rho-associated kinase inhibitors," abbreviated as "ROCK inhibitors," refer to any substance, such as a small molecule, siRNA, miRNA, or antisense RNA, that inhibits or reduces the function of Rho-associated kinase or its signaling pathway in cells. As used herein, "ROCK signaling pathway" may include any signal processor involved in a ROCK-associated signaling pathway, such as the Rho-ROCK-myosin II signaling pathway, its upstream signaling pathway, or its downstream signaling pathway in cells. 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.

[0093] "Cardiac fibroblasts" refers to cardiac fibroblasts, which play an important role in cardiac development, cardiac contractility, and response to disease. There are no specific markers for cardiac fibroblasts; cardiac fibroblasts express a combination of fibroblast- and cardiac-related genes. Cardiac fibroblasts contribute to the production and organization of the extracellular matrix by secreting proteins such as collagen (Col1A1), fibronectin (FN1), periostin (POSTN), and the ECM regulatory proteins matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). The extracellular matrix produced by cardiac fibroblasts serves to organize and support cardiac myocytes during development. Cardiac fibroblasts also secrete cytokines, including growth factors such as interleukin-6 (IL-6), transforming growth factor beta (TGF-B), and connective tissue growth factor (CTGF), in response to injury or disease. Cardiac fibroblasts can be derived from several developmental lineages characterized by the expression of distinct transcription factors, including cardiac progenitor (TBX20+ GATA4+), epicardial (WT1+, TBX18+, TCF21+), neural crest (PAX3+), and endothelial (TIE2+).

[0094] "Stem cell-derived cardiac fibroblasts" refer to cells whose differentiation fate has been engineered to lead to cardiac fibroblasts. Stem cell-derived cardiac fibroblasts may express markers characteristic of fibroblasts, including extracellular matrix proteins, including collagen I, collagen III, fibronectin, and matrix metalloproteinases (MMP2, MMP14). Cardiac fibroblasts may also express transcription factors reflective of their developmental origin. Importantly, cardiac fibroblasts can express various ion channels, including potassium channels and transient receptor potential (TRP) channels, which enable electrical coupling and calcium binding to cardiomyocytes. Cardiac fibroblasts also express connexins, such as connexin 43 (GJA1), which enable electrical coupling of cardiac fibroblasts to cardiomyocytes.

[0095] "Epicardial progenitor cells" refer to multipotent progenitor cells in the outer layer of the heart that give rise to cardiac fibroblast, smooth muscle, and endothelial lineages. Pluripotent stem cell-derived epicardial progenitor cells are derived from cardiac progenitor cells. Epicardial progenitor cells can be cryopreserved. When plated in FGF-containing medium, these cells preferentially differentiate into cardiac fibroblasts that express TE-7, CD29, and CD90. Exemplary epicardial progenitor cell markers include WT-1, TBX18, Tcf21, ALDH1A1, KRT8, KRT19, BNC1, UPK3B, ANAXA8, TJP1, and IGFBP6.

[0096] "Cardiomyocyte" or cardiac muscle cell refers to a muscle cell that constitutes cardiac muscle. Examples of cardiac-specific markers include α-sarcomeric actinin, troponin, myosin heavy chain, or L-type calcium current.

[0097] As used herein, "quiescent fibroblasts" are characterized by low expression of α-smooth muscle actin (αSMA), whereas activated myofibroblasts express high levels of α-smooth muscle actin (αSMA). Myofibroblasts exhibit a contractile phenotype and have an increased ability to produce ECM. In some embodiments, quiescent cardiac fibroblasts are fibroblasts that maintain a biologically relevant, non-activated state. Quiescent cardiac fibroblasts can secrete extracellular matrix and can transition to an activated myofibroblast state upon injury or exposure to profibrotic stimuli (e.g., TGFβ).

[0098] As used herein, "administering" shall mean delivering to have an effect or to produce an effect, using any of a variety of methods and delivery systems known to those skilled in the art. Administration may be, for example, intravenous, oral, via an implant, transmucosally, transdermally, intramuscularly, or subcutaneously. Topical administration is specifically contemplated. "Administering" may also be, for example, one time, multiple times, and / or over one or more extended periods of time.

[0099] "Super donors" are referred to herein as individuals who are homozygous for particular MHC class I and II genes. These homozygous individuals can serve as super donors, and their cells, including the tissues and other materials that make up 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.

[0100] The term "mature" inotropic response is used interchangeably with "physiologically" relevant inotropic response and inotropic response. Previous iPSC-derived cardiomyocytes in 2D culture under standard conditions exhibited immature or erroneous responses to inotropic substances. When exposed to positive inotropic compounds (e.g., isoproterenol, dobutamine), cardiomyocytes increase both contraction frequency and contraction strength, similar to adult human cardiomyocytes. In this embodiment, microtissues containing cardiomyocytes, endothelial cells, and cardiac fibroblasts were formed and cultured for 14 days, resulting in increased cardiomyocyte calcium transient amplitude not observed in 2D cultures, or in microtissues containing only cardiomyocytes.

[0101] "Assembloid" refers to a spheroid composed of purified pre-differentiated cell types, in contrast to an organoid, in which pluripotent stem cell lineages are co-differentiated together under conditions permissive for multiple cell types. In an assembloid, the developmental state and composition of cell types are tightly controlled.

[0102] "Willems' tumor 1+ (WT1+)" is a characteristic marker of the pro-epicardial cell lineage. Epicardial progenitor cells express WT1. WT-1+ is also expressed in the myocardium and has been found in cardiac endothelial cells of small capillaries and large coronary vessels in developing mice and humans.

[0103] As used herein, (CXCR4 low The term "CXCR4 low positive" refers to a population of cells in which less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, or less than 5%) are CXCR4 positive. In addition, the cells may have low signal intensity as measured by flow cytometry.

[0104] III. Pluripotent stem cells In certain embodiments of the present disclosure, methods and compositions are disclosed for providing epicardial progenitor cells and cardiac fibroblasts from pluripotent stem cells, which may be stem cells including, but not limited to, induced pluripotent stem cells and embryonic stem cells.

[0105] In certain aspects, pluripotent stem cells as used herein are human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), which are capable of long-term proliferation in vitro and retain the ability to differentiate into all cell types of the body, including the cardiac progenitor cells of the present disclosure. Thus, these cells potentially provide an unlimited supply of patient-specific functional cardiac progenitor cells for both drug discovery and therapeutic applications.

[0106] A. Embryonic stem cells In a specific embodiment, the pluripotent stem cells are embryonic stem cells (ESCs). ES cells are derived from the inner cell mass of blastocysts and have a high in vitro differentiation potential. ES cells can be dissociated by removing the outer trophectoderm layer of a developing embryo and culturing the inner cell mass on a feeder layer of non-growing cells. The replated cells continue to proliferate, producing new colonies of ES cells, which can be removed, isolated, replated again, and expanded. This process of "passaging" undifferentiated ES cells can be repeated multiple times to generate cell lines containing undifferentiated ES cells (U.S. Patent Nos. 5,843,780; 6,200,806; and 7,029,913). ES cells can be propagated while maintaining pluripotency. For example, ES cells are useful for studying cells and the genes that control cell differentiation. The combination of pluripotency of ES cells with genetic manipulation and selection can be used for in vivo genetic analysis studies through the generation of transgenic, chimeric, and knockout mice.

[0107] Methods for generating mouse ES cells are well known. In one method, preimplantation blastocysts from the 129 mouse strain are treated with mouse antisera to remove the trophectoderm and then cultured on a feeder cell layer of mouse embryonic fibroblasts whose inner cell mass has been chemically inactivated in a medium containing fetal bovine serum. Colonies of undifferentiated ES cells are then passaged on the mouse embryonic fibroblast feeder layer in the presence of fetal bovine serum to generate a population of ES cells. In some methods, mouse ES cells can be grown in the absence of a feeder layer by adding the cytokine leukemia inhibitory factor (LIF) to serum-containing 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).

[0108] Human ES cells can be generated or derived from fertilized eggs or blastocyst-stage mammalian embryos produced by sperm-egg fusion, nuclear transfer, or pathogenesis, or by reprogramming chromatin to generate embryonic cells using previously described methods (Thomson and Marshall, 1998; Reubinoff et al., 2000) and subsequent incorporation of the reprogrammed chromatin into the plasma membrane. In one method, human blastocysts are exposed to anti-human serum, and trophectoderm cells are removed from the inner cell mass, which are lysed and cultured on a feeder layer of mouse embryonic fibroblasts. Furthermore, clumps of cells derived from the inner cell mass are chemically or mechanically dissociated and replated, and colonies with undifferentiated morphology are selected with a micropipette, dissociated, and replated. In some methods, human ES cells can be grown without serum by culturing the ES cells 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 a “conditioned” medium containing basic fibroblast growth factors (Xu et al., 2001).

[0109] ES cells can also be derived from other organisms, including rhesus monkeys and marmosets, by previously described methods (Thomson and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000; U.S. Patent No. 5,843,780), and from established mouse and human cell lines. For example, established human ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14, and ACT30. As a further example, established mouse ES cell lines include the CGR8 cell line established from the inner cell mass of mouse strain 129 embryos; cultures of CGR8 cells can be grown in the presence of LIF without a feeder layer.

[0110] ES stem cells can be detected by protein markers including transcription factor Oct4, alkaline phosphatase (AP), developmental stage-specific embryonic antigen SSEA-1, developmental stage-specific embryonic antigen SSEA-3, developmental stage-specific embryonic antigen SSEA-4, transcription factor NANOG, tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), SOX2, or REX1.

[0111] 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 first achieved using mouse cells in 2006 (Yamanaka et al., 2006) and human cells in 2007 (Yu et al., 2007; Takahashi et al., 2007) by reprogramming somatic cells with 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 may not require lifelong immunosuppressive treatment to prevent graft rejection.

[0112] Any cell, except germ cells, can be used as the starting point for iPSCs. For example, cell types 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. Pat. No. 8,741,648; U.S. Patent Application Publication No. 2015 / 0191697). There are no limitations on the degree of differentiation of the cells or the age of the animal from which the cells are obtained; undifferentiated progenitor cells (including somatic stem 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 can express human ES cell markers, including SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

[0113] Somatic cells can be reprogrammed to generate iPS cells by using methods known to those skilled in the art.Those skilled in the art can easily generate iPS cells.For example, see US Patent Application Publication No. 2009 / 0246875, US Patent Application Publication No. 2010 / 0210014, US Patent Application Publication No. 2012 / 0276636, US Patent No. 8,058,065, US Patent No. 8,129,187, PCT International Publication No. 2007 / 069666 A1, US Patent No. 8,268,620, US Patent No. 8,546,140, ​​US Patent No. 9,175,268, US Patent No. 8,741,648, US Patent Application Publication No. 2011 / 0104125 and US Patent No. 8,691,574, which are incorporated herein by reference. Generally, nuclear reprogramming factors are used to generate 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.

[0114] The mouse and human cDNA sequences for these nuclear reprogramming agents are available by reference to the NCBI accession numbers set forth in WO 2007 / 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 disclosed, for example, in U.S. Patent Nos. 8,268,620, 8,691,574, 8,741,648, 8,546,140, ​​published U.S. Patent Nos. 8,900,871 and 8,071,369, both of which are incorporated herein by reference.

[0115] Once obtained, iPSCs can be cultured in a medium sufficient to maintain pluripotency. iPSCs can be used with a variety of 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. Patent Application Publication No. 2003 / 0211603. Mouse cells are cultured in a standard medium supplemented with leukemia inhibitory factor (LIF) 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 known to those skilled in the art can be used in conjunction with the methods disclosed herein.

[0116] In certain embodiments, non-limiting conditions can be used; for example, pluripotent cells can be cultured on fibroblast feeder cells or in 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 arrest 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).

[0117] Plasmids are designed with several goals in mind: achieving a controlled high copy number to avoid potential sources of bacterial plasmid instability and providing a means of plasmid selection compatible with use in mammalian cells, including human cells. In particular, two requirements for plasmids for use in human cells have been noted. First, the plasmid must be suitable for maintenance and fermentation in Escherichia coli (E. coli), allowing large amounts of DNA to be produced and purified. Second, the plasmid must be safe and suitable for use in human patients and animals. The first requirement is a high-copy-number plasmid that can be relatively easily selected and stably maintained during bacterial fermentation. The second requirement is attention to elements such as selectable markers and other coding sequences. In some embodiments, the marker-encoding plasmid is comprised of: (1) a high copy number origin of replication, (2) a selectable marker, such as, but not limited to, a neo gene for antibiotic selection with kanamycin, (3) a transcription termination sequence including a tyrosinase enhancer, and (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 contain a tyrosinase enhancer or promoter. Numerous plasmid vectors are known in the art for inducing nucleic acids encoding proteins. Plasmid vectors include, but are not limited to, those disclosed in U.S. Pat. Nos. 6,103,470; 7,598,364; 7,989,425; and 6,416,998, and U.S. patent application Ser. No. 12 / 478,154, which are incorporated herein by reference.

[0118] Episomal gene transfer systems can be plasmids, Epstein-Barr virus (EBV)-based episomal vectors (U.S. Patent No. 8,546,140), yeast-based vectors, adenovirus-based vectors, Simian virus 40 (SV40)-based episomal vectors, bovine papillomavirus (BPV)-based vectors, or lentiviral vectors. Viral gene delivery systems can be RNA-based or DNA-based viral vectors (International Application Nos. PCT / JP2009 / 062911 and PCT / JP2011 / 069588).

[0119] C. Embryonic stem cells obtained by somatic cell nuclear transfer Pluripotent stem cells for generating cardiac cells can also be prepared by somatic cell nuclear transfer, in which a donor nucleus is transplanted into a spindle-free oocyte. Stem cells generated by nuclear transfer are genetically identical to the donor nucleus. In one method, donor fibroblast nuclei from rhesus monkey skin fibroblasts are introduced into the cytoplasm of spindle-free mature metaphase II rhesus monkey oocytes by electrofusion (Byrne et al., 2007). The fused eggs are activated by exposure to ionomycin and then incubated until the blastocyst stage. The inner cell mass of selected blastocysts is then cultured to generate embryonic stem cell lines. Embryonic stem cell lines exhibit normal ES cell morphology, express various ES cell markers, and differentiate into multiple cell types both in vitro and in vivo.

[0120] D. MHC haplotype matching The major histocompatibility complex (MHC) is the primary cause of immune rejection in allogeneic organ transplants. There are three major MHC class I haplotypes (A, B, and C) and three major MHC class II haplotypes (DR, DP, and DQ). The HLA locus is highly polymorphic and distributed over a 4-Mb region on chromosome 6. The ability to haplotype HLA genes within this region is clinically important because this region is associated with autoimmune and infectious diseases, and HLA haplotype compatibility between donors and recipients can affect the clinical outcome of transplants. HLA corresponding to MHC class I presents peptides from the interior of cells, while HLA corresponding to MHC class II presents antigens from the exterior of cells to T lymphocytes. MHC haplotype mismatches between the graft and the host trigger an immune response against the graft, leading to its rejection. Therefore, patients can be treated with immunosuppressants to prevent rejection. HLA-matched stem cell lines may overcome the risk of immune rejection.

[0121] Because of the importance of HLA in transplantation, HLA loci are typically typed by serology and PCR to identify suitable donor-recipient pairs. Serological detection of HLA class I and II antigens can be achieved using a complement-mediated lymphocytotoxicity test with purified T or B lymphocytes. This procedure is primarily used to match HLA-A and HLA-B loci. Molecular-based tissue typing can often be more accurate than serological testing. Low-resolution molecular methods, such as the SSOP (sequence-specific oligonucleotide probe) method, in which PCR products are tested against a series of oligonucleotide probes, can identify HLA antigens; these methods are currently the most common method used for class II HLA typing. High-resolution techniques, such as the SSP (sequence-specific primer) method, which utilizes allele-specific primers for PCR amplification, can identify specific MHC alleles.

[0122] The MHC compatibility between the donor and the recipient is significantly increased when the donor cells are HLA homozygous, i.e., contain identical alleles for each antigen-presenting protein. Most individuals are heterozygous for MHC class I and II genes, but certain individuals are homozygous for these genes. These homozygous individuals can function as super donors, and grafts made from their cells can be transplanted into all individuals who are either homozygous or heterozygous for that haplotype. Furthermore, if the homozygous donor cells have a haplotype that is frequently found in the population, these cells can be applied to transplantation therapy for a large number of individuals.

[0123] Thus, in some embodiments, the iPSCs of the present method can be generated from the somatic cells of the subject to be treated or from the somatic cells of another subject with the same or substantially the same HLA type as the patient. In some cases, 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 derived from an MHC homozygous super donor can be used to generate cardiac fibroblasts. Cardiac fibroblasts derived from a super donor can thus be transplanted into a subject who is either homozygous or heterozygous for that haplotype. For example, cardiac fibroblasts can be homozygous for two HLA alleles, such as HLA-A and HLA-B. Thus, cardiac fibroblasts generated from a super donor can be used in the methods disclosed herein to generate cardiac fibroblasts that can potentially be "matched" to a large number of potential recipients.

[0124] Accordingly, certain embodiments of the present disclosure provide a repository (e.g., a library) of HLA homozygous cardiac fibroblasts. The HLA haplotypes represented in the subject library can reflect the most common HLA haplotypes found in the human population, such as common Caucasian HLA haplotypes, common HLA haplotypes found in individuals of African descent, common Asian HLA haplotypes, common Hispanic HLA haplotypes, common Native American HLA haplotypes, etc. For example, a single abundant haplotype may be present in a significant proportion of the population, and a single HLA homozygous cell line may serve as a histocompatible donor for a significant proportion of patients. The library may include one, two, three, four, five, six, seven, eight, nine, ten, ten to fifteen, fifteen to twenty, twenty to twenty-five, twenty to thirty, or more than thirty different types of HLA homozygous cells. The subject library can include first HLA homozygous cells that are homozygous for a first HLA haplotype; and at least a second HLA homozygous cell that is homozygous for a second HLA haplotype. The subject library can include a single cell type or two or more different cell types. The subject library can be cataloged, for example, in a searchable computer database, where information about HLA haplotypes, and optionally additional information such as cell surface markers and karyotype information, is stored and searchable.

[0125] The HLA homozygous cardiac fibroblasts described herein can find use in a wide range of clinical applications, including cell and / or tissue transplantation. The HLA homozygous cardiac fibroblasts are HLA compatible with the recipient and can therefore be introduced into the recipient without, or at least with a reduced need for, immunosuppressive therapy. Standard immunosuppressant drug regimens can cost thousands of dollars per month and can often cause unwanted side effects, including life-threatening infections and cancers that are expensive to treat. Thus, the present HLA homozygous cardiac fibroblasts overcome several obstacles that currently limit the use of human cells for clinical applications.

[0126] IV. Differentiation into cardiac fibroblasts Embodiments of the present disclosure relate to the differentiation of PSCs, particularly iPSCs, into cardiac progenitor cells, including cardiac mesodermal and epicardial cells, as well as cardiomyocytes and cardiac fibroblasts. The schematic in Figure 1A shows an exemplary differentiation process beginning with the use of iPSCs grown in vitronectin-coated vessels containing Essential 8 Medium prior to initiating differentiation, such as large-scale differentiation in a bioreactor.

[0127] A. Aggregate Formation In some embodiments, pluripotent stem cells are differentiated into cardiac fibroblasts by first inducing the formation of aggregates and then initiating differentiation with a Wnt agonist such as CHIR99021. In certain embodiments, differentiation is initiated upon aggregation, and the cells begin to recapitulate embryonic development to a limited extent. The cells cannot form trophectoderm tissue (including the placenta), but can give rise to almost all other cell types present in an organism. The present disclosure can further promote differentiation into the cardiac lineage after aggregate formation.

[0128] Various matrix components can be used for culturing pluripotent cells, including collagen (e.g., collagen IV), laminin, vitronectin, Matrigel™, gelatin, polylysine, thrombospondin (e.g., TSP-1, -2, -3, -4, and / or -5), fibronectin, and / or Pronectin-F™. Combinations of these matrix components may provide additional benefits for promoting cell proliferation and cell viability. In certain embodiments, one, two, three, four, five, six, or more of the above matrix components can be used for culturing cells. In some aspects, pluripotent cells are cultured on a vitronectin-coated surface.

[0129] Pluripotent cells may be allowed to form embryoid bodies or aggregates as part of the differentiation process. The formation of "embryoid bodies" (EBs) or clusters of proliferating cells to induce differentiation generally involves the in vitro aggregation of human pluripotent stem cells into EBs, allowing the spontaneous and random differentiation of human pluripotent stem cells into multiple tissue types representing endodermal, ectodermal, and mesodermal origins.

[0130] 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 stimulate 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 low concentration of about 2 μM. In certain embodiments, the method includes culturing the cells in the presence of about 2 μM (e.g., 1-3, 1-4, or 1-5 μM) of a Wnt agonist during aggregate formation, e.g., from day 0 to day 1, and then culturing in the presence of a higher concentration, e.g., about 7 μM (e.g., 5-10 μM), e.g., on day 1, for mesoderm induction.

[0131] [Table 1]

[0132] ROCK inhibitors can be used in the culture and passage of pluripotent stem cells and / or in stem cell differentiation. Thus, ROCK inhibitors can be present in any cell culture medium in which pluripotent stem cells proliferate, dissociate, form aggregates, or differentiate, for example, in adherent culture and suspension culture. Rho-specific inhibitors, such as botulinum C3 exoenzyme 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.

[0133] An exemplary ROCK-specific inhibitor is Y-27632, which selectively targets ROCK1 (but also inhibits ROCK2) and further inhibits TNF-α and IL-1β. ROCK-specific inhibitors are cell-permeable and inhibit ROCK1 / ROCK2 (IC) by competing with ATP. 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 1-10 μM, e.g., about 1 μM.

[0134] Other non-limiting examples of ROCK inhibitors include antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (e.g., siRNA), competitive peptides, antagonist peptides, inhibitory antibodies, antibody-ScFV fragments, dominant-negative mutants, and expression vectors thereof. Additionally, other small molecule compounds are known as ROCK inhibitors, and such compounds or derivatives thereof can also be used in embodiments (see, e.g., U.S. Patent Application Publication Nos. 20050209261, 20050192304, 20040014755, 20040002508, 20040002507, 20030125344, and 20030087919, and WO 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, and 2004 / 039796, which are incorporated herein by reference). Combinations of one or more ROCK inhibitors can also be used in the present methods.

[0135] According to some embodiments, PSCs can be treated with a ROCK inhibitor in culture medium. Thus, the culture medium used in the disclosed methods may already contain a ROCK inhibitor, or the disclosed methods 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 achieves the desired effect, such as improving stem cell viability. Such ROCK inhibitors, such as Y-27632, HA-1077, or H-1152, can be used at an effective concentration of at least 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 derivable therein. These amounts may refer to the amount of the ROCK inhibitor alone or in combination with one or more other ROCK inhibitors.

[0136] For example, when Y-27632 is used as a ROCK inhibitor, Y-27632 can be used at a concentration of about 0.01 to about 1000 μM, more specifically about 0.1 to about 100 μM, even more specifically about 1.0 to about 30 μM, and most specifically about 2.0 to 20 μM, or any range derivable therein. When Fasudil / HA1077 is used as a ROCK inhibitor, Fasudil / HA1077 can be used at a concentration about twice that of the aforementioned Y-27632. When H1152 is used as a ROCK inhibitor, H1152 can be used at about 1 / 50 of the aforementioned Y-27632 concentration.

[0137] The aggregate formation step is carried out for a period of time sufficient to induce the formation 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, or 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, or 48 hours, or any range derivable therein). In certain embodiments, a period of 1 to 3 days, e.g., about 1 day, is sufficient to induce the formation of cellular aggregates.

[0138] The density of stem cells treated with a ROCK inhibitor is not particularly limited as long as it is a density that can achieve the desired effect, such as improving the survival rate of stem cells. The density of stem cells 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 x 10 3 ~1.0×10 7 cells / ml, most specifically approximately 3.0 × 10 4 ~2.0×10 6 cells / ml.

[0139] In certain embodiments, PSCs are cultured in the presence of a ROCK inhibitor to improve viability, cloning efficiency, or passaging efficiency at low density (dissociated into single cells or small aggregates). In certain embodiments, PSCs are cultured in the absence of feeder cells, feeder cell extract, and / or serum. PSCs can be cultured in the presence of a ROCK inhibitor prior to subcloning or passaging, for example, at least 1 hour prior to subcloning or passaging. Alternatively, or additionally, PSCs are maintained in the presence of a ROCK inhibitor during or after subcloning or passaging.

[0140] Pluripotent stem cells can be seeded into aggregation-promoting medium using any method known in the art of cell culture. For example, pluripotent stem cells can be seeded into aggregation-promoting medium as single colonies or clonal groups, or they 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 connections between the cells and the culture surface and between the cells themselves. Enzymes that can be used to separate pluripotent stem cells for aggregate formation and differentiation can include, but are not limited to, trypsin, its various commercially available preparations, e.g., TrypLE, or a mixture of enzymes such as Accutase®.

[0141] In certain embodiments, pluripotent cells can be added or seeded into culture medium essentially as individual (or dispersed) cells for culture formation on a 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 compatible with standard sterile cell culture methods in the art, e.g., 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 applied to the culture surface before contacting the surface with the cells and medium.

[0142] B. Mesoderm induction Next, pluripotent stem cell aggregates, such as iPS cell aggregates, can be cultured in a medium to promote mesoderm induction. The aggregates can be contacted with a Wnt agonist, and optionally 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 or a different Wnt agonist from the Wnt agonist in the aggregate formation step. 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, e.g., 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 6-8 μM, for example, about 6, 6.5, 7, 7.5, or 8 μM, particularly about 7 μM.

[0143] Activin agonists are compounds that activate the activin / Nodal signaling pathway, for example, by binding to a TGFβ receptor or activin receptor. Examples of activin agonists include activin A, activin B, activin AB, TGFβ1, growth differentiation factor (GDF)-3, BML-284, and Nodal. In certain embodiments, the activin agonist is activin A at a concentration of, for example, about 5 to 20, e.g., 10 to 20 ng / mL, particularly about 12 ng / mL, and particularly about 9 ng / mL. For example, BMPs such as BMP4 can be used at a concentration of 0.1 ng / mL to 10 ng / mL, particularly about 5 ng / mL.

[0144] [Table 2]

[0145] In some embodiments, mesoderm induction involves a first step (e.g., for about 1 day) involving a Wnt agonist, an activin agonist, and a BMP, followed by a second step (e.g., for about 1-3 days, particularly about 2 days) involving an activin agonist and a BMP in the absence of a Wnt agonist.

[0146] [Table 3]

[0147] The basal medium for mesoderm induction can be any medium known in the art for culturing stem cells. Exemplary media include E8, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI 1640, and Fischer's Medium. In certain embodiments, the basal medium is RPMI supplemented with B27 without insulin. In certain embodiments, the medium is insulin-free or essentially insulin-free. In some embodiments, the mesoderm induction medium includes albumin.

[0148] The mesoderm induction step can be for a period sufficient to induce the loss of expression of mesoderm markers such as CXCR4, KDR, PDGFRα, and / or CD56, as well as 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 to 5 days, e.g., about 1, 2, 3, 4, or 5 days. In certain embodiments, the aggregates are cultured for about 2 to 3 days for mesoderm induction.

[0149] In certain embodiments, mesoderm induction in the presence of a Wnt agonist, an activin agonist, and a BMP produces a population comprising NCAM+CXCR4 low cells and NCAM+CXCR4+ cells. In some embodiments, the activin agonist and BMP result in a reduced proportion of CXCR4+ progenitor cells compared to cultures without the activin agonist and BMP (i.e., containing only a Wnt agonist).

[0150] C. Cardiac progenitor cells The mesodermal progenitor cells can then be induced into WT1+ progenitor cells (e.g., epicardial progenitor cells, endothelial progenitor cells, etc.) in the presence of a Wnt inhibitor, for example, for about 1 to 3 days, particularly about 2 days. In certain embodiments, cardiac (e.g., epicardial, endothelial, etc.) progenitor cell induction is in the absence of BMP4, a Wnt agonist (e.g., CHIR99021), GSK3 inhibition, and / or retinoic acid.

[0151] In some embodiments, aggregates at the mesoderm stage can be maintained in suspension culture, or mesoderm cells can be individualized and plated as monolayer cultures before the initiation of epicardial progenitor cell specification. In certain embodiments, aggregates are dissociated before culturing in the presence of a Wnt inhibitor. Dissociation of aggregates can be performed using any known procedure. These procedures include treatment with chelating agents (e.g., EDTA) or enzymes (e.g., trypsin, collagenase), and manipulations such as mechanical dissociation (e.g., pipetting). Cells can be cultured on matrices such as those described above, such as vitronectin-coated surfaces. In certain embodiments, the differentiation process can be serum-free, without the use of drug resistance or metabolic selection. Epicardial cells can be plated on vitronectin-coated plates.

[0152] 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 1-10 μM, e.g., about 1, 2, or 5 mM, particularly about 2 μM.

[0153] [Table 4]

[0154] To support WT1+ cardiac (e.g., epicardial) progenitor cells, the cells can be cultured in the presence of a TGFβ inhibitor, for example, for about 5 to 20 days, to generate a mixture of epicardial progenitor cells and cardiomyocytes. The medium can further contain ascorbic acid. In certain embodiments, the epicardial progenitor cells are cultured in the absence of BMP4, Wnt agonists (e.g., CHIR99021), GSK3 inhibition, and / or retinoic acid. In certain embodiments, the epicardial cells are cultured without Wnt modulation. In certain embodiments, the epicardial cells can be used directly for cardiac fibroblast induction without additional purification steps.

[0155] The TGFβ inhibitor may be SB431542, LDN-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 to 25 mM, e.g., about 5, 10, or 15 mM, particularly about 10 mM. The TGFβ inhibitor, such as SB431542, may be present at a concentration of about 1 to 10 μM, e.g., about 4, 5, or 6 μM, particularly about 5 μM.

[0156] [Table 5]

[0157] D. Cardiac Fibroblast Induction The WT1 cardiac (e.g., epicardial) progenitor cells can then be cultured in the absence of a TGFβ inhibitor for cardiac fibroblast induction. Cardiac fibroblast induction can include, for example, basic FGF (bFGF) at a concentration of 10 to 500 ng / mL, e.g., about 10, 25, 40, 50, 75, 100, 150, 200, 250, 300, 400, or 500 ng / mL. In certain embodiments, cardiac fibroblast induction is performed essentially in the absence of a TGFβ inhibitor.

[0158] In some embodiments, cardiac fibroblast induction is performed in the presence of serum, such as fetal bovine serum (e.g., about 5-15%, particularly about 10%). bFGF can be present at about 150-200 ng / mL. Cells are passaged about 3-5 times to purify cardiac fibroblasts, resulting in quiescent cardiac fibroblasts with greater than 75% TE-7 expression and less than 5% αSMA expression.

[0159] Integrin beta-1 (ITGB1), also known as CD29, is a cell surface receptor encoded by the ITGB1 gene in humans. Cardiac fibroblasts differentiated by this method can increase CD29 expression, such as at least 75% or 85% CD29 expression. The integrin beta-1D isoform of CD29 is specifically expressed in cardiac and skeletal muscles.

[0160] [Table 6]

[0161] In another embodiment, cardiac fibroblast induction is performed in the absence of serum. bFGF can be present at about 5 to 200 ng / mL, particularly about 40 ng / mL. The culture can further contain BSA, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), hydrocortisone, and heparin. The cells are passaged about 3 to 5 times to purify cardiac fibroblasts, resulting in quiescent cardiac fibroblasts with greater than 75% TE-7 expression and less than 5% α-SMA expression.

[0162] [Table 7]

[0163] Cardiac fibroblasts can be cryopreserved, such as at passages 5 to 15. Cryopreserved cardiac fibroblasts can be used for cardiac triple co-culture microtissues and other assays. Cardiac fibroblasts can be cultured in the presence of cardiomyocytes and endothelial cells. The co-culture medium can contain BSA, VEGF, bFGF, EGF, and / or IGF. In some embodiments, the medium does not contain VEGF.

[0164] [Table 8]

[0165] Cardiac fibroblasts can be activated in the presence of TGFβ, such as on vitronectin-coated plates, and can be characterized by measuring the secretion of fibronectin.

[0166] E. Cell culture conditions The culture conditions according to the present disclosure are appropriately defined 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 its basal medium. Examples of basal media that can be used include E8, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI 1640, and Fisher's medium, as well as any combination thereof. However, the medium is not particularly limited to these, as long as it can be used for culturing animal cells.

[0167] In certain embodiments, the medium according to the present disclosure is a serum-free medium. A serum-free medium refers to a medium that does not contain raw or unpurified serum and thus may include a medium containing purified blood-derived components or animal tissue-derived components (e.g., growth factors). The medium according to the present disclosure may or may not contain any substitute for serum. Serum substitutes may include substances that suitably contain albumin (e.g., lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Serum substitutes can be prepared, for example, by the methods disclosed in WO 98 / 30679. Alternatively, any commercially available substance can be used for greater convenience. Commercially available materials include Knockout Serum Replacer (KSR), Chemically Defined Fat Concentrate (Gibco), and Glutamax (Gibco).

[0168] The medium of the present disclosure may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffering agents, 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 appropriate for culturing stem cells.

[0169] Culture vessels used to culture stem cells include, but are not limited to, flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, tubes, trays, CellSTACK® chambers, culture bags, roller bottles, and bioreactors, such as PBS 500 and / or PBS 3, provided that the stem cells can be cultured therein. Stem cells can be cultured in 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 of volumes derivable therein, depending on the needs of the culture. In certain embodiments, the culture vessel can be a bioreactor, which can refer to any device or system that supports a biologically active environment. The bioreactor can have a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.

[0170] Culture vessels can be cell-adhesive or non-adhesive, and can be selected according to the purpose. Cell-adhesive culture vessels can be coated with any cell-adhesive substrate, such as an extracellular matrix (ECM), to improve cell adhesion to the vessel surface. The cell-adhesive substrate can be any material intended to adhere stem cells or feeder cells (if used). Cell-adhesive substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin, as well as mixtures thereof, such as Matrigel™, and dissolved cell membrane preparations (Klimanskaya et al., 2005).

[0171] Other culture conditions can also be defined appropriately. For example, the culture temperature can be about 30 to 40°C, for example, at least about 31, 32, 33, 34, 35, 36, 37, 38, or 39°C, but is not 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 about 1, 5, 8, 10, or 20%, or any range derivable therein.

[0172] The disclosed method can also be used for stem cell suspension culture, including suspension culture on a carrier (Fernandes et al., 2007) or gel / biopolymer encapsulation (US Patent Application Publication No. 20070116680). The term stem cell suspension culture refers to the culture of stem cells in a medium under non-adherent conditions on a culture vessel or feeder cells (if used). Stem cell suspension culture includes dissociated stem cell culture and aggregated stem cell suspension culture. The term dissociated stem cell culture refers to the culture of suspended stem cells, and includes the dissociated culture of a single stem cell or the dissociated culture of small cell aggregates consisting of multiple stem cells (e.g., approximately 2-400 cells). Continuing the dissociated culture allows the cultured dissociated cells to form larger stem cell aggregates, which can then be subjected to aggregate suspension culture. Aggregate suspension cultures include embryoid body culture methods (see Keller et al., 1995) and SFEB methods (Watanabe et al., 2005; U.S. Patent Application Publication No. 2005 / 123902). The methods disclosed herein can significantly improve the survival rate and / or differentiation efficiency of stem cells in suspension culture.

[0173] Bioreactors can be classified according to general classifications, including static bioreactors, stirred flask bioreactors, rotating wall vessel bioreactors, hollow fiber bioreactors, and direct perfusion bioreactors. Within the bioreactors, cells can be seeded, either free or immobilized, on a porous three-dimensional scaffold (hydrogel). In certain embodiments, the bioreactor is a suspension bioreactor for efficient mixing and low shear stress with homogenous particle suspensions.

[0174] The methods disclosed and utilized herein can be scaled to multiple (e.g., 3 L) bioreactor manufacturing batches using all GMP-compliant materials to achieve the purity and cell numbers required for the development of cardiac cell therapies.

[0175] F. Cardiac Fibroblast Characterization Cells obtained according to this method can be characterized according to a number of phenotypic criteria. Quiescent cardiac fibroblasts can be characterized by expression of the fibroblast markers TE-7, CD29, and / or CD90. Quiescent cardiac fibroblasts are specifically negative for α-smooth muscle actin by qPCR and flow cytometry. In some cases, there may be less than 5% α-smooth muscle actin-positive cells. Alpha-smooth muscle actin-positive cells may have a spindle morphology with multiple processes emanating from the cell body and a prominent round nucleus.

[0176] Cardiomyocytes and progenitor cells derived from pluripotent stem cell lines often have morphological characteristics of cardiomyocytes from other sources. Cardiomyocytes and progenitor cells can be spindle-shaped, round, triangular, or polygonal, and can exhibit striated sarcomeric structures detectable by immunostaining. Cardiomyocytes and progenitor cells can form flat sheets of cells or aggregates that remain attached to a substrate or float in suspension, and exhibit typical sarcomeres and atrial granules when examined by electron microscopy.

[0177] Pluripotent stem cell-derived cardiac fibroblasts and their precursors typically possess at least one of the following cardiomyocyte-specific markers: cardiac troponin I (cTnI), cardiac troponin T (cTnT), subunits of the troponin complex that provide a calcium-sensitive molecular switch for the regulation of striated muscle contraction; or Nkx2.5, a cardiac transcription factor expressed in the cardiac mesoderm during early mouse embryonic development and persisting in the developing heart. The cells also typically express at least one (and often at least three, five, or more) of the following markers: atrial natriuretic factor (ANF), myosin heavy chain (MHC), particularly the cardiac-specific β-chain, MLC, titin, tropomyosin, α-sarcomeric actinin, and desmin. ANF is a hormone expressed in the developing heart and fetal cardiomyocytes but downregulated in adults. ANF is considered an excellent marker for cardiomyocytes because it is highly specifically expressed in cardiomyocytes but not in skeletal muscle cells. Additional markers include MEF-2A, MEF-2B, MEF-2C, and MEF-2D (transcription factors expressed in cardiac mesoderm and persisting in the developing heart), N-cadherin, which mediates adhesion between cardiac cells, connexin 43, which forms gap junctions between cardiomyocytes, β1-adrenergic receptor (β1-AR), which is elevated in serum after myocardial infarction, creatine kinase MB (CK-MB), and myoglobin, α-cardiac actin, early growth response-I, cyclin D2, and GATA-4, transcription factors highly expressed in cardiac mesoderm and persisting in the developing heart. ANF regulates many cardiac genes and is involved in heart development.

[0178] In this study, pluripotent stem cell-derived cardiac fibroblasts expressed characteristic fibroblast markers such as vimentin (VIM), extracellular matrix proteins such as collagens (COL1A1 and COL1A2), and genes regulating procollagen processing collagen, such as secretory protein acidic and cysteine-rich (SPARC). Collagen 1A and fibronectin secretion were measured by ELISA under resting and TGFβ-activated conditions. Because cardiac fibroblasts play a role in maintaining the cardiac extracellular matrix, pluripotent stem cell-derived cardiac fibroblasts expressed matrix metalloproteinases such as MMPs (MMP1, MMP2, MMP14) and highly expressed tissue inhibitors of matrix metalloproteinases (TIMP1 and TIMP3). iPSC-derived cardiac fibroblasts generated by this method were over 85% THY-1 (CD90) positive by flow cytometry. Additional markers include MEF-2A, MEF-2C, MEF-2D (transcription factors expressed in cardiac mesoderm and persisting in the developing heart), N-cadherin (CDH2), which mediates adhesion between cardiac cells, and connexin 43 (GJA1), which is important for forming gap junctions between cardiomyocytes and cardiac fibroblasts throughout the heart.

[0179] Further investigation of this method demonstrated the heterogeneity of cardiac fibroblast populations through single-cell RNA sequencing. During embryonic development, cardiac fibroblasts can arise from up to four different sources: epicardial progenitors, endocardial / endothelial progenitors, neural crest progenitors, and secondary heart field progenitors. Cardiac fibroblasts possess the transcription factor codes of their developmental lineage. Single-cell data from cardiac fibroblasts generated by this AB method demonstrated a heterogeneous population. In these studies, bulk RNA sequencing demonstrated the expression of discoidin domain receptor 2 (DDR2) and periostin (POSTN) in a subset of our iPSC-derived cardiac fibroblasts. Approximately 50% of these cardiac fibroblasts expressed GATA-4, a transcription factor highly expressed in cardiac mesoderm and persisting in the developing heart. GATA-4 regulates many cardiac genes and is involved in heart development. Approximately 50% of cardiac fibroblasts were also shown to express the secondary heart field marker HAND2. We also observed that 33% of cardiac fibroblasts expressed the endocardial / endothelial fibroblast progenitor marker Tie2 (TEK). 10–20% of cardiac fibroblasts expressed WT1, SNAI1, TBX18, and TBX20, indicating their epicardial lineage origin. Expression of the cardiac transcription factor WT1 was observed at early passages, but WT1 expression decreased with passage. Fewer than 3% of cardiac fibroblasts generated by this method were positive for the neural crest marker Pax3. In addition, cardiac transcription factors Nkx2-5 and Isl1 were expressed in less than 2% of the cells. Furthermore, only less than 2% of cardiac fibroblasts expressed the characteristic quiescent cardiac fibroblast marker TCF21.

[0180] Interestingly, 66% of the cardiac fibroblast population was observed to express sinoatrial node-associated markers, including T-box transcription factor 3 (TBX3), and a subpopulation of these also expressed connexin 45 (GCA1), which connects cardiac fibroblasts with cardiomyocytes in the sinoatrial node.

[0181] In some embodiments, cardiac fibroblasts are cell surface marker positive for PDGFRa and PDGFRb, indicating that these cells may have pericyte morphology, constituting two subpopulations of cardiac fibroblasts.

[0182] Gene expression analysis and flow cytometry of cardiac fibroblasts for α-smooth muscle actin revealed a very small, <5% positive population. Total RNA sequencing showed that ACTA2 levels were comparable in adult primary cardiac fibroblasts, fetal primary cardiac fibroblasts, and iPSC-derived cardiac fibroblasts. TCF21 levels were lower in iPSC-derived cardiac fibroblasts than in their primary counterparts. Extracellular matrix protein levels were similar in all fibroblast cell types. Single-cell RNA sequencing revealed a 38% population of α-smooth muscle actin cells with expression levels greater than zero. As shown in the literature, there were two populations of ACTA2 cells. The high-expressing population clustered with activation markers and comprised 5-10% of the cells.

[0183] Tissue-specific markers can be detected using any appropriate immunological technique, such as flow immunocytometry or affinity adsorption for cell surface markers, immunocytochemistry (e.g., of fixed cells or tissue sections) for intracellular or cell surface markers, Western blot analysis of cell extracts, and enzyme-linked immunosorbent assays of cell extracts or cell products secreted into the medium. Antibodies that distinguish cardiac markers such as GATA4 and HAND2 or fibroblast markers such as vimentin, CD90, or DDR2 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 significantly detectable amount of antibody binds to the antigen in a standard immunocytochemistry or flow cytometry assay, optionally after fixation of the cells, using an optionally labeled secondary antibody.

[0184] Expression of tissue-specific gene products can also be detected at the mRNA level by Northern blot analysis, dot blot hybridization analysis, RNA sequencing (e.g., single cells), or by reverse transcriptase-initiated polymerase chain reaction (RT-PCR) using sequence-specific primers with standard amplification methods using publicly available sequence data (GenBank). Expression of a tissue-specific marker detected at the protein or mRNA level is considered positive if the level is at least or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold, particularly more than 10-fold, more than 20-fold, more than 30-fold, more than 40-fold, or more than 50-fold, of the level of control cells, such as undifferentiated pluripotent stem cells or other unrelated cell types.

[0185] Tissue-specific markers can be detected using any appropriate immunological technique, such as flow immunocytometry or affinity adsorption for cell surface markers, immunocytochemistry (e.g., of fixed cells or tissue sections) for intracellular or cell surface markers, Western blot analysis of cell extracts, and enzyme-linked immunosorbent assays of cell extracts or cell products secreted into the medium. Antibodies that distinguish fibroblast 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 significantly detectable amount of antibody binds to the antigen in a standard immunocytochemistry or flow cytometry assay, optionally after fixation of the cells, using an optionally labeled secondary antibody.

[0186] Expression of tissue-specific gene products can also be detected at the mRNA level by Northern blot analysis, dot blot hybridization analysis, or reverse transcriptase-initiated polymerase chain reaction (RT-PCR) using sequence-specific primers with standard amplification methods using publicly available sequence data (GenBank). Expression of a tissue-specific marker detected at the protein or mRNA level is considered positive if the level is at least or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold, particularly more than 10-fold, more than 20-fold, more than 30-fold, more than 40-fold, or more than 50-fold, higher than that of control cells, such as undifferentiated pluripotent stem cells or other unrelated cell types.

[0187] Once markers have been identified on the cell surface of the desired phenotype, these markers can be used in immunoselection to further enrich the population by techniques such as immunopanning or antibody-mediated fluorescence-activated cell sorting.

[0188] While functional attributes provide a way to characterize cells and their precursors in vitro, they may not be necessary for some of the uses mentioned in this disclosure. For example, mixed cell populations enriched for cells that possess some of the above markers, but not all of the functional or electrophysiological properties, could be of considerable therapeutic benefit if they could be transplanted into damaged cardiac tissue and acquire the functional properties necessary in vivo to compensate for cardiac function.

[0189] 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 the line from which they were derived. This means that the chromosomal DNA between pluripotent stem cells and cardiac cells is more than 90% identical, which can be inferred if cardiac cells are derived from an undifferentiated lineage through the normal process of mitosis. The characteristic that cardiomyocytes are derived from a parent cell population is important in several respects. In particular, the undifferentiated cell population can be used to generate additional cells with a shared genome (such as further batches of cardiac cells or other cell types that may be useful for therapy), for example, as a population that can pre-tolerize patients to a histocompatible type of cardiac allograft (U.S. Patent Application Publication No. 2002 / 0086005; WO 03 / 050251).

[0190] V. How to use Cardiac fibroblasts provided by certain embodiment methods and compositions can be used in a variety of applications, including, but not limited to, in vivo cell transplantation or implantation, in vitro screening for cytotoxic compounds, carcinogens, mutagens, growth / regulatory factors, pharmaceutical compounds, and the like, elucidating mechanisms of cardiac disease and injury, studying mechanisms by which drugs and / or growth factors act, diagnosing and monitoring cancer in patients, gene therapy, and producing biologically active products.

[0191] The cardiac fibroblasts of the present disclosure can be used commercially to screen factors (e.g., solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (e.g., culture conditions or manipulations) that affect the properties of such cells and their various progeny.

[0192] Cardiac fibroblasts can be used in three-species co-culture assays with endothelial cells and fibroblasts for in vitro disease modeling, drug discovery, and toxicity testing.

[0193] In some embodiments, cardiac fibroblasts are used to screen for factors that promote maturation into late-stage cardiac cells or terminally differentiated cells, or to promote the growth and maintenance of such cells in long-term culture. For example, candidate maturation or growth factors are tested by adding them to different wells of cells and then determining any resulting phenotypic changes according to desired criteria for further culture and use of the cells.

[0194] Another screening application of the present disclosure relates to testing pharmaceutical compounds for their effects on cardiac fibrosis and / or heart failure. Screening can be performed because the compound is designed to have a pharmacological effect on cells, or because compounds designed to have an effect elsewhere may have unintended side effects on cells of this tissue type. Screening can be performed using either progenitor cells or terminally differentiated cells of the present disclosure.

[0195] The reader is generally referred to the standard texts "In Vitro Methods in Pharmaceutical Research," Academic Press, 1997, and U.S. Patent No. 5,030,015. Evaluating the activity of a candidate pharmaceutical compound generally involves combining differentiated cells of the present disclosure with the candidate compound alone or in combination with other drugs. Researchers determine any changes in cell morphology, marker phenotype, or functional activity resulting from the compound (compared to untreated cells or cells treated with an inactive compound), and then correlate the effect of the compound with the observed changes.

[0196] Cytotoxicity can be primarily determined by its effects on cell viability, survival rate, morphology, and expression of specific markers and receptors. Drug effects on chromosomal DNA can be determined by measuring DNA synthesis or repair. 3Incorporation of [H]-thymidine or BrdU, particularly at unscheduled times in the cell cycle or at levels above those required for cell replication, is consistent with a drug effect. Undesirable effects may also include abnormal rates of sister chromatid exchange determined by metaphase spreads. The reader is referred to Vickers (pp 375-410 in In Vitro Methods in Pharmaceutical Research, Academic Press, 1997) for further details.

[0197] The effect on cell function can be assessed using any standard assay for observing cardiomyocyte phenotype or activity, e.g., marker expression, receptor binding, contractile activity, or electrophysiology, either in cell culture or in vivo. Drug candidates can also be tested for their effect on contractile activity, such as increasing or decreasing the degree or frequency of contractions. If an effect is observed, the concentration of the compound can be titrated to determine the median effective dose (ED 50 ) can be determined.

[0198] The present disclosure further provides a method for screening for drugs that have an effect on cardiomyocytes, endothelial cells, and cardiac fibroblasts. The method involves contacting cells from one of the cell populations described herein with a candidate drug and determining whether the drug has an effect on the cell population. The drug to be tested can be a single compound or mixture, natural or synthetic, a small molecule or polymer, including polypeptides, polysaccharides, and polynucleotides, an antibody or fragment thereof, a compound from a library of natural or synthetic compounds, a compound derived from rational drug design, cell culture conditions, or any drug whose effect on a cell population can be assessed using assays known in the art. The effect on a cell population can be determined by any standard assay for phenotype or activity, including, for example, assays for marker expression, receptor binding, contractile activity, electrophysiology, cell viability, survival rate, morphology, or DNA synthesis or repair. Standard proliferation and differentiation assays are described in U.S. Patent No. 6,110,739. Such drugs are useful for controlling cell proliferation, differentiation, and viability in vivo and in vitro, as well as for tissue maintenance, regeneration, and repair.

[0199] A. Pharmaceutical Compositions The present disclosure further provides compositions comprising a population of cardiac fibroblasts. The compositions may further comprise a population of cardiomyocytes. The compositions may comprise a pharmaceutically acceptable carrier and diluent. The compositions may further comprise components that facilitate engraftment. Compositions comprising these populations are useful for cell and tissue replacement and repair, and for generating cardiac fibroblast populations in vitro and in vivo. The compositions can be formulated as drugs or delivery devices for treating cardiac disease.

[0200] The cardiac fibroblasts of the present disclosure can be provided in the form of a pharmaceutical composition containing an isotonic excipient prepared under sufficiently sterile conditions for administration to humans. In certain embodiments, it may be desirable to disperse the cardiac fibroblasts into a suspension of single cells or smaller clusters using a protease or by gentle mechanical manipulation. To reduce the risk of cell death during engraftment, the cells can be heat-shocked or cultured with approximately 0.5 U / mL erythropoietin approximately 24 hours prior to administration.

[0201] 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 selection of the cell excipient and any accompanying components of the composition is adapted according to the route and device used for administration. The composition may also contain or be accompanied by one or more other components that promote engraftment or functional recruitment of cardiomyocytes. Suitable components include cardiac fibroblast-derived matrix proteins that support or promote adhesion of cardiomyocytes, or complementary cell types, particularly endothelial cells.

[0202] The present disclosure also includes reagent systems containing sets or combinations of cells that are present at all times during manufacturing, shipping, or use. Cell sets include any combination of two or more cell populations described herein, exemplified but not limited to differentiated cell types (e.g., cardiac progenitor cells, epicardial cells, and cardiac fibroblasts) often sharing the same genome, combined with undifferentiated pluripotent stem cells or other differentiated cell types (e.g., cardiomyocytes and endothelial cells). Each cell type in the set can be packaged together or in separate containers, at the same or different times, in the same facility or at different locations, under the control of the same company or different companies that share a business relationship.

[0203] The pharmaceutical compositions of the present disclosure can optionally be packaged in a suitable container along with written instructions for a desired purpose, such as reconstituting a mixture of cardiomyocytes, cardiac fibroblasts, and endothelial cells to ameliorate a disease state or abnormality of the myocardium.

[0204] B. Therapeutic Uses 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 diseases such as myocardial infarction, cardiomyopathy, congestive heart failure, ventricular septal defect, atrial septal defect, congenital heart defects, ventricular aneurysms, cardiac diseases of pediatric origin, ventricular aneurysms, or cardiac diseases requiring ventricular reconstruction.

[0205] In human treatment, the dose is generally about 10 8 ~10 12 cells, and typically about 2 x 10 8 ~1×10 9 The dose of the cells will be adjusted according to the subject's weight, the nature and severity of the affliction, and the replicative capacity of the administered cells. The ultimate responsibility for determining the method of treatment and appropriate dosage rests with the supervising clinician.

[0206] Certain aspects also provide for the use of cardiac fibroblasts to enhance myocardial tissue maintenance or repair for any recognized need, such as congenital abnormalities of metabolic function, the effects of disease states, or the consequences of severe trauma.

[0207] To determine the suitability of a cell composition for therapeutic administration, the cells can first be tested in an appropriate animal model. At one level, the ability of the cells to survive in vivo and maintain their phenotype is assessed. The cell composition is administered to an immunodeficient animal (e.g., nude rats, or animals rendered immunodeficient by chemical or irradiation). Tissues are harvested after a period of engraftment and assessed for the presence of pluripotent stem cell-derived cells.

[0208] Other methods for tracking cells in vivo include (e.g., BrdU or 3This may be by administration of cells expressing a detectable label (e.g., green fluorescent protein or β-galactosidase) that have been pre-labeled (with [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 render amplification specific for human polynucleotides according to published sequence data.

[0209] Compatibility can also be determined by assessing the degree of cardiac recovery resulting from treatment with a population of cardiac fibroblasts derived from pluripotent stem cells. Several animal models are available for such testing. For example, hearts can be cryoinjured by placing a pre-chilled aluminum rod in contact with the surface of the anterior wall of the left ventricle (Murry et al., 1996; Reinecke et al., 1999; US Pat. 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 N2 on the anterior wall of the left ventricle 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 cardiac tissue is examined by histology for the presence of cells in the damaged area. Cardiac function can be monitored by determining parameters such as left ventricular end-diastolic pressure, developed pressure, rate of pressure rise, and rate of pressure fall.

[0210] After appropriate testing, the differentiated cells of the present disclosure can be used to reconstruct or regenerate tissue in human patients or other subjects in need of such treatment. The cells are administered in a manner that allows them to engraft or migrate 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 to the desired location within the ventricle, pericardium, or myocardium.

[0211] If desired, patients receiving allografts of pluripotent stem cell-derived cardiac fibroblasts can be treated to reduce immune rejection of the transplanted cells. Potential methods include administering conventional immunosuppressants such as cyclosporine A (Dunn et al., Drugs 61:1957, 2001) or inducing immune tolerance using a matched population of pluripotent stem cell-derived cells (WO 02 / 44343; U.S. Patent No. 6,280,718; WO 03 / 050251). Another approach is to adapt the cardiac fibroblast cell population, for example by treating it with allopurinol, to reduce the amount of uric acid produced by the cells upon transplantation into a subject. Alternatively, or in combination, patients can be primed by administering allopurinol or an enzyme that metabolizes uric acid, such as urate oxidase (International Application PCT / US04 / 42917).

[0212] Suitable patients for receiving regenerative medicine according to the present methods include those with a variety of acute and chronic heart diseases, such as coronary heart disease, cardiomyopathy, endocarditis, and congenital cardiovascular abnormalities, and congestive heart failure. The effectiveness of treatment can be monitored by clinically accepted criteria, such as a reduction in the area occupied by scar tissue or revascularization of scar tissue, and a decrease in the frequency and severity of angina pectoris; or an improvement in incident pressure, systolic pressure, end-diastolic pressure, patient mobility, and quality of life.

[0213] In another embodiment, the present disclosure provides methods of cell replacement and tissue replacement useful for treating disorders characterized by insufficient cardiac function, including, for example, congenital heart disease, coronary heart disease, cardiomyopathy, endocarditis, and congestive heart failure. Cardiac fibroblasts and cardiovascular progenitor cells are useful for replacement therapy. Methods for engineering cardiac tissue are known in the art and are reviewed, for example, by Birla in "Stem Cell Therapy and Tissue Engineering for Cardiovascular Repair," Springer, 2006. In a preferred embodiment, the subject is a human. The composition can be administered by a route of delivery or transfer to cardiac tissue, including, for example, injection or implantation, under conditions that result in the alleviation of at least one adverse effect or symptom or disorder.

[0214] The treatment methods of the present disclosure are not intended to limit the administration of cardiac fibroblasts to a mammal to a particular mode, dosage, or frequency of administration; the present disclosure contemplates all modes of administration, including intramuscular, intravenous, intra-arterial, intralesional, subcutaneous, or other routes sufficient to provide an appropriate dose for disease prevention or treatment. Cardiac fibroblasts can be administered to a mammal in a single dose or multiple doses. If multiple doses are administered, each dose can be separated from the others by, for example, one week, one month, one year, or ten years. One or more growth factors, hormones, interleukins, cytokines, small molecules, or other cells can also be administered before, during, or after administration of the cells to further bias them toward a particular cell type. [Example]

[0215] VI. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. Those of skill in the art will appreciate that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those of skill in the art will, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0216] Example 1 - Cardiac fibroblast development In this study, we developed a differentiation method to generate a highly pure population of iPSC-derived quiescent cardiac fibroblasts from a population of (WT1+) epicardial cells by adding serum-containing or serum-free medium in the presence of basic FGF (Figure 1).

[0217] The starting population of iPSCs was formed into aggregates in E8 medium (Table 1) containing a ROCK inhibitor and a low concentration of GSK3 inhibitor. On day 1, iPSCs were treated with a high concentration of the GSK3 inhibitor (CHIR99021) in the presence of activin A and BMP4 growth factors in RPMI / B27 minus insulin-containing medium for cardiac mesoderm induction for 1 day. On day 2, the activin A and BMP4 growth factors were refreshed without the GSK3 inhibitor. This is referred to as ABC mesoderm induction.

[0218] By day 3, the use of CHIR alone generated a distinct mesodermal progenitor population (NCAM+ / CXCR4+) compared to the generation of a population of NCAM+ / CXCR4-low mesodermal progenitors using activin A and BMP4 in combination with CHIR (ABC) (Figure 2A). The use of activin A and BMP4 in addition to CHIR during mesoderm induction resulted in a decreased percentage of CXCR4+ progenitors compared to CHIR alone. For CHIR alone, 7 μM CHIR99021 was added. For ABC conditions, 9 ng / mL activin A, 5 ng / mL BMP4, and 7 μM CHIR99021 (Table 2) were added on day 1, and activin A and BMP4 alone (Table 3) were added on day 2 of differentiation.

[0219] Cardiac mesoderm cells were then dissociated and exposed to a low concentration of the Wnt inhibitor XAV939 (Table 4) for 2 days to specify the WT1 epicardial lineage (Figure 3A). The percentage of epicardial cells was measured under single CHIR, ABC, and double CHIR conditions. Epicardial induction by single CHIR or ABC steps generated a mixed population of epicardial cells and cardiomyocytes. The percentage of WT1 epicardial cells ranged from 30% to 90% WT1. These epicardial progenitor cells were used directly for cardiac fibroblast induction without additional passaging or purification of epicardial cells.

[0220] Dose-escalating doses of CHIR were administered to iPSCs. We found that increasing the dose of CHIR on day 1 in the single CHIR regimen did not increase the proportion of WT1+ epicardial cells on day 9 (Figure 3B). Administration of 6 μM CHIR on day 1 generated a population of 60% WT1 epicardial progenitor cells with characteristic cobblestone morphology by day 14 (Figure 3C).

[0221] Single-CHIR-treated epicardial cells were observed to have a flat cobblestone morphology and prominent, round, large nuclei (Figure 3C). When activin and BMP were added along with single-CHIR, epicardial cells maintained their flat cobblestone morphology and dark, round nuclei. The cell boundaries became less pronounced. There was no need to passage epicardial cells. Notably, applying a second CHIR at the epicardial stage dramatically expanded epicardial progenitor cells. This was confirmed by an increase in cell number, a decrease in cell size, and a more elongated morphology. Flow cytometry at D12 showed that the WT-1 purity of double-CHIR epicardial cells was over 90%, whereas in ABC single-CHIR at D9, the WT-1 purity was lower, ranging from 30 to 80%, more similar to CHIR alone. In these studies, ABC progenitor cells did not proliferate extensively and required passaging like double-CHIR cells.

[0222] Cardiac fibroblasts were induced in serum-containing conditions on day 25. Cells were fed with DMEM-low glucose, 10% FBS, ascorbic acid, and 200 ng / mL basic FGF and purified by serial passage (Table 6). Cardiac fibroblasts were >85% positive for TE-7 and CD29 and were quiescent (i.e., <5% αSMA) (Figure 4A).

[0223] It was observed that increasing the concentration of basic FGF increased the percentage of CD29-positive cells during cardiac fibroblast differentiation of epicardial progenitor cells from the ABC method (Figure 4B).

[0224] Seven independent iPSC lines were differentiated into cardiac fibroblasts using ABC conditions in serum-containing medium with high concentrations of basic FGF during mesoderm induction and cardiac fibroblast specification. All cardiac fibroblasts had high fibroblast purity (TE-7) and were quiescent with less than 5% alpha-smooth muscle actin purity (aSMA) (Figure 4C).

[0225] Alternatively, cardiac fibroblasts were differentiated from ABC epicardial progenitor cells under serum-free conditions (Table 7). Cardiac fibroblasts were induced from epicardial cells by culturing for five passages in serum-free medium without TGFβ inhibitors. Cardiac fibroblasts induced in serum-free medium had high TE-7 purity (>85%) and were quiescent (aSMA <5%) by flow cytometry (Figure 4D). Cardiac fibroblasts were cryopreserved at passages 5–7.

[0226] Thaw cardiac fibroblasts and culture at 10k / cm. 2 Cardiac fibroblasts were plated onto vitronectin-coated plates at 100°C and activated with TGFβ. TGFβ was added at 20 or 40 ng / mL to activate cardiac fibroblasts on day 2, and the medium was changed on day 4. On day 5, the culture supernatant was collected and analyzed for fibronectin secretion using the Human FN ELISA Kit Cat #BMS2028. On days 3 and 5, cardiac fibroblasts were harvested and counted using a ViCell-XR. After 5 days, cells were harvested for flow cytometry. The emergence of an α-smooth muscle actin-expressing population was confirmed in the presence of TGFβ (Figure 4E).

[0227] In the cardiac triple coculture microtissue assay, cryopreserved iPSC-derived cell types were combined at specific ratios and formed into microtissues in microwell plates (Figure 5A). Figure 5B shows a microtissue containing 65% iCell cardiomyocytes, 20% iCell endothelial cells, and 15% iPSC-derived cardiac fibroblasts. After 1–2 days, the microtissues had contracted.

[0228] To support all three cell types, the microtissues were supplied with co-culture medium (Table 8). Fourteen days after construction, the microtissues were observed to respond to inotropic compound exposure. Isogenic triple-coculture cardiac microtissues demonstrated inotropic responses to the β-adrenergic agonist isoproterenol in the iPSC-cardiac fibroblast clone, multiple cardiac fibroblasts, and donor strains (Figure 6A). Microtissues were generated from three donor strains, 11713, 01279, and 01434. Microtissues contained either iPSC-derived cardiomyocytes alone or triple-cocultures containing isogenic iPSC-derived cardiomyocytes, iPSC-derived endothelial cells, and iPSC-derived cardiac fibroblasts. All microtissues were assayed for response to isoproterenol at day 14 of culture. Increases in calcium transient amplitude were observed only in triple-coculture microtissues, not in cardiomyocyte-only microtissues. This response occurred in triple coculture microtissues from all three donor lines, in microtissues containing cardiac fibroblasts from two independently generated lots of cardiac fibroblasts from donor 01434, and from two independently derived clones (11713.008 and 11713.847) from donor 11713.

[0229] An increase in the inotropic response was observed with an increase in cardiac fibroblasts in the 3D coculture microtissues (Figure 6B). The concentration of cardiac fibroblasts was found to affect the ionotropic response of the 3D coculture microtissues. The responses to dobutamine (DOB), digoxin (DIG), isoproterenol (ISO), and epinephrine (EPI) were assessed at DIV 14 in cardiac 3D coculture microtissues consisting of a total of 10,000 cells, 20% iCell endothelial cells and iCell cardiomyocytes, and various concentrations of iPSC-derived cardiac fibroblasts (CFs). Increasing CF content in cardiac 3D microtissues resulted in increased calcium amplitude inotropic responses to dobutamine, digoxin, and epinephrine (Figure 6B).

[0230] Screening of compound libraries can be performed using cardiac triple coculture microtissues for cardiovascular drug discovery. Small numbers of cells are used in multiwell plates (e.g., 96, 384, or 1536) in a number of high-throughput assay platforms (Figure 7A).

[0231] Hypoxia was observed to result in a disease phenotype in the dilated cardiomyopathy (CM) model LMNAL35P microtissues. Triple-species coculture microtissues were generated from healthy control (NHC), isogenic LMNA-corrected, and isogenic LMNA-L35P iPSC-derived cell types. After cell percussion, cells were placed in hypoxic (5% O2) or normoxia (20% O2). Healthy control microtissues and isogenic LMNA mutation-corrected microtissues exhibited similar beating rates in normoxia and hypoxia, whereas LMNA mutant triple-species coculture MTs exhibited significantly slower beating rates in hypoxia (Figure 7A).

[0232] Microtissues derived from dilated cardiomyopathy mutant LMNAL35P cells were unresponsive to inotropic compounds. Triple-culture microtissues were generated from healthy control (NHC) and isogenic LMNA-L35P iPSC-derived cell types. 14 days after microtissue formation, microtissues were exposed to isoproterenol. Microtissues derived from healthy donor iPSCs responded with an increase in calcium transient amplitude at 100 nM isoproterenol. Disease model microtissues were unresponsive to increased isoproterenol (Figure 8).

[0233] Thus, this study demonstrated that a highly pure population of cryopreserved quiescent cardiac fibroblasts (>75% TE-7 and <5% aSMA) could be generated from a population of (WT1+) epicardial cells under serum or serum-free conditions. These cardiac fibroblasts were observed to have an isotropic response to isoproterenol when cocultured with cardiomyocytes and endothelial cells.

[0234] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made in the methods described herein and in the steps or sequence of steps thereof without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. 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. 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. Archer, 2018 Byrne et al., Nature, 450(7169):497-502, 2007. Dunn et al., Drugs 61:1957, 2001. Fernandes, et al., J. Biotechnology, 132(2):227-236, 2007. Guo and Pu et al., 2020 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. Ravenscroft, 2016 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 12 / 478,154 U.S. Patent 8,546,140 U.S. Patent 5,843,780 U.S. Patent 5,843,780 US Patent 6,103,470 US Patent 6,110,739 US Patent 6,200,806 US Patent 6,280,718 US Patent 6,416,998 US Patent 7,029,913 US Patent 7,442,548 US Patent 7,598,364 US Patent 7,989,425 US Patent 8,058,065 US Patent 8,071,369 US Patent 8,129,187 US Patent 8,183,038 US Patent 8,268,620 US Patent 8,268,620 US Patent 8,546,140 US Patent 8,546,140 US Patent 8,691,574 US Patent 8,691,574 US Patent 8,741,648 US Patent 8,741,648 US Patent 8,741,648 US Patent 8,900,871 US Patent 9,175,268 US Patent Publication No. 2003 / 0211603 US Patent Publication 2009 / 0246875 US Patent Publication No. 2010 / 0210014 US Patent Publication No. 2011 / 0104125 US Patent Publication No. 2012 / 0276636 US Patent Publication No. 2015 / 0191697 US 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 generating human pluripotent stem cell (PSC)-derived cardiac fibroblast progenitor cells, comprising: (a) culturing PSC aggregates in a medium containing a Wnt agonist, an activin agonist, and BMP4 for mesoderm induction; (b) further culturing the PSC aggregates in a medium comprising an activin agonist and BMP4, and essentially free of a Wnt agonist, to generate a population of mesodermal progenitor cells; and (c) culturing the mesodermal progenitor cells in a medium containing a Wnt inhibitor to generate a population of cardiac fibroblast progenitor cells.

2. 2. The method of claim 1, wherein the medium in step (b) does not contain a Wnt agonist.

3. The mesodermal progenitor cells are NCAM-positive and CXCR4 (NCAM+ / CXCR4 ｌｏｗ 2. The method of claim 1, wherein the gene encoding the nucleotide sequence of the present invention has low expression of the nucleotide sequence of the present invention.

4. The method of claim 1 or 3, wherein less than 10% of the mesodermal progenitor cells are CXCR4 positive.

5. 5. The method of claim 1, wherein the cardiac fibroblast progenitor cells comprise epicardial progenitor cells, endothelial fibroblast progenitor cells, secondary heart field progenitor cells, and / or neural crest progenitor cells.

6. The method of any one of claims 1 to 5, wherein approximately 30 to 70% of the cardiac fibroblast progenitor cells are secondary heart field progenitor cells.

7. The method of any one of claims 1 to 5, wherein about 50% of the cardiac fibroblast progenitor cells are secondary heart field progenitor cells.

8. The method according to claim 6 or 7, wherein the secondary heart field progenitor cells are GATA4-positive and / or HAND2-positive.

9. The method of any one of claims 1 to 8, wherein approximately 10-50% of the cardiac fibroblast precursor cells are endothelial fibroblast precursor cells.

10. The method of any one of claims 1 to 8, wherein approximately 30-40% of the cardiac fibroblast precursor cells are endothelial fibroblast precursor cells.

11. The method of claim 9 or 10, wherein the endothelial fibroblast precursor cells are TEK positive.

12. The method of any one of claims 1 to 11, wherein about 5-40%, about 10-20% of the cardiac fibroblast progenitor cells are epicardial progenitor cells.

13. The method of any one of claims 1 to 11, wherein about 5-40%, about 10-20% of the cardiac fibroblast progenitor cells are epicardial progenitor cells.

14. The method of claim 13 or 14, wherein the epicardial progenitor cells are WT1-positive, SNAI1-positive, TBX19-positive, and / or TBX20-positive.

15. The method of any one of claims 1 to 14, wherein the cardiac fibroblast progenitor cells are oligopotent.

16. 10. The method of claim 1, wherein step (c) is further defined as generating a mixed population of cardiac fibroblast progenitor cells and cTNT+ cardiomyocytes.

17. 13. The method of claim 1 or 12, further comprising generating PSC-derived cardiac fibroblasts comprising further differentiating the cardiac fibroblast progenitor cells in a medium comprising basic FGF (bFGF) to generate a population of cardiac fibroblasts.

18. 18. The method of claim 17, wherein the differentiation is carried out in the absence of a TGFβ inhibitor.

19. 19. The method of claim 17 or 18, wherein the cardiac fibroblasts express vimentin (VIM), COL1A1, COL1A2, and / or secreted protein acidic and rich in cysteine ​​(SPARC).

20. The method of any one of claims 17 to 19, wherein the cardiac fibroblasts express discoidin domain receptor 2 (DDR2) and / or periostin (POSTN).

21. The method of any one of claims 17 to 20, wherein at least 50% of the cardiac fibroblasts are GATA-4 positive.

22. The method of any one of claims 17 to 21, wherein at least 85% of the cardiac fibroblasts are CD90 positive.

23. The method of any one of claims 1 to 17, wherein the PSCs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

24. 24. The method of claim 23, wherein the iPSCs are derived from a healthy subject or a subject with a genotype for a genetic disease.

25. 24. The method of claim 23, wherein the iPSCs are engineered to contain a disease-associated mutation for a cardiovascular disease.

26. 25. The method of claim 24, wherein the iPSCs are derived from a subject with dilated cardiomyopathy and contain the LMNA-L35P mutation.

27. 27. The method of any one of claims 1 to 26, wherein the Wnt agonist in step (a) is CHIR99021, SB216763, CHIR98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, or IM-12.

28. 28. The method of any one of claims 1 to 27, wherein the Wnt agonist is CHIR99021.

29. 29. The method of claim 28, wherein the CHIR99021 is present in step (a) at a concentration of about 5 μM to 10 μM.

30. 30. The method of any one of claims 1 to 29, wherein the culture medium of steps (a) to (c) is free or essentially free of insulin.

31. The method of any one of claims 1 to 30, wherein the activin agonist is activin A.

32. 32. The method of any one of claims 1 to 31, wherein the medium in step (a) further comprises albumin.

33. The method according to any one of claims 1 to 32, wherein the medium in any of steps (a) to (c) further comprises albumin.

34. 34. The method of any one of claims 1 to 33, wherein the PSC aggregates of step (a) are obtained by culturing PSCs in the presence of a Wnt agonist and a survival agent.

35. 35. The method of claim 34, wherein the Wnt agonist is CHIR99021, SB216763, CHIR98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, or IM-12.

36. 35. The method of claim 34, wherein the Wnt agonist is CHIR99021.

37. 37. The method of claim 36, wherein the CHIR99021 is present in step (a) at a concentration of about 1 μM to 3 μM.

38. The method of any one of claims 34 to 37, wherein the survival agent is a Rho-associated kinase (ROCK) inhibitor or a myosin II inhibitor.

39. The method of claim 38, wherein the ROCK inhibitor is H1152 or Y-27632.

40. 39. The method of claim 38, wherein the myosin II inhibitor is H1152.

41. 41. The method of any one of claims 1 to 40, wherein the mesodermal progenitor cells are dissociated into essentially single cells prior to step (c).

42. 42. The method of any one of claims 1 to 41, wherein the culture medium in step (c) is free or essentially free of BMP4, Wnt agonists, and retinoic acid.

43. 43. The method of any one of claims 1 to 42, wherein 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.

44. 44. The method of any one of claims 1 to 43, wherein the Wnt inhibitor is XAV939.

45. 45. The method of claim 44, wherein the XAV939 is present in the medium at a concentration of 1 μM to 5 μM.

46. 46. ​​The method of any one of claims 1 to 45, wherein the population of cardiac fibroblast progenitor cells in step (c) comprises at least 30% WT1+ progenitor cells.

47. 47. The method of any one of claims 1 to 46, wherein the population of cardiac fibroblast progenitor cells in step (c) comprises less than 80% WT1+ progenitor cells.

48. 48. The method of any one of claims 1 to 47, which does not include a purification step between step (c) and differentiating the cells into cardiac fibroblasts.

49. 49. The method of claim 48, wherein the purification step is further defined as cell sorting.

50. 50. The method of any one of claims 1 to 49, wherein the TGFβ inhibitor is SB431542, LDN-193189, LY2157299, LY2109761, SB525334, SIS HCl, SB505124, GW788388, or LY364947.

51. 51. The method of any one of claims 1 to 50, wherein the TGFβ inhibitor is SB431542.

52. 52. The method of claim 51, wherein the SB431542 is present in the medium at a concentration of 1 μM to 10 μM.

53. 43. The method of any one of claims 17 to 42, wherein the culture medium in step (a) is free or essentially free of Wnt agonists and Wnt inhibitors.

54. 54. The method of any one of claims 17 to 53, wherein the culture medium of step (b) is free or essentially free of TGFβ inhibitors.

55. 55. The method of any one of claims 17 to 54, wherein the culture medium in step (b) further comprises serum.

56. 56. The method of claim 55, wherein the bFGF is present in the medium at a concentration of 50 to 200 ng / mL.

57. 55. The method of any one of claims 17 to 54, wherein the culture medium of step (b) is serum-free or essentially serum-free.

58. 58. The method of claim 57, wherein the bFGF is present in the medium at a concentration of 10 to 100 ng / mL.

59. 59. The method of claim 57 or 58, wherein the medium further comprises VEGF, EGF, and IGF.

60. 60. The method of any one of claims 17 to 59, wherein the population of cardiac fibroblasts comprises at least 75% TE-7, CD29, and / or CD90 positive cells.

61. The method of any one of claims 17 to 60, wherein the population of cardiac fibroblasts comprises at least 85% TE-7, CD29, and / or CD90 positive cells.

62. 62. The method of any one of claims 17 to 61, wherein the cardiac fibroblasts are quiescent cardiac fibroblasts.

63. 63. The method of any one of claims 17 to 62, wherein the population of cardiac fibroblasts comprises less than 5% alpha-smooth muscle actin (αSMA) positive cells.

64. 64. The method of any one of claims 17 to 63, wherein the population of cardiac fibroblasts comprises at least 85% TE-7 and CD29 positive cells and less than 5% αSMA positive cells.

65. 65. The method of any one of claims 17 to 64, further comprising cryopreserving the population of cardiac fibroblasts.

66. 66. The method of any one of claims 17 to 65, further comprising culturing the population of cardiac fibroblasts in the presence of TGFβ to produce a population of activated cardiac fibroblasts.

67. 67. The method of claim 66, wherein the TGFβ is present at a concentration of 10 to 100 ng / mL.

68. The method of claim 64 or 67, wherein the activated fibroblast population exhibits increased fibronectin secretion and / or increased αSMA expression compared to the cardiac fibroblast population before culturing in the presence of TGFβ.

69. 60. The method of any one of claims 1 to 59, wherein the culturing is carried out in a defined medium.

70. 70. The method of any one of claims 1 to 69, which is GMP (good manufacturing practice) compliant.

71. 1. An in vitro method for generating human pluripotent stem cell (PSC)-derived epicardial progenitor cells, comprising: (a) culturing PSC aggregates in a medium containing a Wnt agonist, an activin agonist, and BMP4 for mesoderm induction; (b) further culturing the PSC aggregates in a medium comprising an activin agonist and BMP4, and essentially free of a Wnt agonist, to generate a population of NCAM+ / CXCR4+ mesodermal progenitor cells; and (c) culturing the mesodermal progenitor cells in a medium containing a Wnt inhibitor to generate a population of WT1+ epicardial progenitor cells.

72. 72. The method of claim 71, wherein step (b) does not include a Wnt agonist.

73. 72. The method of claim 71, wherein step (c) is further defined as producing a mixed population of WT1+ epicardial progenitor cells and cTNT+ cardiomyocytes.

74. (a) culturing the WT1+ epicardial progenitor cells in a medium containing a TGFβ inhibitor; and 74. The method of claim 71 or 73, further comprising (b) generating PSC-derived cardiac fibroblasts, comprising differentiating the WT1+ epicardial progenitor cells to generate a population of cardiac fibroblasts in a medium comprising basic FGF (bFGF).

75. 75. The method of any one of claims 71 to 74, wherein the PSCs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

76. 76. The method of claim 75, wherein the iPSCs are derived from a healthy subject or a subject with a genotype for a genetic disease.

77. 76. The method of claim 75, wherein the iPSCs are engineered to contain a disease-associated mutation for a cardiovascular disease.

78. 77. The method of claim 76, wherein the iPSCs are derived from a subject with dilated cardiomyopathy and contain the LMNA-L35P mutation.

79. 79. The method of any one of claims 71 to 78, wherein the Wnt agonist in step (a) is CHIR99021, SB216763, CHIR98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, or IM-12.

80. 80. The method of any one of claims 71 to 79, wherein the Wnt agonist is CHIR99021.

81. 81. The method of claim 80, wherein the CHIR99021 is present in step (a) at a concentration of about 5 μM to 10 μM.

82. 82. The method of any one of claims 71 to 81, wherein the culture medium of steps (a) to (c) is free or essentially free of insulin.

83. The method of any one of claims 71 to 82, wherein the activin agonist is activin A.

84. 84. The method of any one of claims 71 to 83, wherein the medium of step (a) further comprises albumin.

85. 85. The method of any one of claims 71 to 84, wherein the culture medium in any of steps (a) to (c) further comprises albumin.

86. 86. The method of any one of claims 71 to 85, wherein the PSC aggregates of step (a) are obtained by culturing PSCs in the presence of a Wnt agonist and a survival agent.

87. 87. The method of claim 86, wherein the Wnt agonist is CHIR99021, SB216763, CHIR98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, or IM-12.

88. 87. The method of claim 86, wherein the Wnt agonist is CHIR99021.

89. 89. The method of claim 88, wherein the CHIR99021 is present in step (a) at a concentration of about 1 μM to 3 μM.

90. 90. The method of any one of claims 86 to 89, wherein the survival agent is a Rho-associated kinase (ROCK) inhibitor or a myosin II inhibitor.

91. 91. The method of claim 90, wherein the ROCK inhibitor is H1152 or Y-27632.

92. 91. The method of claim 90, wherein the myosin II inhibitor is H1152.

93. 93. The method of any one of claims 71 to 92, wherein the mesodermal progenitor cells are dissociated into essentially single cells prior to step (c).

94. 94. The method of any one of claims 71 to 93, wherein the culture medium in step (c) is free of or essentially free of BMP4, Wnt agonists, and retinoic acid.

95. 95. The method of any one of claims 71 to 94, wherein 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.

96. 96. The method of any one of claims 71 to 95, wherein the Wnt inhibitor is XAV939.

97. 97. The method of claim 96, wherein the XAV939 is present in the medium at a concentration of 1 μM to 5 μM.

98. 98. The method of any one of claims 71 to 97, wherein the population of WT1+ epicardial progenitor cells in step (c) comprises at least 30% positive WT1+ epicardial progenitor cells.

99. 99. The method of any one of claims 71 to 98, wherein the population of WT1+ epicardial progenitor cells in step (c) comprises less than 80% positive WT1+ epicardial progenitor cells.

100. 100. The method of any one of claims 71 to 99, which does not include a purification step between step (c) and differentiating the cells into cardiac fibroblasts.

101. 101. The method of claim 100, wherein said purification step is further defined as cell sorting.

102. 102. The method of any one of claims 71 to 101, wherein the TGFβ inhibitor is SB431542, LDN-193189, LY2157299, LY2109761, SB525334, SIS HCl, SB505124, GW788388, or LY364947.

103. The method of any one of claims 71 to 102, wherein the TGFβ inhibitor is SB431542.

104. 104. The method of claim 103, wherein the SB431542 is present in the medium at a concentration of 1 μM to 10 μM.

105. 95. The method of any one of claims 74 to 94, wherein the culture medium of step (a) is free or essentially free of Wnt agonists and Wnt inhibitors.

106. 106. The method of any one of claims 74 to 105, wherein the culture medium of step (b) is free or essentially free of TGFβ inhibitors.

107. 107. The method of any one of claims 74 to 106, wherein the culture medium in step (b) further comprises serum.

108. 108. The method of claim 107, wherein the bFGF is present in the medium at a concentration of 50 to 200 ng / mL.

109. 107. The method of any one of claims 74 to 106, wherein the culture medium of step (b) is serum-free or essentially serum-free.

110. 110. The method of claim 109, wherein the bFGF is present in the medium at a concentration of 10 to 100 ng / mL.

111. 111. The method of claim 109 or 110, wherein the medium further comprises VEGF, EGF, and IGF.

112. The method of any one of claims 74 to 111, wherein the population of cardiac fibroblasts comprises at least 75% TE-7 and / or CD29 / CD90 positive cells.

113. The method of any one of claims 74 to 112, wherein the population of cardiac fibroblasts comprises at least 85% TE-7 and / or CD29 / CD90 positive cells.

114. 114. The method of any one of claims 74 to 113, wherein the cardiac fibroblasts are quiescent cardiac fibroblasts.

115. 115. The method of any one of claims 74 to 114, wherein the population of cardiac fibroblasts comprises less than 5% alpha-smooth muscle actin (αSMA) positive cells.

116. The method of any one of claims 74 to 115, wherein the population of cardiac fibroblasts comprises at least 85% TE-7 and CD29 positive cells and less than 5% αSMA positive cells.

117. 117. The method of any one of claims 74 to 116, further comprising cryopreserving the population of cardiac fibroblasts.

118. 118. The method of any one of claims 74 to 117, further comprising culturing the population of cardiac fibroblasts in the presence of TGFβ to produce a population of activated cardiac fibroblasts.

119. 119. The method of claim 118, wherein the TGFβ is present at a concentration of 10 to 100 ng / mL.

120. The method of claim 116 or 119, wherein the activated fibroblast population exhibits increased fibronectin secretion and / or increased αSMA expression compared to the cardiac fibroblast population before culturing in the presence of TGFβ.

121. The method of any one of claims 71 to 111, wherein the culturing is carried out in a defined medium.

122. 122. The method of any one of claims 71 to 121, which is GMP (good manufacturing practice) compliant.

123. A population of cardiac progenitor cells, epicardial progenitor cells, or cardiac fibroblasts produced by the method of any one of claims 1 to 122.

124. A composition comprising a population of PSC-derived cardiac fibroblasts that express TE-1 and CD29 at least 75% and αSMA at less than 5%.

125. The composition of claim 124, wherein the population of cardiac fibroblasts comprises at least 85% TE-7 and / or CD29 positive cells.

126. The composition of claim 124, wherein the population of cardiac fibroblasts comprises at least 85% TE-7 and CD29 positive cells and less than 5% αSMA positive cells.

127. The composition of claim 124, wherein the cardiac fibroblasts are produced by the method of any one of claims 71 to 122.

128. 128. The composition of any one of claims 124 to 127, wherein the population of cardiac fibroblasts is GMP compliant.

129. The composition of any one of claims 124 to 128, which is a pharmaceutical composition.

130. 130. A method of treating a cardiac disease in a subject, comprising administering to a subject in need thereof an effective amount of cardiac fibroblasts according to any one of claims 124 to 129.

131. 131. The method of claim 130, wherein the cardiac fibroblasts are administered directly to the heart.

132. 132. The method of claim 131, wherein the administration is by use of an intramyocardial catheter.

133. 133. The method of claim 132, wherein the cells are administered in a suspension comprising human albumin.

134. 134. The method of claim 133, wherein the human albumin is present at a concentration of 1% to 10%.

135. 139. The method of claim 138, wherein the human albumin is present at a concentration of 5%.

136. The method of any one of claims 130 to 135, wherein the subject is a human.

137. 137. The method of any one of claims 130 to 136, wherein the cardiac disease is fibrosis, myocardial infarction, cardiomyopathy, congestive heart failure, ventricular septal defect, atrial septal defect, congenital heart defect, ventricular aneurysm, cardiac disease of pediatric origin, ventricular aneurysm, or cardiac disease requiring ventricular reconstruction.

138. A co-culture comprising cardiac fibroblasts, endothelial cells, and cardiomyocytes according to any one of claims 124 to 129.

139. 139. The co-culture of claim 138, comprising approximately 40% cardiomyocytes, approximately 5% endothelial cells, and approximately 10% cardiac fibroblasts.

140. 139. The co-culture of claim 138, comprising approximately 60% cardiomyocytes, approximately 20% endothelial cells, and approximately 20% cardiac fibroblasts.

141. 139. The co-culture of claim 138, comprising approximately 75% cardiomyocytes, approximately 15% endothelial cells, and approximately 15% cardiac fibroblasts.

142. 139. The co-culture of claim 138, comprising approximately 75% cardiomyocytes, approximately 30% endothelial cells, and approximately 30% cardiac fibroblasts.

143. 140. The co-culture of claim 138 or 139, further defined as a microtissue.

144. 144. The co-culture of claim 143, in a microwell plate.

145. 145. The co-culture of claim 143 or 144, which does not include a scaffold.

146. 146. The co-culture of any one of claims 138 to 145, wherein the cardiomyocytes, the endothelial cells, and the cardiac fibroblasts are isogenic.

147. 147. The co-culture of any one of claims 138 to 146, which exhibits an inotropic response to the β-adrenergic agonist isoproterenol.

148. 130. A method of screening a test compound comprising introducing the test compound into a cardiac fibroblast population according to any one of claims 124 to 129.

149. 149. The method of claim 148, further comprising measuring cardiac fibroblast viability, cardiotoxins, and / or cardiomyocyte function.

150. A kit comprising the cardiac fibroblast population of any one of claims 124 to 129.

151. 151. The kit of claim 150, further comprising endothelial cells and / or cardiomyocytes.

152. 151. Use of the kit of claim 150 for disease modeling or disease screening.

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