Cell therapy

Abstract

Cell populations for the treatment of cardiac diseases and methods of making the same.

Description

1 Background Technology

[0001] It is estimated that 64 million people worldwide suffer from heart failure (HF), with a known prevalence of HF of approximately 1% to 2% of the total adult population. HF results in more than one million hospitalizations annually in the United States and Europe[1]. Once diagnosed, HF patients are hospitalized an average of once a year[2]. Due to a combination of population growth, aging, and increased prevalence of comorbidities, the absolute number of HF hospitalizations is expected to increase in the future, potentially by as much as 50% within 25 years[2]. HF with a low ejection fraction (LVEF) (i.e., <40%) accounts for approximately half of all HF cases[3,4].

[0002] The main drug treatments for HF include beta-blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor neprilysin inhibitors, SGLT2 inhibitors, mineralocorticoid receptor antagonists, as well as diuretics for fluid retention and hydralazine / nitrate therapy for selected patients[5].

[0003] Although existing standards of care for cardiac HF include pharmacological treatment with several available classes of drugs, devices, and heart transplants, all of these have certain limitations, and significant unmet medical needs remain due to the 1-year mortality rate of 25% to 75% in patients with advanced HF [2]. Guidelines have recently been updated to include, for example, SGLT2 inhibitors and angiotensin receptor neprilysin inhibitors, but improving cardiac function and prognosis remains challenging. Many therapies are also under development for HF, but options for patients with advanced HF remain limited.

[0004] Foo et al. [6] described a method for generating ISL1-positive human ventricular progenitor cells (“HVPs”) from human embryonic stem cells (ESCs) capable of differentiating into ventricular cardiomyocytes in vivo. The HVP population was harvested on day 6 of differentiation, and Foo et al. identified the HVP population as the optimal differentiation window [6].

[0005] WO 2016 / 029122 A1[7] describes a population of human cardiac ventricular progenitor cells derived from ESCs and harvested on day 6 of differentiation and which have been contacted with one or more reagents that are reactive to Jagged 1 (JAG1) and / or Frizzled 4 (FZD4).

[0006] WO 2017 / 172086 A1[8] describes genetic markers JAG1, FZD4, LIFR, FGFR3 and / or TNFSF9 for identifying implantable ventricular progenitor cells. HVPs were harvested from ESCs on day 6 of differentiation.

[0007] WO 2018 / 100433 A1[9] describes a method for isolating human cardiac ventricular progenitor cells harvested on days 5 through 7 of differentiation.

[0008] WO 2019 / 038587 A1

[10] describes a method for isolating human cardiac ventricular progenitor cells, the method comprising contacting a human cell culture containing cardiac progenitor cells with one or more reagents having reactivity with neurocilia protein-1 (NRP1). 2. Summary of the Invention

[0009] One risk associated with cells derived from pluripotent stem cells is teratoma formation. This disclosure, supported by data presented for the first time herein, is based on a novel ventricular progenitor cell differentiation protocol that reduces the risk of teratoma formation and provides a cell population suitable for use as a cell therapy product for patient administration. 3. Description of the attached figures

[0010] Figure 1 Cardiac progenitor cell differentiation protocol

[0011] Differentiation of cell populations suitable for repairing cardiac ventricular tissue involves obtaining a pluripotent stem cell population from a culture. On day 0, cells are cultured in the presence of a GSK inhibitor (e.g., CHIR). On day 1, the GSK inhibitor is removed. On day 3, cells are cultured in the presence of a WNT inhibitor. On day 5, the WNT inhibitor is removed. On day 8, differentiated progenitor cells are harvested and TRA-1-60 sorting is performed to eliminate cells expressing TRA-1-60. PSC: pluripotent stem cells; VPC: cardiac ventricular progenitor cells; MACS: magnetically activated cell sorting. Foo et al. described a method for early differentiation of ventricular progenitor cells (harvested on day 6).

[0012] Figure 2 Gene expression in cardiac progenitor cells

[0013] PCA plots from RNAseq data from day 0 (pluripotent cells), day 5 through day 10 (VPC), and day 15 (immature cardiomyocytes).

[0014] Figure 3 Teratoma formation in cardiac progenitor cell populations implanted in mouse kidneys by day 6.

[0015] Unsorted day 6 cardiac progenitor cells were injected into the renal capsule and grew outwards on the mouse kidneys. Left: Macroscopic image of teratoma; Right: Histological appearance of teratoma (HE).

[0016] Figure 4 Cells harvested on days 6 and 8 showed OCT4 and TRA-1-60 expression.

[0017] Cell populations harvested on days 6 and 8 were sorted for TRA-1-60 (cells expressing TRA-1-60 were removed).

[0018] Figure 5 Residual pluripotent cells were detected by qPCR, FACS, and HEC assays.

[0019] HEC: High-efficiency culture. FACS: Fluorescence-activated cell sorting; qPCR: Quantitative polymerase chain reaction.

[0020] Figure 6 Expression of undifferentiated biomarkers in differentiated VPC populations harvested on day 8 after incorporation.

[0021] All cell populations were derived from H9 cells. H9 = Human embryonic stem cell line WIC-WA09

[0022] Figure 7 Effects of AZD6414 (derived from H9) on adverse cardiac remodeling in mice after myocardial infarction (MI)

[0023] In immunocompromised mice, the effects of VPC cells derived from H9 (AZD6414; n=15) or mediators (n=14) on cardiac remodeling and function following permanent ligation of the left anterior descending artery (LAD). Cells or mediators were injected immediately after LAD ligation, and cardiac function was assessed by echocardiography at 24 hours, 28 days, and 49 days after myocardial infarction (MI). Echocardiographic analysis was performed using the Simpson method. Values ​​are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism 7.1. Mixed-effects analysis was performed with p-values ​​set at p<0.05. H9 = human embryonic stem cell line WIC-WA09; VPC = ventricular progenitor cells; LAD = left anterior descending artery; MI = myocardial infarction; n = number of mice treated; SEM = standard error of the mean.

[0024] Figure 8 The impact of HVP treatment on cardiac function over time a) Study protocol. b) Effect of HVP cells on left ventricular ejection fraction over time. c) Effect of HVP cells on overall longitudinal strain over time. d) Effect of HVP cells on left ventricular stroke volume over time. e) Left ventricular wall motion score index. f) Infarct volume over time. Mean ± SEM; mediator, n=6;

[100] million, n=7;

[300] million, n=7;

[600] million, n=6; two-way ANOVA mixed effect, followed by Dunnett's multiple comparison test. *, p < 0.05 relative to mediator. Day 0 and 21 after MI; Day 30, 60, and 90 after epicardial HVP cell injection.

[0025] Figure 9 HVP cells with a mature ventricular cardiomyocyte phenotype persisted in porcine myocardium for 3 months after implantation. .

[0026] a) HVP cells in the boundary region of infarcted porcine myocardium (the depicted area) express the ventricular cardiomyocyte protein MLC2v. b) The same tissue area stained with hematoxylin and eosin (H&E) as in (a) confirms the absence of inflammatory infiltration in the area of ​​implanted cells. Black arrows indicate the extent of human cell transplantation. c) High-power images of HVP cells show diffuse cytoplasmic expression of the ventricular cardiomyocyte protein MLC2v (*) and the presence of cross-stripes (arrows), providing evidence that HVP cells differentiate into a mature cardiomyocyte phenotype. d) Similar immunostaining patterns of N-cadherin in human and porcine cardiomyocytes (*) provide evidence of adhesion junctions in HVP cells. Note the multiple tightly juxtaposed areas in human and porcine cardiomyocytes (black arrows). 4. Detailed Implementation

[0027] 4.1 Cell population

[0028] This disclosure relates to a cell population in which at least 70% of the cells express cardiac progenitor cell (CPC)-related markers. One percent or less of the cells in the cell population express octamer transcription factor 4 (OCT4, also known as OCT4 / OCT3).

[0029] This disclosure also relates to a formulation comprising ≤1% OCT4+ cells and ≥70% ISL1+ cells.

[0030] 4.2 Cell population characteristics

[0031] The cell population disclosed herein may comprise dissociated cells. The cell population disclosed herein may consist of dissociated cells. Cell dissociation may be performed according to standard protocols and is a standard technique in cell culture, such as EDTA dissociation. The cell population may consist of cells in a suspension. Cells may consist of cells in a suspension. Cells in the suspension do not adhere to a surface. The cell population may not be in the form of a 2D adherent monolayer. The cell population may not be in the form of a monolayer. The cell population may not be in the form of an adherent monolayer. The cell population may not be in the form of a 3D culture. The cell population may be derived from a 2D adherent monolayer. The cell population may be cells dissociated from a 2D adherent monolayer. The cell population may not adhere to a scaffold or extracellular matrix, such as matrix gel, but may be derived from cells grown on a scaffold or extracellular matrix such as matrix gel. Cells in the suspension may not adhere to each other or to tissue culture surfaces, such as tissue culture flasks or tissue culture dishes. Cells in the suspension may be precipitated by centrifugation. The cell population may comprise or consist of live cells. Most cells in a cell population may be viable cells (e.g., cells that are not yet fixed). A cell population may have 50% viability. A cell population may have 55% viability. A cell population may have 60% viability. A cell population may have 65% viability. A cell population may have 70% viability. A cell population may have 75% viability. A cell population may have 80% viability. A cell population may have 85% viability. A cell population may have 90% viability. A cell population may have 95% viability. A cell population may have 96% viability. A cell population may have 97% viability. A cell population may have 98% viability. A cell population may have 99% viability. Cell viability can be measured or assessed by methods known in the art, such as methods for detecting ATP activity.

[0032] A cell population refers to a group, set, or collection of cells. This disclosure relates to a formulation containing cells in the form of a cell population or a formulation composed of cells in the form of a cell population. A cell population may contain at least 500 cells. A cell population may contain at least 1000 cells.

[0033] Cell populations can be heterogeneous. Cells within a cell population can be genetically identical. Cell populations can be pluripotent. Cell populations can contain adherent cells or be composed of adherent cells.

[0034] 4.3 Expression of pluripotency-related genes

[0035] 4.3.1OCT4 (POU5F1)

[0036] As used herein, OCT4 (also known as OCT3 / 4 or OCT3) refers to the protein encoded by the gene POU5F1 (POU class 5 homeobox 1) (gene ID: 5460, HUGO Gene Nomenclature Committee, HGNC). According to this disclosure, less than 1% of the cells in a cell population express OCT4. Less than 1% of the cells in a cell population express OCT4. Less than 0.9% of the cells in a cell population express OCT4. Less than 0.8% of the cells in a cell population express OCT4 or OCT3 / OCT4. Less than 0.7% of the cells in a cell population express OCT4. Less than 0.6% of the cells in a cell population express OCT4. Less than 0.5% of the cells in a cell population express OCT4. Less than 0.4% of the cells in a cell population express OCT4. Less than 0.3% of the cells in a cell population express OCT4. Less than 0.2% of the cells in a cell population express OCT4. Less than 0.1% of the cells in the cell population express OCT4.

[0037] The percentage (%) of cells expressing OCT4 in a cell population can be measured by flow cytometry. The percentage (%) of cells expressing OCT4 in a cell population can also be measured by immunofluorescence. Finally, the percentage (%) of cells expressing OCT4 in a cell population can be measured by high-throughput immunofluorescence.

[0038] According to this disclosure, as measured by flow cytometry, less than 1% of the cells in the cell population express OCT4 or OCT3 / OCT4. Less than 1% of the cells in the cell population express OCT4 as measured by flow cytometry. Less than 0.9% of the cells in the cell population express OCT4 as measured by flow cytometry. Less than 0.8% of the cells in the cell population express OCT4 as measured by flow cytometry. Less than 0.7% of the cells in the cell population express OCT4 as measured by flow cytometry. Less than 0.6% of the cells in the cell population express OCT4 or OCT3 / OCT4 as measured by flow cytometry. Less than 0.5% of the cells in the cell population express OCT4 or OCT3 / OCT4 as measured by flow cytometry. Less than 0.4% of the cells in the cell population express OCT4 or OCT3 / OCT4 as measured by flow cytometry. As measured by flow cytometry, less than 0.3% of the cells in the cell population express OCT4 or OCT3 / OCT4. As measured by flow cytometry, less than 0.2% of the cells in the cell population express OCT4 or OCT3 / OCT4. As measured by flow cytometry, less than 0.1% of the cells in the cell population express OCT4 or OCT3 / OCT4. As measured by flow cytometry, approximately 0.1% of the cells in the cell population express OCT4 or OCT3 / OCT4.

[0039] No cells in the cell population expressed OCT4 or OCT3 / 4. No cells in the cell population expressed OCT4, as measured by flow cytometry. Antibodies specific for OCT4 used for immunofluorescence and flow cytometry (FACS) are commercially available; see examples (catalog number 130-117-709, Miltenyi Biotec). OCT4 expression in the cell population may be below the detection limit, as measured by flow cytometry. OCT4 expression in the cell population may be below the detection limit, as measured by microarray, RNA sequencing, or quantitative polymerase chain reaction (PCR).

[0040] As measured by high-efficiency culture (HEC) assays, cell populations can produce less than one OCT4 per million cells. + Colonies. Cell populations may be substantially devoid of cells expressing OCT4. As measured by Western blotting, a cell population may not express OCT4. OCT4 can be human OCT4.

[0041] 4.3.2TRA-1-60

[0042] This disclosure also relates to cell populations in which 1.5% or fewer cells express T-cell receptor α locus 1-60 (TRA-1-60). In some embodiments, less than 1.5% of the cells in the cell population may express TRA-1-60. Less than 1.4% of the cells in the cell population may express TRA-1-60. Less than 1.3% of the cells in the cell population may express TRA-1-60. Less than 1.2% of the cells in the cell population may express TRA-1-60. Less than 1.1% of the cells in the cell population may express TRA-1-60. Less than 1% of the cells in the cell population may express TRA-1-60. Less than 0.9% of the cells in the cell population may express TRA-1-60. Less than 0.8% of the cells in the cell population may express TRA-1-60. Less than 0.7% of the cells in the cell population may express TRA-1-60. Less than 0.6% of the cells in the cell population may express TRA-1-60. Less than 0.5% of cells in the cell population express TRA-1-60. Less than 0.4% of cells in the cell population express TRA-1-60. Less than 0.3% of cells in the cell population express TRA-1-60. Less than 0.2% of cells in the cell population express TRA-1-60. The percentage of cells expressing TRA-1-60 in the cell population can be measured by flow cytometry using an antibody specific to TRA-1-60. 1.5% or less of the cells in the population express T cell receptor α locus 1-60 (TRA-1-60).

[0043] The percentage (%) of cells expressing TRA-1-60 in a cell population can be measured or determined by flow cytometry. The percentage (%) of cells expressing TRA-1-60 in a cell population can also be measured or determined by immunofluorescence. Antibodies specific to TRA-1-60 for use in immunofluorescence and flow cytometry (FACS) are commercially available.

[0044] TRA-1-60 expression in a cell population may be below the detection limit, as measured by flow cytometry, microarray, RNA sequencing, or quantitative polymerase chain reaction (PCR).

[0045] As measured by Western blotting, the cell population may not express TRA-1-60. The cell population may be substantially devoid of cells expressing TRA-1-60. TRA-1-60 may be human TRA-1-60.

[0046] 4.3.3NANOG

[0047] NANOG is expressed by 1% or less of the cells in the population. Less than 0.9% of the cells in the population express NANOG. Less than 0.8% of the cells in the population express NANOG. Less than 0.7% of the cells in the population express NANOG. Less than 0.6% of the cells in the population express NANOG. Less than 0.5% of the cells in the population express NANOG. Less than 0.4% of the cells in the population express NANOG. Less than 0.3% of the cells in the population express NANOG. Less than 0.2% of the cells in the population express NANOG. The percentage (%) of cells expressing NANOG in the cell population can be measured or determined by flow cytometry. The percentage (%) of cells expressing NANOG in the cell population can be measured or determined by immunofluorescence. NANOG-specific antibodies for immunofluorescence and flow cytometry (FACS) are commercially available.

[0048] As measured by flow cytometry, NANOG expression in a cell population may be below the detection limit. As measured by microarray, NANOG expression in a cell population may be below the detection limit. As measured by RNA sequencing, NANOG expression in a cell population may be below the detection limit. As measured by quantitative polymerase chain reaction (PCR), NANOG expression in a cell population may be below the detection limit.

[0049] As measured by Western blotting, a cell population may not express NANOG. A cell population may essentially contain no cells expressing NANOG. NANOG may be human NANOG.

[0050] 4.3.4 SOX2

[0051] 3% or less of the cells in the population express SOX2. Less than 2.9% of the cells in the population express SOX2. Less than 2.8% of the cells in the population express SOX2. Less than 2.7% of the cells in the population express SOX2. Less than 2.6% of the cells in the population express SOX2. Less than 2.5% of the cells in the population express SOX2. Less than 2.4% of the cells in the population express SOX2. Less than 2.3% of the cells in the population express SOX2. SOX2 expression in the cell population may be below the detection limit. The percentage (%) of cells expressing SOX2 in the cell population may be measurable by flow cytometry or determined by flow cytometry. The percentage (%) of cells expressing SOX2 in the cell population may be measurable by immunofluorescence or determined by immunofluorescence. Antibodies specific to SOX2 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0052] If measured by Western blotting, the cell population may not express SOX2. If measured by flow cytometry, SOX2 expression in the cell population may be below the detection limit. If measured by immunofluorescence, SOX2 expression in the cell population may be below the detection limit. If measured by RNA sequencing, SOX2 expression in the cell population may be below the detection limit. If measured by microarray, SOX2 expression in the cell population may be below the detection limit. If measured by quantitative polymerase chain reaction (PCR), SOX2 expression in the cell population may be below the detection limit. The cell population may be substantially free of cells expressing SOX22. SOX2 can be human SOX2.

[0053] 4.4 Expression of genes related to cardiac progenitor cells

[0054] At least 75% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 76% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 77% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 78% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 79% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 81% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 80% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 82% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 83% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 84% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 85% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 86% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 87% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 88% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 89% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 90% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 91% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 92% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 93% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 94% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 95% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 96% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 97% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 98% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. At least 99% of the cells in the cell population expressed cardiac progenitor cell (CPC) related markers. Approximately all cells in the cell population expressed cardiac progenitor cell (CPC) related markers.

[0055] 4.4.1 ISL1

[0056] A CPC-related marker can be insulin gene enhancer protein (ISL1). The following cell populations must express ISL1: at least 75%; at least 76%; at least 77%; at least 78%; at least 79%; at least 80%; at least 81%; at least 82%; at least 85%; at least 83%; at least 84%; at least 90%; at least 85%; at least 86% At least 87% of the cells in the cell population express ISL1. At least 88% of the cells in the cell population express ISL1. At least 89% of the cells in the cell population express ISL1. At least 90% of the cells in the cell population express ISL1. At least 91% of the cells in the cell population express ISL1. At least 92% of the cells in the cell population express ISL1. At least 93% of the cells in the cell population express ISL1. At least 94% of the cells in the cell population express ISL1. At least 95% of the cells in the cell population express ISL1. At least 96% of the cells in the cell population express ISL1. At least 97% of the cells in the cell population express ISL1. At least 98% of the cells in the cell population express ISL1. At least 99% of the cells in the cell population express ISL1. Approximately all cells in the cell population express ISL1.

[0057] The percentage (%) of cells expressing ISL1 in a cell population can be measured or determined by flow cytometry. The percentage (%) of cells expressing ISL1 in a cell population can be measured or determined by immunofluorescence. Antibodies specific to ISL1 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6]. As measured by Western blotting, a cell population may express ISL1. ISL1 may be human ISL1.

[0058] 4.4.2 Markers related to ventricular progenitor cells

[0059] Cells in the cell population expressed ventricular progenitor cell-related markers. Ventricular progenitor cell-related markers were described in Foo et al. [6]. Cells in the cell population expressed ISL1. Cells in the cell population expressed PDGFRA. Cells in the cell population expressed TBX1. Cells in the cell population expressed HAND1. Cells in the cell population expressed TBX5. Cells in the cell population expressed SMARCD3. Cells in the cell population expressed Jagged-1 (JAG1). Cells in the cell population expressed Frizzled-4 (FZD4). Cells in the cell population expressed fibroblast growth factor receptor 3 (FGFR3). Cells in the cell population expressed leukemia inhibitory factor receptor (LIFR). Cells in the cell population expressed TNF superfamily member 9 (TNFSF9). Cells in the cell population expressed ISL1 and LIFR. Cells in the cell population expressed PDGFRA and ISL1. Cells in the cell population expressed TBX1. Cells in the cell population expressed HAND1 and ISL1. Cells in the cell population expressed TBX5. Cells in the cell population express SMARCD3 and ISL1. Cells in the cell population express Jagged-1 (JAG1) and ISL1. Cells in the cell population express Frizzled-4 (FZD4) and ISL1. Cells in the cell population express fibroblast growth factor receptor 3 (FGFR3) and ISL1. Cells in the cell population express leukemia inhibitory factor receptor (LIFR). Cells in the cell population express TNF superfamily member 9 (TNFSF9). Cells in the cell population express ISL1 and LIFR.

[0060] The percentage (%) of cells expressing PDGFRA in a cell population can be measured or determined by flow cytometry. The percentage (%) of cells expressing PDGFRA in a cell population can be measured or determined by immunofluorescence. Antibodies specific to PDGFRA for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0061] The percentage (%) of cells expressing TBX1 in a cell population can be measured or determined by flow cytometry. The percentage (%) of cells expressing TBX1 in a cell population can be measured or determined by immunofluorescence. Antibodies specific to TBX1 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0062] The percentage (%) of cells expressing HAND1 in a cell population can be measured or determined by flow cytometry. The percentage (%) of cells expressing HAND1 in a cell population can be measured or determined by immunofluorescence. Antibodies specific to HAND1 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0063] The percentage (%) of cells expressing SMARCD3 in a cell population can be measured or determined by flow cytometry. The percentage (%) of cells expressing SMARCD3 in a cell population can be measured or determined by immunofluorescence. Antibodies specific to SMARCD3 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0064] At least 70% of the cells in the cell population express LIFR and ISL1. At least 71% of the cells in the cell population express LIFR and ISL1. At least 72% of the cells in the cell population express LIFR and ISL1. At least 73% of the cells in the cell population express LIFR and ISL1. At least 74% of the cells in the cell population express LIFR and ISL1. At least 75% of the cells in the cell population express LIFR and ISL1. At least 76% of the cells in the cell population express LIFR and ISL1. At least 77% of the cells in the cell population express LIFR and ISL1. At least 78% of the cells in the cell population express LIFR and ISL1. At least 79% of the cells in the cell population express LIFR and ISL1. At least 80% of the cells in the cell population express LIFR and ISL1. At least 81% of the cells in the cell population express LIFR and ISL1. At least 82% of the cells in the cell population express LIFR and ISL1. At least 83% of the cells in the cell population express LIFR and ISL1. At least 84% of the cells in the cell population express LIFR and ISL1. At least 85% of the cells in the cell population express LIFR and ISL1. At least 85% of the cells in the cell population express LIFR and ISL1. At least 86% of the cells in the cell population express LIFR and ISL1. At least 87% of the cells in the cell population express LIFR and ISL1. At least 89% of the cells in the cell population express LIFR and ISL1. At least 70% of the cells in the cell population express LIFR and ISL1. At least 90% of the cells in the cell population express LIFR and ISL1. At least 91% of the cells in the cell population express LIFR and ISL1. At least 92% of the cells in the cell population express LIFR and ISL1. At least 93% of the cells in the cell population express LIFR and ISL1. At least 94% of the cells in the cell population express LIFR and ISL1. At least 95% of the cells in the cell population express LIFR and ISL1. At least 96% of the cells in the cell population express LIFR and ISL1. At least 97% of the cells in the cell population express LIFR and ISL1. At least 98% of the cells in the cell population express LIFR and ISL1. Approximately all cells in the cell population express LIFR and ISL1.

[0065] The percentage (%) of cells expressing LIFR and ISL1 in a cell population can be measured or determined by flow cytometry. The percentage (%) of cells expressing LIFR and ISL1 in a cell population can be measured or determined by immunofluorescence. Antibodies specific to LIFR and ISL1 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0066] 4.5 Cell Types

[0067] All cells in a cell population can be human cells. A cell population may contain human cells. A cell population may consist primarily of human cells. A cell population may be AZD6414. A cell population may contain AZD6414. A cell population may contain cardiac progenitor cells. A cell population may contain human ventricular progenitor cells (HVP). A cell population may contain ventricular progenitor cells (VPC). A cell population may contain hPSC-pan-mesoderm-cardiac mesoderm-pancreatic islet ventricular progenitor cells.

[0068] 80% or more of the cells in the cell population can be HVP. 81% or more of the cells in the cell population can be HVP. 82% or more of the cells in the cell population can be HVP. 83% or more of the cells in the cell population can be HVP. 84% or more of the cells in the cell population can be HVP. 85% or more of the cells in the cell population can be HVP. 86% or more of the cells in the cell population can be HVP. 87% or more of the cells in the cell population can be HVP. 88% or more of the cells in the cell population can be HVP. 89% or more of the cells in the cell population can be HVP. 90% or more of the cells in the cell population can be HVP. 91% or more of the cells in the cell population can be HVP. 92% or more of the cells in the cell population can be HVP. 93% or more of the cells in the cell population can be HVP. 94% or more of the cells in the cell population can be HVP. 95% or more of the cells in the cell population can be HVP. 96% or more of the cells in the cell population can be HVP. 97% or more of the cells in a cell population can be HVPs. 98% or more of the cells in a cell population can be HVPs. 99% or more of the cells in a cell population can be HVPs. Approximately all the cells in a cell population can be HVPs.

[0069] 80% or more of the cells in the cell population can be VPCs. 81% or more of the cells in the cell population can be VPCs. 82% or more of the cells in the cell population can be VPCs. 83% or more of the cells in the cell population can be VPCs. 84% or more of the cells in the cell population can be HVPs. 85% or more of the cells in the cell population can be VPCs. 86% or more of the cells in the cell population can be VPCs. 87% or more of the cells in the cell population can be VPCs. 88% or more of the cells in the cell population can be VPCs. 89% or more of the cells in the cell population can be HVPs. 90% or more of the cells in the cell population can be VPCs. 91% or more of the cells in the cell population can be VPCs. 92% or more of the cells in the cell population can be VPCs. 93% or more of the cells in the cell population can be VPCs. 94% or more of the cells in the cell population can be HVPs. 95% or more of the cells in the cell population can be VPCs. 96% or more of the cells in the cell population can be VPCs. 97% or more of the cells in a cell population can be VPCs. 98% or more of the cells in a cell population can be VPCs. 99% or more of the cells in a cell population can be VPCs. Approximately all the cells in a cell population can be VPCs.

[0070] 5% or fewer of the cells in a cell population may be stem cells. 4% or fewer of the cells in a cell population may be stem cells. 3% or fewer of the cells in a cell population may be stem cells. 2% or fewer of the cells in a cell population may be stem cells. 1% or fewer of the cells in a cell population may be stem cells. No stem cells may be present in the cell population.

[0071] 5% or fewer of the cells in a cell population may be pluripotent. 4% or fewer of the cells in a cell population may be pluripotent. 3% or fewer of the cells in a cell population may be pluripotent. 2% or fewer of the cells in a cell population may be pluripotent. 1% or fewer of the cells in a cell population may be pluripotent. No more pluripotent cells are present in the cell population.

[0072] 10% or fewer of the cells in the cell population may be fully differentiated. 9% or fewer of the cells in the cell population may be fully differentiated. 8% or fewer of the cells in the cell population may be fully differentiated. 7% or fewer of the cells in the cell population may be fully differentiated. 6% or fewer of the cells in the cell population may be fully differentiated. 5% or fewer of the cells in the cell population may be fully differentiated. 4% or fewer of the cells in the cell population may be fully differentiated. 3% or fewer of the cells in the cell population may be fully differentiated. 2% or fewer of the cells in the cell population may be fully differentiated. 1% or fewer of the cells in the cell population may be fully differentiated. No cells in the cell population may be fully differentiated.

[0073] 10% or fewer of the cells in the cell population may be terminally differentiated. 9% or fewer of the cells in the cell population may be fully differentiated. 8% or fewer of the cells in the cell population may be fully differentiated. 7% or fewer of the cells in the cell population may be terminally differentiated. 6% or fewer of the cells in the cell population may be terminally differentiated. 5% or fewer of the cells in the cell population may be terminally differentiated. 4% or fewer of the cells in the cell population may be terminally differentiated. 3% or fewer of the cells in the cell population may be terminally differentiated. 2% or fewer of the cells in the cell population may be terminally differentiated. 1% or fewer of the cells in the cell population may be terminally differentiated. No cells in the cell population may be terminally differentiated.

[0074] A cell population may contain or consist of somatic cells. A cell population may contain or consist of adult cells. A cell population may not contain cardiac atrial progenitor cells.

[0075] Ventricular progenitor cells (VPCs) can be classified into cardiac lineages. VPCs can differentiate into all three cardiac lineages (cardiomyocytes, endothelial cells, and smooth muscle cells). Cultures of human cardiac progenitor cells can be obtained, for example, by culturing stem cells under conditions that predispose them to differentiate into cardiac lineages.

[0076] 4.6 Functions

[0077] The cell population can be classified as a cardiac lineage. Cells within the cell population can be classified as a ventricular lineage. Cells within the cell population may further differentiate into cardiac tissue or ventricular cardiac tissue.

[0078] Upon delivery to the ventricular wall of a subject, the cell population may not form a teratoma. The subject can be a human. The subject can be a pig. The subject can be a mouse. The subject can be a miniature pig. The cell population may contain cells engineered to be low in immunogenicity or consist of cells engineered to be low in immunogenicity. The cell population may contain cells engineered to be low in allogeneicity or consist of cells engineered to be low in allogeneicity. The cell population may contain cells that can differentiate into cardiomyocytes in vitro. The cell population may contain cells that can differentiate into beating cardiomyocytes in vitro.

[0079] After at least 15 days of in vitro differentiation, at least 70% of the cells in the cell population express cardiac troponin T. Upon delivery to the heart of a subject, the cell population differentiates into cardiac tissue. Upon delivery to the ventricular wall of a subject, the cell population differentiates into ventricular wall tissue. Upon delivery to the ventricular wall of a subject, the cell population differentiates into beating myocardium. Upon delivery to a damaged heart of a subject, the cell population repairs damaged cardiac tissue. The cell population is suitable for repairing damaged cardiac tissue. The subject can be a human. The subject can be a pig. The subject can be a mouse. The subject can be a miniature pig. The cell population is suitable for repairing damaged myocardium. Upon delivery to the heart of a subject, the cell population can form a vascularized, electrically responsive ventricular myocardial patch that secretes extracellular matrix. Upon delivery to a tissue (e.g., kidney or heart tissue), the cell population may be able to produce laminin. Upon delivery to a tissue (e.g., kidney or heart tissue), the cell population may be able to produce cardiac laminin.

[0080] 4.7 Disclaimer

[0081] The cell population may not have been sorted for Jagged 1 (JAG1) expression. The cell population may not have been sorted for Frizzled 4 expression. The cell population may not have been sorted for neurociliin-1 (NRP1) expression. The cell population may not have been exposed to reagents that bind Jagged 1. The cell population may not have been exposed to reagents that bind Frizzled 4. The cell population may not have been exposed to reagents that bind NRP1. The cells in the cell population may not have been isolated from postnatal myocardium.

[0082] The cell population may not have originated from cells separated from the heart tissue.

[0083] 4.8 Expression of myocardial related genes

[0084] Cells in a cell population may not express cardiac-related markers. Less than 10% of cells in a cell population may express cardiac-related markers. Less than 9% of cells in a cell population may express cardiac-related markers. Less than 8% of cells in a cell population may express cardiac-related markers. Less than 7% of cells in a cell population may express cardiac-related markers. Less than 6% of cells in a cell population may express cardiac-related markers. Less than 5% of cells in a cell population may express cardiac-related markers. Less than 4% of cells in a cell population may express cardiac-related markers. Less than 3% of cells in a cell population may express cardiac-related markers. Less than 2% of cells in a cell population may express cardiac-related markers. Less than 1% of cells in a cell population may express cardiac-related markers. Cardiac-related markers may include TNNT2. Cardiac-related markers may include TNNC1. Cardiac-related markers may include MYL2. Cardiac-related markers may include MYL7. Cardiac-related markers may include MYH6. Cardiac-related markers may include IRX4. Cardiac-related markers may include SSEA-3.

[0085] 4.8.1.1TNNT2

[0086] Less than 10% of the cells in the cell population express TNNT2. Less than 9% of the cells in the cell population express TNNT2. Less than 8% of the cells in the cell population express TNNT2. Less than 7% of the cells in the cell population express TNNT2. Less than 6% of the cells in the cell population express TNNT2. Less than 5% of the cells in the cell population express TNNT2. Less than 4% of the cells in the cell population express TNNT2. Less than 3% of the cells in the cell population express TNNT2. Less than 2% of the cells in the cell population express TNNT2. Less than 1% of the cells in the cell population express TNNT2. Less than 0.9% of the cells in the cell population express TNNT2. Less than 0.8% of the cells in the cell population express TNNT2. Less than 0.7% of the cells in the cell population express TNNT2. Less than 0.6% of the cells in the cell population express TNNT2. Less than 0.4% of the cells in the cell population express TNNT2. Less than 0.3% of the cells in the cell population express TNNT2. Less than 0.2% of the cells in the cell population express TNNT2. Less than 0.1% of the cells in the cell population express TNNT2.

[0087] TNNT2 expression in a cell population may be below the detection limit. The percentage (%) of cells expressing TNNT2 in a cell population may be measurable or determined by flow cytometry. The percentage (%) of cells expressing TNNT2 in a cell population may be measurable or determined by immunofluorescence. Antibodies specific to TNNT2 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0088] If measured by Western blotting, the cell population may not express TNNT2. If measured by flow cytometry, TNNT2 expression in the cell population may be below the detection limit. If measured by immunofluorescence, TNNT2 expression in the cell population may be below the detection limit. If measured by RNA sequencing, TNNT2 expression in the cell population may be below the detection limit. If measured by microarray, TNNT2 expression in the cell population may be below the detection limit. If measured by quantitative polymerase chain reaction (PCR), TNNT2 expression in the cell population may be below the detection limit. The cell population may be substantially devoid of cells expressing TNNT2. TNNT2 may be human TNNT2.

[0089] 4.8.1.2TNNC1

[0090] Less than 10% of the cells in the cell population express TNNC1. Less than 9% of the cells in the cell population express TNNC1. Less than 8% of the cells in the cell population express TNNC1. Less than 7% of the cells in the cell population express TNNC1. Less than 6% of the cells in the cell population express TNNC1. Less than 5% of the cells in the cell population express TNNC1. Less than 4% of the cells in the cell population express TNNC1. Less than 3% of the cells in the cell population express TNNC1. Less than 2% of the cells in the cell population express TNNC1. Less than 1% of the cells in the cell population express TNNC1. Less than 0.9% of the cells in the cell population express TNNC1. Less than 0.8% of the cells in the cell population express TNNC1. Less than 0.7% of the cells in the cell population express TNNC1. Less than 0.6% of the cells in the cell population express TNNC1. Less than 0.4% of the cells in the cell population express TNNC1. Less than 0.3% of the cells in a cell population express TNNC1. Less than 0.2% of the cells in a cell population express TNNC1. Less than 0.1% of the cells in a cell population express TNNC1. A cell population may be substantially devoid of cells expressing TNNC1.

[0091] TNNC1 expression in a cell population may be below the detection limit. The percentage (%) of cells expressing TNNC1 in a cell population may be measurable or determined by flow cytometry. The percentage (%) of cells expressing TNNC1 in a cell population may be measurable or determined by immunofluorescence. Antibodies specific to TNNC1 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0092] If measured by Western blotting, the cell population may not express TNNC1. If measured by flow cytometry, TNNC1 expression in the cell population may be below the detection limit. If measured by immunofluorescence, TNNC1 expression in the cell population may be below the detection limit. If measured by RNA sequencing, TNNC1 expression in the cell population may be below the detection limit. If measured by microarray, TNNC1 expression in the cell population may be below the detection limit. If measured by quantitative polymerase chain reaction (PCR), TNNC1 expression in the cell population may be below the detection limit. TNNC1 can be human TNNC1.

[0093] 4.8.1.3MYL2

[0094] Less than 10% of the cells in the cell population express MYL2. Less than 9% of the cells in the cell population express MYL2. Less than 8% of the cells in the cell population express MYL2. Less than 7% of the cells in the cell population express MYL2. Less than 6% of the cells in the cell population express MYL2. Less than 5% of the cells in the cell population express MYL2. Less than 4% of the cells in the cell population express MYL2. Less than 3% of the cells in the cell population express MYL2. Less than 2% of the cells in the cell population express MYL2. Less than 1% of the cells in the cell population express MYL2. Less than 0.9% of the cells in the cell population express MYL2. Less than 0.8% of the cells in the cell population express MYL2. Less than 0.7% of the cells in the cell population express MYL2. Less than 0.6% of the cells in the cell population express MYL2. Less than 0.4% of the cells in the cell population express MYL2. Less than 0.3% of the cells in the cell population express MYL2. Less than 0.2% of the cells in the cell population express MYL2. Less than 0.1% of the cells in the cell population express MYL2.

[0095] MYL2 expression in a cell population may be below the detection limit. The percentage (%) of cells expressing MYL2 in a cell population may be measurable or determined by flow cytometry. The percentage (%) of cells expressing MYL2 in a cell population may be measurable or determined by immunofluorescence. Antibodies specific to MYL2 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0096] If measured by Western blotting, the cell population may not express MYL2. If measured by flow cytometry, MYL2 expression in the cell population may be below the detection limit. If measured by immunofluorescence, MYL2 expression in the cell population may be below the detection limit. If measured by RNA sequencing, MYL2 expression in the cell population may be below the detection limit. If measured by microarray, MYL2 expression in the cell population may be below the detection limit. If measured by quantitative polymerase chain reaction (PCR), MYL2 expression in the cell population may be below the detection limit.

[0097] The cell population may contain virtually no cells expressing MYL2. MYL2 may be human MYL2.

[0098] 4.8.1.4MYL7

[0099] Less than 10% of the cells in the cell population express MYL7. Less than 9% of the cells in the cell population express MYL7. Less than 8% of the cells in the cell population express MYL7. Less than 7% of the cells in the cell population express MYL7. Less than 6% of the cells in the cell population express MYL7. Less than 5% of the cells in the cell population express MYL7. Less than 4% of the cells in the cell population express MYL7. Less than 3% of the cells in the cell population express MYL7. Less than 2% of the cells in the cell population express MYL7. Less than 1% of the cells in the cell population express MYL7. Less than 0.9% of the cells in the cell population express MYL7. Less than 0.8% of the cells in the cell population express MYL7. Less than 0.7% of the cells in the cell population express MYL7. Less than 0.6% of the cells in the cell population express MYL7. Less than 0.4% of the cells in the cell population express MYL7. Less than 0.3% of the cells in the cell population express MYL7. Less than 0.2% of the cells in the cell population express MYL7. Less than 0.1% of the cells in the cell population express MYL7.

[0100] MYL7 expression in a cell population may be below the detection limit. The percentage (%) of cells expressing MYL7 in a cell population may be measurable or determined by flow cytometry. The percentage (%) of cells expressing MYL7 in a cell population may be measurable or determined by immunofluorescence. Antibodies specific to MYL7 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0101] If measured by Western blotting, the cell population may not express MYL7. If measured by flow cytometry, MYL7 expression in the cell population may be below the detection limit. If measured by immunofluorescence, MYL7 expression in the cell population may be below the detection limit. If measured by RNA sequencing, MYL7 expression in the cell population may be below the detection limit. If measured by microarray, MYL7 expression in the cell population may be below the detection limit. If measured by quantitative polymerase chain reaction (PCR), MYL7 expression in the cell population may be below the detection limit.

[0102] The cell population may be essentially devoid of cells expressing MYL7. MYL7 can be human MYL7.

[0103] 4.8.1.5MYH6

[0104] Less than 10% of the cells in the cell population express MYH6. Less than 9% of the cells in the cell population express MYH6. Less than 8% of the cells in the cell population express MYH6. Less than 7% of the cells in the cell population express MYH6. Less than 6% of the cells in the cell population express MYH6. Less than 5% of the cells in the cell population express MYH6. Less than 4% of the cells in the cell population express MYH6. Less than 3% of the cells in the cell population express MYH6. Less than 2% of the cells in the cell population express MYH6. Less than 1% of the cells in the cell population express MYH6. Less than 0.9% of the cells in the cell population express MYH6. Less than 0.8% of the cells in the cell population express MYH6. Less than 0.7% of the cells in the cell population express MYH6. Less than 0.6% of the cells in the cell population express MYH6. Less than 0.4% of the cells in the cell population express MYH6. Less than 0.3% of the cells in the cell population express MYH6. Less than 0.2% of the cells in the cell population express MYH6. Less than 0.1% of the cells in the cell population express MYH6.

[0105] MYH6 expression in a cell population may be below the detection limit. The percentage (%) of cells expressing MYH6 in a cell population may be measurable or determined by flow cytometry. The percentage (%) of cells expressing MYH6 in a cell population may be measurable or determined by immunofluorescence. Antibodies specific to MYH6 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0106] If measured by Western blotting, the cell population may not express MYH6. If measured by flow cytometry, the expression of MYH6 in the cell population may be below the detection limit. If measured by immunofluorescence, the expression of MYH6 in the cell population may be below the detection limit. If measured by RNA sequencing, the expression of MYH6 in the cell population may be below the detection limit. If measured by microarray, the expression of MYH6 in the cell population may be below the detection limit. If measured by quantitative polymerase chain reaction (PCR), the expression of MYH6 in the cell population may be below the detection limit.

[0107] The cell population may be substantially devoid of cells expressing MYH6. MYH6 can be human MYH6.

[0108] 4.8.1.6IRX4

[0109] Less than 10% of the cells in the cell population express IRX4. Less than 9% of the cells in the cell population express IRX4. Less than 8% of the cells in the cell population express IRX4. Less than 7% of the cells in the cell population express IRX4. Less than 6% of the cells in the cell population express IRX4. Less than 5% of the cells in the cell population express IRX4. Less than 4% of the cells in the cell population express IRX4. Less than 3% of the cells in the cell population express IRX4. Less than 2% of the cells in the cell population express IRX4. Less than 1% of the cells in the cell population express IRX4. Less than 0.9% of the cells in the cell population express IRX4. Less than 0.8% of the cells in the cell population express IRX4. Less than 0.7% of the cells in the cell population express IRX4. Less than 0.6% of the cells in the cell population express IRX4. Less than 0.4% of the cells in the cell population express IRX4. Less than 0.3% of the cells in the cell population express IRX4. Less than 0.2% of the cells in the cell population express IRX4. Less than 0.1% of the cells in the cell population express IRX4.

[0110] IRX4 expression in a cell population may be below the detection limit. The percentage (%) of cells expressing IRX4 in a cell population may be measurable or determined by flow cytometry. The percentage (%) of cells expressing IRX4 in a cell population may be measurable or determined by immunofluorescence. Antibodies specific to IRX4 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0111] If measured by Western blotting, the cell population may not express IRX4. If measured by flow cytometry, IRX4 expression in the cell population may be below the detection limit. If measured by immunofluorescence, IRX4 expression in the cell population may be below the detection limit. If measured by RNA sequencing, IRX4 expression in the cell population may be below the detection limit. If measured by microarray, IRX4 expression in the cell population may be below the detection limit. If measured by quantitative polymerase chain reaction (PCR), IRX4 expression in the cell population may be below the detection limit.

[0112] The cell population may be essentially devoid of cells expressing IRX4. IRX4 may be human IRX4.

[0113] 4.8.1.7SSEA-3

[0114] Less than 10% of the cells in the cell population express SSEA-3. Less than 9% of the cells in the cell population express SSEA-3. Less than 8% of the cells in the cell population express SSEA-3. Less than 7% of the cells in the cell population express SSEA-3. Less than 6% of the cells in the cell population express SSEA-3. Less than 5% of the cells in the cell population express SSEA-3. Less than 4% of the cells in the cell population express SSEA-3. Less than 3% of the cells in the cell population express SSEA-3. Less than 2% of the cells in the cell population express SSEA-3. Less than 1% of the cells in the cell population express SSEA-3. Less than 0.9% of the cells in the cell population express SSEA-3. Less than 0.8% of the cells in the cell population express SSEA-3. Less than 0.7% of the cells in the cell population express SSEA-3. Less than 0.6% of the cells in the cell population express SSEA-3. Less than 0.4% of the cells in the cell population expressed SSEA-3. Less than 0.3% of the cells in the cell population expressed SSEA-3. Less than 0.2% of the cells in the cell population expressed SSEA-3. Less than 0.1% of the cells in the cell population expressed SSEA-3.

[0115] SSEA-3 expression in a cell population may be below the detection limit. The percentage (%) of cells expressing SSEA-3 in a cell population may be measurable or determined by flow cytometry. The percentage (%) of cells expressing SSEA-3 in a cell population may be measurable or determined by immunofluorescence. Antibodies specific to SSEA-3 for use in immunofluorescence and flow cytometry (FACS) are commercially available [6].

[0116] If measured by Western blotting, the cell population may not express SSEA-3. If measured by flow cytometry, SSEA-3 expression in the cell population may be below the detection limit. If measured by immunofluorescence, SSEA-3 expression in the cell population may be below the detection limit. If measured by RNA sequencing, SSEA-3 expression in the cell population may be below the detection limit. If measured by microarray, SSEA-3 expression in the cell population may be below the detection limit. If measured by quantitative polymerase chain reaction (PCR), SSEA-3 expression in the cell population may be below the detection limit. The cell population may be substantially free of cells expressing SSEA-3. SSEA-3 may be human SSEA-3.

[0117] 4.9 Cell source

[0118] A cell population may contain cells derived from stem cells or consist of cells derived from stem cells. Stem cells may be embryonic stem cells (ESCs). A cell population may contain cells derived from stem cell lines or consist of cells derived from stem cell lines. A stem cell line may be H9 cells. GMP MCB H9 is derived from the source cell line WiCell WA (WiCell Institute) 09, also known as H9. A cell population may contain cells derived from induced pluripotent stem cells (iPSCs) or consist of cells derived from induced pluripotent stem cells (iPSCs). A cell population may not contain any cells derived from totipotent cells.

[0119] Cell populations can originate from cells containing stem cells. Cell populations can be derived from stem cells. Cell populations can be derived from pluripotent cells. Cell populations can be derived from cells containing embryonic stem cells. Cell populations can be derived from cells containing induced pluripotent stem cells. Cell populations can be derived from cells containing mesoderm cells. Cells can be derived from cells containing intermediate mesoderm cells.

[0120] 4.10 Non-natural products

[0121] The cell population may be derived from one or more cells that have been expanded in vitro. The cell population may be derived from one or more cells isolated from a subject. The cell population may be artificial. The cell population may not exist in nature. The cell population may contain one or more cells that have been genetically modified or gene-edited. The cell population may be isolated from the body. The cell population may be included in a pharmaceutical composition. The cell population may be isolated from the body and included in a pharmaceutical composition.

[0122] Cells in a cell population can be, for example, autologous, allogeneic, or xenogeneic.

[0123] A cell population may be derived from one or more cells that have been expanded in vitro. A cell population may be derived from one or more cells isolated from a subject. A cell population may be artificial or composed of artificial cells. A cell population may not exist in nature, or may be composed of cells that do not exist in nature. A cell population may contain modified cells, such as those modified through CRISPR gene editing or genetic modification. A cell population may be an isolated cell population. A cell population may contain engineered cells. A cell population may contain one or more cells that have been modified or engineered in vitro or in vitro.

[0124] Cell populations according to any of the preceding claims, wherein the cell population comprises cells that have been modified or engineered in vitro. Cell populations wherein the cell populations are isolated from the body and contained in the pharmaceutical composition.

[0125] This disclosure also relates to a method for preparing a pharmaceutical composition, the method comprising combining a cell population of the present disclosure with a pharmaceutical excipient. This disclosure further relates to a method for preparing a pharmaceutical composition, the method comprising combining a cell population with a pharmaceutical excipient, wherein at least 70% of the cells in the cell population express cardiac progenitor cell (CPC)-related markers, and 1% or less of the cells in the cell population express octamer transcription factor 4 (OCT4). This disclosure further relates to a method for preparing a pharmaceutical composition, the method comprising combining a cell population with a pharmaceutical excipient, wherein at least 90% of the cells in the cell population express cardiac progenitor cell (CPC)-related markers, and 1% or less of the cells in the cell population express octamer transcription factor 4 (OCT4). The cardiac progenitor cell-related marker may be ISL1.

[0126] This disclosure also relates to the use of isolated cell populations in which at least 70% of the cells express cardiac progenitor cell (CPC)-related markers and 1% or less of the cells express octamer transcription factor 4 (OCT4). This disclosure further relates to the use of isolated cell populations in which at least 90% of the cells express cardiac progenitor cell (CPC)-related markers and 1% or less of the cells express octamer transcription factor 4 (OCT4). The cardiac progenitor cell-related marker may be ISL1.

[0127] This disclosure also relates to a composition or formulation comprising the cell population of this disclosure. The formulation may be non-natural. This disclosure also relates to a cryopreservation comprising the cell population of this disclosure. This disclosure also relates to an in vitro cell culture comprising the cell population of this disclosure.

[0128] 4.11 Process

[0129] This disclosure also relates to a method for producing the cell population of this disclosure, the method comprising harvesting cells from a cell culture, wherein the harvested cells are derived from stem cells cultured for 8 to 12 days under conditions suitable for cardiomyogenesis. This disclosure also relates to a method for producing the cell population of this disclosure, the method comprising harvesting cells from a cell culture, wherein the harvested cells are derived from stem cells cultured for 8 to 10 days under conditions suitable for cardiomyogenesis. Cardiomyogenesis conditions can be conditions suitable for stem cells to differentiate into cardiac ventricular cells. This disclosure also relates to a method for producing the cell population of this disclosure, the method comprising harvesting cells from a cell culture, wherein the harvested cells are derived from stem cells cultured for approximately 8 days under conditions suitable for cardiomyogenesis. This disclosure also relates to a method for producing the cell population of this disclosure, the method comprising harvesting cells from a cell culture, wherein the harvested cells are derived from stem cells cultured for approximately 9 days under conditions suitable for cardiomyogenesis. This disclosure also relates to a method for producing the cell population of this disclosure, the method comprising harvesting cells from a cell culture, wherein the harvested cells are derived from stem cells cultured for 10 days under conditions suitable for cardiomyogenesis.

[0130] A method for producing a cell population comprising cardiac ventricular progenitor cells, the method comprising harvesting the cell population, wherein the harvested cells are derived from stem cells cultured for 8 to 12 days under conditions suitable for cardiomyogenesis. Cardiomyogenesis conditions can be conditions suitable for stem cells to differentiate into cardiac ventricular progenitor cells.

[0131] Conditions suitable for myocardial development may include:

[0132] a. On day 0, activate Wnt / Wnt / β-catenin signaling in the culture.

[0133] b. From day 3 to day 5, Wnt / β-catenin signaling in the culture was inhibited to generate a ventricular progenitor cell (VPC) population.

[0134] Wnt / Wnt / β-catenin signaling can be activated by GSK3 inhibitors. An example of a GSK3 inhibitor is CHIR-98014. Wnt / β-catenin signaling can be inhibited by Wnt inhibitors. A Wnt inhibitor could be Wnt-C59.

[0135] Methods for generating cell populations may include knocking out pluripotent cells from harvested cells to produce a purified VPC population. Knockout of pluripotent cells from harvested cells may include removing cells expressing pluripotency markers from harvested cells. A VPC population knocked out of pluripotent cells from harvested cells may include a cell population expressing TRA-1-60. A VPC population knocked out of pluripotent cells from harvested cells may include a cell population expressing TRA-1-60 by magnetically activated cell sorting. The stem cells used in this method may be induced pluripotent cells or embryonic stem cells. In the cell population produced by this method, at least 70% of the cells express cardiac progenitor cell (CPC)-related markers, and 1% or less of the cells express octamer transcription factor 4 (OCT4).

[0136] The methods disclosed herein may include various manufacturing steps that require manipulation of cells, such as inoculation, feeding, dissociation of adherent cultures, or washing.

[0137] 4.12 Process Abandonment

[0138] This disclosure also relates to a method for generating the cell population of this disclosure, wherein the method does not include sorting cells for NRP1 expression. The method may not include sorting cells for Jagged 1 expression. The method may not include sorting cells for Frizzled 4 expression. The method may not include using an agent that binds neuropilin-1 (NRP1). The method may not include using an agent that binds Jagged 1. The method may not include using an agent that binds Frizzled 4. The method may not include seeding cells onto a substrate including LN-521, LN-51 1, or LN-221. Therefore, the method of this disclosure may exclude the unnecessary step of sorting for markers selected from Jagged, Frizzled, or NRP1. As demonstrated in the examples, the method for producing a cell population suitable for a cell therapy product for treating, for example, heart failure requires differentiation for at least 8 days. Cells can then be sorted to eliminate cells expressing TRA-1-60, and sorting for NRP1, Jagged, or Frizzled expression is not required. Cell populations expressing ISL1 and containing 1% or less of cells expressing OCT4 lack the capacity for pluripotency (e.g., as measured by HEC assays) and are therefore suitable for use as cell therapy products.

[0139] 4.13 Products characterized by processes

[0140] This disclosure also relates to a cell population produced by a method including harvesting a cell population, wherein the harvested cells are derived from stem cells and have been cultured for 8 to 12 days under conditions suitable for cardiomyogenesis. The cardiomyogenesis conditions can be those suitable for stem cells to differentiate into cardiac ventricular progenitor cells.

[0141] This disclosure also relates to a cell population comprising cardiac ventricular progenitor cells, wherein the cell population is produced by a method comprising harvesting cells from a cell culture, wherein the harvested cells are derived from stem cells, and wherein the cells have been cultured for 8 to 12 days under conditions suitable for cardiomyogenesis prior to harvesting. Cardiomyogenesis conditions can be conditions suitable for stem cells to differentiate into cardiac ventricular progenitor cells.

[0142] Conditions suitable for myocardial development may include:

[0143] a. On day 0, activate Wnt / Wnt / β-catenin signaling in the culture.

[0144] b. From day 3 to day 5, Wnt / β-catenin signaling in the culture was inhibited to generate a ventricular progenitor cell (VPC) population.

[0145] Wnt / Wnt / β-catenin signaling can be activated by GSK3 inhibitors. An example GSK3 inhibitor is CHIR-98014. Wnt / β-catenin signaling can be inhibited by Wnt inhibitors. A Wnt inhibitor could be Wnt-C59.

[0146] Methods for generating cell populations may include knocking out pluripotent cells from harvested cells to produce a purified VPC population. Knocking out pluripotent cells from harvested cells may include knocking out cells that express pluripotency markers. Knocking out pluripotent cells from the harvested CPC population includes knocking out cell populations expressing TRA-1-60. Knocking out pluripotent cells from the harvested CPC population includes knocking out cell populations expressing TRA-1-60 through magnetically activated cell sorting. The stem cells used in this method may be induced pluripotent cells or embryonic stem cells. In the cell population produced by this method, at least 70% of the cells express cardiac progenitor cell (CPC)-related markers, and 1% or less of the cells express octamer transcription factor 4 (OCT4).

[0147] Cells can be harvested using methods known in the art, such as trypsin dissociation. Once harvested, the cells will no longer be in a 2D monolayer culture, but may be in suspension (e.g., in cell culture medium) or in the form of a cell pellet. The harvested cells may be contained in vials or tubes.

[0148] 4.14 Treatment Methods

[0149] This disclosure also relates to a method of treating or preventing a disease in a subject, the method comprising administering to a subject in need an effective amount of the cell population of this disclosure, wherein at least 70% of the cells in the cell population express cardiac progenitor cell (CPC)-related markers, and 1% or less of the cells in the cell population express octamer transcription factor 4 (OCT4) or OCT4 / OCT3. The cell population may be allogeneic or allogeneic relative to the subject. The cell population may comprise or consist of cells that are allogeneic or allogeneic relative to the subject. The cell population may be administered in conjunction with immunosuppressive therapy. The cell population may comprise or consist of cells derived from autologous cells relative to the subject. The cell population may be administered to the subject without immunosuppressive therapy. The cell population may comprise cells derived from one or more cells not isolated from the subject. The cell population may comprise cells derived from one or more cells isolated from the subject. The disease may be heart failure. The subject may be a person. The subject may be an adult. The subject may have heart disease. The disease may be heart failure. The disease can be chronic ischemic cardiomyopathy. Subjects may have chronic ischemic cardiomyopathy. The disease can be myocardial infarction. Subjects may have myocardial infarction. Subjects may be receiving immunosuppressive therapy. This disclosure also relates to the use of cell populations for the prevention or treatment of diseases associated with myocardial loss and / or fibrotic scarring.

[0150] This disclosure also relates to a method of treating or preventing a disease in a subject, the method comprising: a) producing a cell population according to this disclosure in vitro, and b) administering the cell population to the subject, wherein at least 70% of the cells in the cell population express cardiac progenitor cell (CPC)-related markers, and 1% or less of the cells in the cell population express octamer transcription factor 4 (OCT4) or OCT4 / OCT3.

[0151] 4.15 Pharmaceutical Composition

[0152] This disclosure also relates to a pharmaceutical composition comprising the cell population and pharmaceutical excipient of this disclosure. This disclosure further relates to a pharmaceutical composition comprising a cell population and pharmaceutical excipient, wherein at least 70% of the cells in the cell population express cardiac progenitor cell (CPC)-related markers, and 1% or less of the cells in the cell population express octamer transcription factor 4 (OCT4) or OCT4 / OCT3.

[0153] The pharmaceutical composition may contain a freezing medium.

[0154] 4.16 Container

[0155] This disclosure also relates to a container comprising a pharmaceutical composition of this disclosure or a cell population of this disclosure, wherein at least 70% of the cells in the cell population express cardiac progenitor cell (CPC)-related markers, and 1% or less of the cells in the cell population express octamer transcription factor 4 (OCT4) or OCT4 / OCT3. The container may be a storage vial, a cryogenic vial, an injection device, or a syringe.

[0156] 4.17EPC 2000

[0157] This disclosure also relates to a pharmaceutical composition in a method for treating or preventing a disease in a subject of need, wherein the pharmaceutical composition comprises the cell population of this disclosure. This disclosure further relates to a pharmaceutical composition in a method for treating or preventing a disease in a subject, wherein the method of treating or preventing the disease includes administering the pharmaceutical composition to the subject, wherein at least 70% of the cells in the cell population express cardiac progenitor cell (CPC)-related markers, and 1% or less of the cells in the cell population express octamer transcription factor 4 (OCT4) or OCT4 / OCT3. The disease can be any disease disclosed herein.

[0158] 4.18Swiss type

[0159] This disclosure also relates to the use of the cell population of this disclosure in the manufacture of a medicament for treating or preventing a disease in a subject. The cell population is wherein at least 70% of the cells express cardiac progenitor cell (CPC)-related markers, and 1% or less of the cells express octamer transcription factor 4 (OCT4) or OCT4 / OCT3. The disease can be any disease disclosed herein.

[0160] 5 terms

[0161] 5.1AZD6414

[0162] AZD6414 cells were derived from hESCs using a differentiation protocol (adding a GSK3 inhibitor on day 0 of differentiation and a WNT antagonist on day 3 of differentiation). Cells were harvested on day 8 of differentiation and then negative magnetic activation cell sorting was used to eliminate potential pluripotent stem cells (cells expressing the pluripotency-associated protein TRA-1-60 were eliminated).

[0163] The starting material used for the production of AZD6414 is GMP MCB H9 derived from the original cell line H9 (WIC-WA09) (human embryonic cell line).

[0164] 5.2 Cellular efficacy

[0165] Pluripotent cells are cells that can self-renew and proliferate while remaining undifferentiated and can be induced to differentiate into specialized cell types under appropriate conditions. As used herein, the term “pluripotent cell” encompasses embryonic stem cells and other types of stem cells, including fetal, amniotic, or adult stem cells. Exemplary human stem cell lines include the H9 human embryonic stem cell line. Other exemplary stem cell lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection.

[11]

[0166] Pluripotent stem cells have the potential to differentiate into any of the following three germ layers: endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue). As used herein, the term “pluripotent stem cell” also encompasses “induced pluripotent stem cell” or “iPSC” (a type of pluripotent stem cell derived from non-pluripotent cells). Examples of parental cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype in various ways. Such “iPS” or “iPSC” cells can be generated by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods for inducing iPS cells are known in the art [12-15].

[0167] The ability to produce offspring that, under appropriate conditions, can differentiate into cell types that collectively exhibit characteristics associated with cell lineages from all three germ layers (endoderm, mesoderm, and ectoderm) is a characteristic of pluripotent stem cells. The expression or absence of certain combinations of molecular markers is also a characteristic of pluripotent stem cells. For example, human pluripotent stem cells express at least some of the following markers, and may express all of them: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, SOX2, E-cadherin, UTF-1, OCT4, Rexl, and NANOG.

[0168] Pluripotent cells can produce a limited number of other specific cell types. For example, induced pluripotent cells can form endoderm cells. Additionally, pluripotent hematopoietic stem cells can differentiate themselves into several types of blood cells, including lymphocytes, monocytes, and neutrophils. Oligopotent cells are adult stem cells that can differentiate into only a few different cell types. For example, lymphoid or bone marrow stem cells can form cells of the lymphoid or bone marrow lineage, respectively. Unipotent cells form a single cell type. For example, spermatogonial stem cells can only form sperm cells. Totipotent cells have the ability to form the entire organism. For example, in mammals, only the zygote and the first cleavage blastomeres are totipotent. Differentiated cells include, but are not limited to, pluripotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells.

[0169] Non-pluripotent cells are not pluripotent cells. Examples of such cells include differentiated cells and progenitor cells. Examples of differentiated cells include, but are not limited to, cells from tissues selected from bone marrow, skin, skeletal muscle, adipose tissue, and peripheral blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T cells. The starting cells used to generate induced pluripotent cells, endoderm progenitor cells, and hepatocytes can be non-pluripotent cells.

[0170] 5.3 Cardiac cells

[0171] The term cardiomyocyte refers to the muscle cells of the heart (e.g., cardiac myocytes). Cardiomyocytes typically express one or more heart-specific markers on their cell surface and / or in their cytoplasm. Suitable cardiomyocyte-specific markers include, but are not limited to, cardiac troponin I, cardiac troponin-C, tropomyosin, caveolin-3, GATA-4, myosin heavy chain, myosin light chain-2a, myosin light chain-2v, rennetine receptor, and atrial natriuretic factor.

[0172] 5.4 Cell dissociation

[0173] Cell dissociation is a process during cell passage in which cells detach from the surface of a tissue culture. Dissociated cells do not adhere to each other or to a cell surface. Dissociated cells may be in a suspension. Dissociated cells may be single cells in a suspension. Cell passage of adherent cells involves cell passage. The adherent cells can then be dissociated from the substrate or flask (e.g., by using a protease, such as trypsin or collagenase), a medium may be added, optional washing may be performed (e.g., by centrifugation), and the cells can then be replated or reseeded into one or more culture vessels. Other methods for removing non-adherent cells include non-enzymatic treatment steps (e.g., using EDTA).

[0174] 5.5 Conditions suitable for myocardial development

[0175] Methods suitable for differentiating stem cells into cardiac ventricular progenitor cells are described in Foo et al. [6], WO 2016 / 029122 A1 [7], WO 2017 / 172086 A1 [8], WO 2018 / 100433 A1 [9] and WO 2019 / 038587 A1

[10] .

[0176] 5.6 genes

[0177] Table 1: Gene Identifiers

[0178]

[0179] Expression can refer to gene expression (e.g., as measured by transcript, mRNA analysis, or RNA seq) or protein expression (e.g., as measured by immunofluorescence, Western blotting, or flow cytometry).

[0180] 5.7 Harvesting Cells

[0181] The process of harvesting cells refers to the separation of cells or cell populations from growth or culture media. Cell populations growing in cultures can be harvested by dissociating and resuspending cells.

[0182] 5.8 High-efficiency culture assay for evaluating stem cell contamination in cell therapy products

[0183] Undifferentiated hPSCs have the potential to form teratomas, and therefore, residual undifferentiated hPSCs are one of the major risks of tumor formation from hPSC-derived cell therapy products. Among currently available assays, the high-efficiency culture (HEC) assay has been reported to be one of the most sensitive for detecting residual undifferentiated hPSCs [16-18]. Cell therapy products are cultured for 7 days under conditions optimized for stem cell proliferation. Colonies of proliferating undifferentiated stem cells are identified by staining with fluorescent antibodies targeting undifferentiated stem cell marker proteins. Oct4-positive colonies with more than 7 cells (representing 3 divisions within 7 days) are considered positive colonies.

[0184] International validation trials conducted under the HESI CT-TRACS initiative showed that, in high-efficiency culture assays, approximately 50% of undifferentiated cells incorporated into differentiated populations would be detected as colonies

[18] .

[0185] 5.9 Octameric transcription factor 4 (OCT4)

[0186] OCT4 (gene POU5F1 (POU class 5 homeobox 1) (gene ID: 5460, HUGO Gene Nomenclature Committee, HGNC)) (also known as OCT3 or OCT3 / 4) is a protein encoded by the POU5F1 gene in humans. It was initially identified as a DNA-binding protein that activates gene transcription via a cis-element containing an octamer motif. It is expressed in pluripotent embryonic stem cells and germ cells. This protein is closely associated with maintaining the self-renewal and pluripotency of undifferentiated embryonic stem cells. OCT4 is not only a major regulator of pluripotency controlling lineage typing, but it is also the first and most recognized biomarker for identifying pluripotent ES cells. It has been associated with tumorigenesis in adult germ cells.

[0187] 5.10 Stem Cells

[0188] To avoid any doubt, the human embryonic cells used in this disclosure may be derived from human oocytes activated by parthenogenesis.

[0189] Stem cells can be pluripotent. Stem cells do not have to be pluripotent.

[0190] 5.11 Teratoma

[0191] Teratomas are tumors containing tissues from all embryonic germ tracts (endoderm, ectoderm, and mesoderm).

[0192] 5.12TRA-1-60 (podocyte marker protein)

[0193] TRA-1-60 is a cell surface antigen expressed in human embryonic stem cells, embryonic cancer cells and induced pluripotent stem cells (iPS)

[19] .

[0194] 5.13 Ventricular progenitor cells

[0195] Ventricular progenitor cells are cells that, when delivered to the ventricular wall of the heart, have the ability to differentiate into functional, beating ventricular cardiomyocytes.

[0196] 6. Example 1: Materials and Methods

[0197] 6.1 Cardiac progenitor cell differentiation protocol

[0198] A schematic diagram of a protocol for differentiating cardiac progenitor cells from stem cells is shown in Figure 1 middle.

[0199] 6.2 VPC harvesting and TRA-1-60 magnetically activated cell sorting

[0200] VPC harvesting and TRA-1-60 sorting were performed according to the standard protocol. Equipment and reagents are provided in Tables 2 and 3.

[0201] Table 2: Equipment used for cell harvesting and TRA-1-60 magnetic cell sorting

[0202]

[0203] Table 3: Reagents used for TRA-1-60 magnetic cell sorting in cell harvesting

[0204]

[0205] 6.2.1 Cell Harvesting

[0206] In short, ventricular progenitor cells were harvested on day 8. To isolate the cells, Accutase was administered at 0.1 mL / cm². 2 The concentration was added to the adherent cell culture and incubated at 37°C for 2 to 5 minutes. Cold AutoMACS running buffer (0.1 mL / cm³) was then added. 2 Add the 1,000 μL of Accutase to the culture vessel to dilute the 1,000 μL. Resuspend the isolated cells to produce a single-cell suspension and transfer them to centrifuge tubes. Centrifuge the single-cell suspension at 300 x g for 6 minutes. Discard the supernatant and resuspend the cell pellet in the single-cell suspension in AutoMACS running buffer to a concentration corresponding to 0.1 mL / cm³. 2 The volume of the collection container was determined. The single-cell suspension was then centrifuged at 300 x g for 6 minutes. The supernatant was discarded, and the cell pellet was resuspended in the single-cell suspension in AutoMACS running buffer to a concentration corresponding to 0.01 mL / cm³. 2 The volume of the collection container. Cell counting was performed using an NC-202.

[0207] 6.2.2 TRA-1-60 cell sorting

[0208] Calculate the total number of unsorted ventricular progenitor cells to add the correct amount of anti-TRA-1-60 conjugated microbeads / 1 mL. Centrifuge the single-cell suspension at 300 x g for 6 minutes at 4 °C. Set the pellet at 66.7 x 10⁻⁶ cm⁻¹. 6 Resuspend cells / mL (total cells) in cold AutoMACS running buffer. Add 667 μL of anti-TRA-1-60 conjugated beads / mL and incubate at 4°C on an AutoMACS cooling rack (50 rpm in a low-temperature environment) for 10 minutes. After incubation, divide the cells into 40 x 10⁻⁶ cells / mL tubes. 6 Add 1 mL of AutoMACS running buffer to each cell and centrifuge at 300 x g for 6 minutes at 4°C. Then centrifuge the cells at 40 x 10⁻⁶ cm⁻¹. 6 Resuspend the total cells in cold AutoMACS run buffer at / mL. Then place the sample in an AutoMACS Pro separator. After sorting, the sorted cell population can be frozen.

[0209] 6.3 Highly Efficient Culture (HEC) Assay for Assessing Stem Cell Contamination in Cell Therapy Products

[0210] Perform HEC assays according to standard protocols. In short, thaw and / or dissociate the cell therapy product to be evaluated. Transfer 9 million cells to 50 ml tubes. A 9 million cell assay will allow prediction of up to 220 cells out of approximately 1 billion doses, assuming a 50% colony formation rate. Seed cells into 6-well plates and incubate in full E8 without Rock inhibitors for 7 days, changing the medium every 2 to 3 days.

[0211] Cells were then fixed with PFA according to standard protocol and stained with alkaline phosphate stain (Vector, SK-5300) and antibody stain. The plates were imaged on ImageXpress microplates. Materials used in the HEC assay are shown in Table 4.

[0212] Table 4: Reagents used for HEC determination

[0213]

[0214] 6.4 Flow Cytometry

[0215] Flow cytometry was performed according to the standard protocol. Reagents are shown in Table 5.

[0216] Table 5: Reagents used in flow cytometry

[0217]

[0218] 6.5 RNA sequencing

[0219] RNA sequencing was performed according to the standard protocol. In short, frozen cells were thawed and cell counts were determined using standard trypan blue exclusion assays. After assessing cell counts and viability, scRNA sequencing was performed using the Chromium Next GEM Single Cell 3' Kit v3.1 (10xGenomics). In short, cells and reagents were captured using the Chromium Controller according to the manufacturer's recommendations, followed by reverse transcription, cDNA amplification, and library construction. Libraries were sequenced using a NovaSeq 6000 (Illumina) instrument with 28, 8, and 91 cycles.

[0220] 6.6 RNA isolation, RT-PCR and RT-qPCR .

[0221] RT-PCR was performed according to the standard protocol. In short, a cell pellet containing approximately one million live cells was lysed using RLT buffer (Qiagen) and stored at -80°C or processed immediately. RNA was isolated from the cell lysate using column-based separation with on-column DNA digestion (Qiagen, Rneasy Mini Kit), and RNA concentration was determined using a spectrophotometer (Nanodrop). A two-step RT-qPCR was then performed. For cDNA generation, 1 μg of RNA was typically reverse transcribed into cDNA, and the results were measured using TaqMan master mix and TaqMan RT-PCR assay. Subsequent qPCR was performed using 12 ng of cDNA. qPCR was performed using a QuantStudio 7 Flex instrument and analyzed using QuantStudio software. Reagents: TaqMan ™ Fast Advanced Master Mixture (2X), Catalog No.: 4444557; High-Capacity cDNA Reverse Transcription Kit, Catalog No.: 4368814; Instrument Type: QuantStudio ™ 7 Flex.

[0222] 6.7 Primer and probe sequences

[0223] The primers and probes used in the methods of this disclosure are known in the art. See also Table 6.

[0224] Table 6: Example Primers and Probes

[0225]

[0226] 6.8H9 cells

[0227] GMP MCB H9 is derived from the source cell line WiCell WA (WiCell Institute) 09, also known as H9. This material consists of the hESC line first described by Thomson et al.

[20] , which was selected based on the standard selection of these cells based on their developmental potential to form trophoblasts and derivatives of all three germ layers (endoderm, ectoderm, and mesoderm) after growth without a feeder layer. H9 cells have been used in clinical research [21,22]. In 2009, the current GMP library of H9 cells was established by Waidman Biomanufacturing (WiCell Analytical Qualification) from passage 27 cells.

[0228] Example 2: Differentiation window of cardiac progenitor cells

[0229] 7.1 Introduction

[0230] Foo et al. [6] described the generation of ventricular progenitor cells (HVPs) derived from human pluripotent stem cells (hPSCs). Foo et al. specifically described a method in which HVPs were harvested and enriched by knockout of cells positive for pluripotency markers on day 6 of differentiation from embryonic stem cells (ESCs). [6] Foo et al. selected day 6 as the harvest window for HVPs based on peak ISL1 expression and the lack of teratoma formation after implantation of day 6 HVPs into mouse kidneys, with or without a pre-sorting step of knockout of pluripotent cells from the cells. Foo et al. specifically identified day 6 of differentiation as a unique developmental window for HVP implantation.

[0231] 7.2 Reassessing Cardiac Progenitor Cell Differentiation

[0232] The inventors have confirmed that, under suitable conditions, populations differentiated from stem cells into cardiomyocytes exhibit favorable gene expression changes from a pluripotent state to a differentiated cardiomyocyte population. Figure 2 ).

[0233] However, the inventors surprisingly found that, as recommended by Foo et al., the cardiac progenitor cell population harvested on day 6 from stem cell differentiation still expressed unacceptable levels of pluripotency-related genes. Teratomas were also found in the kidneys of mice injected with unsorted cell populations from days 0, 5, and 6 (Table 7). Figure 3 ).

[0234] Table 7: Teratoma formation via implanted cardiac progenitor cell populations

[0235]

[0236] With T175cm 2 Flask-formed cells; grafts formed under all conditions; TRA-1-60: a marker of undifferentiated stem cells. Grafts were injected subrenally into mice and evaluated 2 months post-transplantation.

[0237] An alternative differentiation protocol was designed in which cardiac progenitor cell populations were harvested from cell cultures on day 8. FACS analysis revealed that on day 8, the cardiac ventricular progenitor cell (VPC) population retained the expression of the cardiac progenitor-associated marker (ILS1) and had reduced expression of pluripotency markers (including TRA-1-60, OCT4, NANOG, and SOX2) (Table 8). Even after TRA-1-60 sorting, cells expressing OCT4 remained present at high levels in the cells harvested on day 6. Figure 4 In contrast, the percentage of OCT4-expressing cells harvested on day 8 and knocked out by TRA-1-60 was almost undetectable (approximately 0.1%). Figure 4In other words, unlike the group on day 6, on day 8 the cells are still in the state of cardiac progenitor cells, but have transitioned from a dangerous pluripotent state.

[0238] Table 8: Gene Expression Analysis

[0239]

[0240] ISL1: a marker of cardiac progenitor cells; TRA-1-60, OCT4, NANOG, and SOX2: markers of undifferentiated stem cells. Expression percentage as assessed by FACS.

[0241] 7.3 Detection of residual pluripotency in cell cultures

[0242] To better understand the contamination of undifferentiated cells in cell populations harvested from day 6 cells, further analysis of undifferentiated stem cell markers was performed using a high-efficiency culture (HEC) assay. The HEC assay is considered one of the most sensitive methods for detecting residual undifferentiated pluripotent stem cells in cell populations for cell therapy

[23] . The HEC assay is suitable for detecting low numbers of undifferentiated stem cell contamination within differentiated cell populations. The HEC assay can detect colonies formed by PSCs using a high-efficiency culture system that is conducive to PSC growth, and the limit of detection (LOD) has been reported to be 0.0002% [23,24].

[0243] The expression of pluripotency-related genes (OCT4 and NANOG) was assessed using HEC assays to evaluate cell populations before and after TRA-1-60 knockout. As a positive control, pluripotent (undifferentiated) stem cells (H9) were incorporated into the cell population on day 8 (Table 9).

[0244] Table 9: Pluripotent gene expression in harvested cardiac progenitor cell populations

[0245]

[0246] Inclusion criteria: Each colony contained at least 8 cells, representing 3 cell divisions within 7 days. OCT 4+ cells were measured by FACS; all cell populations were derived from H9 cells. ~D5 cells were estimated because colonies were too close together to be definitively identified as individual colonies.

[0247] The results of HEC assays showed that, as measured by OCT4 and / or NANOOG expression, day 8 colonies of harvested cardiac progenitor cell populations after TRA-1-60 cell removal had very low levels of residual undifferentiated pluripotent stem cells (LSCs). Figure 5 ).

[0248] Embryonic stem cell-associated genes (ESRG) and long intergenic nonproteins encoding RNA 678 (LINC00678) and Lin28A have also been identified as markers of undifferentiated stem cells in differentiated cell therapy products

[25] . The inventors evaluated whether the expression of these genes predicted colony formation in HEC assays. The mRNA levels of ESRG, NANOG, and LINC00678 (as measured by qPCR) were able to detect the incorporation of undifferentiated stem cells in cultures (0.0001% H9 incorporation), but did not predict colony formation in HEC assays ( Figure 5 In other words, ESRG, NANOG, or LINC00678 qPCR are less sensitive than HEC assays as assays for residual pluripotency. In contrast, OCT4 mRNA levels measured by qPCR predict colony formation in HEC assays. Figure 5 Lin28A qPCR could not distinguish between sorted and unsorted cardiac ventricular progenitor cells.

[0249] Surprisingly, TRA-1-60 FACS for both sorted and unsorted cell populations failed to predict HEC colony formation. In other words, TRA-1-60 FACS is less sensitive than HEC assays as a measure of residual pluripotency. Figure 5 Specifically, the TRA-1-60 FACS of cells harvested on day 5 of sorting could not predict the HEC colony-forming potential of that population. In contrast, the OCT4 FACS value predicted HEC colony formation. Figure 5 (Table 9).

[0250] In further studies, OCT4 expression (as measured by qPCR and FACs) showed an increase with dose-dependent incorporation of heart progenitor cell cultures on day 8 of harvest (Table 10), demonstrating the sensitivity of qPCR and FACs assays.

[0251] Table 10: Expression of undifferentiated markers in differentiated cell populations harvested on day 8 after incorporation.

[0252]

[0253] VPC: Ventricular progenitor cells

[0254] 7.4 Summary

[0255] In summary, measuring OCT4 levels using both mRNA and FACS to predict HEC assay results, with HEC colony formation considered the most sensitive assay for assessing residual pluripotency in cell therapy products, is appropriate. Therefore, measuring OCT4 levels in cell therapy products using either mRNA or FACS is considered a suitable method for assessing the risk of teratoma formation in harvested cardiac progenitor cell populations. Furthermore, HEC assay-validated OCT4 FAC and qPCR analyses demonstrated the superiority of the day 8 cardiac progenitor cell population over the day 6 population (Table 8). Figure 5 ).

[0256] Example 3: Manufacturing of AZD6414

[0257] Based on the novel differentiation scheme described in Example 2, the inventors have designed a method for producing a cell therapy product (AZD6414) comprising a population of cardiac ventricular progenitor cells for treating heart disease.

[0258] The starting material for AZD6414 was produced in GMP MCB H9 cells derived from the source cell line H9 (WIC-WA09), a human embryonic cell line. Manufacturing began with thawing H9 cells from the MCB, followed by cell culture (expansion and differentiation) in 2D adherent monolayers using tissue culture flasks. Differentiation cultures containing cardiac progenitor cells were then harvested, purified, and cryopreserved.

[0259] Expansion begins with thawing frozen cells from the MCB. Cells are then washed and expanded in 2D monolayers (tissue culture flasks). Monitoring during expansion includes cell viability and live cell count, as well as microscopic examination to determine hESC morphology and cell confluence according to standard operating procedures. Culture medium is changed daily.

[0260] The differentiation phase begins with seeding the expanded cells into the culture medium at a controlled cell density. The culture medium is changed daily, and cells (morphology and confluence) are monitored according to standard operating procedures.

[0261] On the second day after vaccination, cardiac differentiation begins as follows:

[0262] • Day 0 GSK3 inhibition: Replace the growth medium (mTeSR1) with differentiation medium (RPMI 1640 / B27 with insulin removed and containing CHIR-98014) (a cell-permeable GSK3 inhibitor).

[0263] • Day 1 GSK3 inhibition removal: Replace differentiation medium with insulin-free RPMI 1640 / B27.

[0264] • Day 3 WNT suppression: Replace the culture medium with RPMI 1640 / B27 containing Wnt-C59 and insulin removed.

[0265] • Day 5 WNT inhibition removal: Replace the culture medium with insulin-free RPMI 1640 / B27.

[0266] • On day 7, replace the culture medium with insulin-free RPMI 1640 / B27.

[0267] • On day 8, cells were harvested, cardiac progenitor cells (AZD6414) were purified, formulated, and cryopreserved.

[0268] The purification phase begins with harvesting differentiated cells, followed by washing and incubation with anti-TRA-1-60 immunomagnetic beads. Purification is achieved by using magnetically activated cell sorting to eliminate cells expressing the pluripotency-associated protein TRA-1-60.

[0269] The purified cells are then concentrated, and the culture medium from the purification stage is removed. Next, the concentrated cells are reconstituted in cryogenic medium. The drug product is then filled into cryovials, visually inspected and labeled, and then transferred to a controlled-rate freezer for deep cryopreservation. Once deep cryopreservation is complete, the frozen drug product vials are transferred to gaseous liquid nitrogen for long-term storage.

[0270] Example 4: In vivo toxicology of AZD6414

[0271] Cardiac delivery of AZD6414 was evaluated using a toxicology procedure consisting of rodent studies, large animal studies, and in vitro assessments. Key safety concerns regarding AZD6414 are related to teratoma and teratoma formation.

[0272] 9.1 Assess the risk of teratomas and malignant teratomas

[0273] As described in Example 2, a key risk associated with pluripotent stem cell-derived cells is teratoma formation (tumors containing tissues from all embryonic germline [endoderm, ectoderm, and mesoderm]). The H9 hESC line was used as the source material in the manufacture of AZD6414, and the manufacturing process was designed to remove residual pluripotent cells (TRA-1-60 marker) using magnetically activated cell sorting after differentiation into cardiac progenitor cells via negative sorting. Furthermore, residual levels of pluripotent cells were controlled using pluripotency-related markers (e.g., OCT3 / 4) at the time of drug product release.

[0274] Combinatorial analysis showed that in vitro analysis provided greater sensitivity and more transformable data to define pluripotent stem cell contamination levels compared to in vivo studies (Table 11). As described in Example 2, in vitro HEC assays are considered highly sensitive and more likely to identify potential pluripotent cell contaminants than conventional in vivo studies

[23] . HEC assays have been reported to detect iPSCs incorporated into primary human mesenchymal stem cells or human neurons at colony-forming rates of 0.001% to 0.01%. This is a higher sensitivity than that achievable in vivo.

[0275] Table 11: Results of in vitro and in vivo studies of potential teratomas and malignant teratomas

[0276]

[0277] hESC = Human embryonic stem cells; HEC = High-efficiency culture; NA = Not applicable; SC = Subcutaneous; TNC = Too many to count.

[0278] Example 5: Study of AZD6114 in the heart of mice after MI (Study BS002651-08)

[0279] To investigate the effects of H9-derived AZD6414 on rodent cardiac function, immunocompromised mice (NOD / SCIDγ) were subjected to myocardial infarction (MI) through permanent occlusion of the left ventricular artery (LAD). Two million AZD6414 cells were directly injected into the border zone of the infarcted heart (two injection sites), and cardiac function was assessed by echocardiography on days 1, 28, and 49. AZD6414 attenuated adverse left ventricular remodeling (end-diastolic volume and end-systolic volume) and tended to improve LVEF at 49 days post-injection. Figure 7 Immunohistochemical analysis of human nucleoli revealed small clusters of cells injected into the myocardium on day 49. No evidence of teratoma or teratoma formation due to AZD6414 derived from H9 was observed.

[0280] Example 6: GLP toxicology study.

[0281] 6.1 Materials and Methods

[0282] 6.1.1 Subcutaneous injection of HVP cells into NSG mice On day 1, five groups of NSG mice (strain: NOD.Cg-Prkdc) were injected subcutaneously. scid Il2rg tm1Wjl(SzJ, from Jackson Lab, USA) Placebo (15 animals), HVP cells only (15 animals), HVP cells with undifferentiated human embryonic stem cells (0.3% or 1%, 7 animals), or pluripotent cells (positive control, 5 animals) were administered once in the diluent control (Matrigel). The target dose level for HVP cells with or without impurities was 20 million cells per animal in a volume of 0.2 mL. Mice were sacrificed at 6 months or earlier due to large tumor growth (positive control animals only). Tissues were examined microscopically to assess evidence of HVP cell toxicity and tumorigenicity.

[0283] 6.1.2 Pig experiments used for GLP toxicology / efficacy studies .

[0284] 6.1.2.1 Myocardial infarction and immunosuppression in pigs Male and female Göttingen miniature pigs (25kg to 30kg) were obtained from the Sinclair Research Center in the United States. After anesthesia, small incisions were made in the carotid artery and jugular vein, a small opening was made in the artery, and a sheath was inserted. Heparin was administered. to To maintain an activated clotting time, which is twice the baseline activated clotting time level, the guiding catheter is advanced into the mouth of the LAD. The balloon catheter is introduced by advancing it through the guiding catheter into the LAD and positioned below the first diagonal branch of the LAD, then inflated to occlude the artery for 90 minutes in males and 120 minutes in females. It is then deflated to reperfusion the ischemic area. All procedures and treatments were performed according to appropriate animal welfare standards and in accordance with the regulatory guidelines of Charles River, Mattawan, Michigan (MI, USA) (IACUC 1974-061). Two weeks after the myocardial infarction procedure, pre-drug cardiac MRI was performed between day -5 and day -1 to assess left ventricular ejection fraction (LVEF). Animals deemed suitable for assignment to the study were selected based on infarction survival and meeting the inclusion criteria of LVEF ≤ 45% and / or a reduction in LVEF of at least 15% relative to baseline. Animals meeting the inclusion criteria were randomly assigned to either the vector group or the HVP cell group (low dose, 1.0 × 10⁻⁶). 8 10 cells; medium dose, 3.0 x 10 8 Cells; high dose, 6.0 x 10⁻⁶ 8(cells). For immunosuppression, a combination of cyclosporine A, methylprednisolone, and abatacept was used (REF, Romagnuolo et al. Stem Cell Reports 12, 967-981; Nat Cell Biol paper). From D-6 to D84, except for D1, cyclosporine A was administered orally twice daily ( to On the morning of day 1, the dose of cyclosporine A was [missing value]. Intravenous infusion. Starting on day 1, administer methylprednisolone (…). Until D3, then gradually reduced to (D4 to D6); gradually decrease to (D7 to D9) and gradually decrease from D10 to The remainder of the study will continue. Starting on day 1, abatacept will be administered via intravenous infusion over 30 minutes every two weeks. Blood samples were collected for monitoring cyclosporine A levels, ranging from [specific range not provided]. to Except for collection immediately after cyclosporine A infusion.

[0285] 6.1.2.2 After MI, HVP cell epicardial grafts were transplanted into the pig heart. Three weeks after the initial MI, incisions were made in the femoral artery and vein under anesthesia, with an opening in the artery, and a sheath was introduced to monitor blood pressure. A midline incision was made above the sternum, allowing the skin and underlying muscle tissue to retract. The pericardium was opened, and sutures were passed through the chest wall and adhered to it to form a suspensory band. Glass beads were used to indicate injection sites. Using a 30G needle, 300 μl of mediator or HVP cells were injected at each site in the left ventricular myocardium (n=6, mediator; n=7, HVP cells - low; n=7, HVP cells - medium; and n=6, HVP cells - high): five injections in the borderline area and five injections in the infarcted area. After the final injection, the heart was returned to the pericardial suspensory band and the sutures were removed. The pig was maintained and euthanized after 8 or 90 days. Tissue sections from each injection site were stained with hematoxylin and eosin, examined microscopically by a committee-certified pathologist, and the presence of human cells (confirmed by anti-human nucleolar immunostaining) and any other histopathological changes, including the presence of teratomas, were recorded. All HVP-derived grafts were teratoma-negative at 3 months. Lung, liver, heart, kidney, spleen, brain, thyroid, adrenal gland, pituitary, prostate, and lymph nodes were collected. Human hemoglobin B was not detected by ddPCR using the probe HS00758889_s1 HBB FAM, and there was no indication of extracardiac HVP-derived cells.

[0286] 6.1.2.3 Cardiac Magnetic Resonance ImagingcMRI was performed under anesthesia using a 1.5 T MRI scanner (Philips Intera platform R12 software) before MI surgery; day -7 (after MI and before treatment); and at D30, D60, and D90 after HVP cell injection. Short-axis and long-axis images with a 1 cm intersegmentation were obtained. Data were analyzed using Circle Cardiovascular Imaging cvi42 (V5.12). Three-dimensional (3D) volume was calculated as the sum of (area × (segment thickness + distance between segments)) of all short-axis segments. Ejection fraction was calculated as 100 × (end-diastolic volume - end-systolic volume) / end-diastolic volume. Infarct area was quantified as a percentage of volume (%) using late gadolinium-enhanced MRI. Global longitudinal strain (GLS) was also analyzed. Animals were sacrificed after 12 weeks. The heart was removed and perfused with lactated Ringer's solution. The left ventricle containing all injection sites was fixed in formalin for 48±12 hours, then transferred to 70% ethanol and embedded in paraffin.

[0287] 6.1.3 Immunohistochemistry of HVP cells Paraffin-embedded hearts were sectioned into 4 μm sections. Immunohistochemical methods and protocols were established on an automated Ventana Discovery Ultra stainer (V12.5.4; Roche). Immunohistochemistry for detecting the desired epitopes was performed according to the manufacturer's recommendations (Supplementary Table 1), and all reagents except antibodies were Ventana products (Roche). Antigen retrieval was performed before the addition of the primary antibody, followed by antibody blocking and secondary antibody detection with rabbit reagent or anti-mouse horseradish peroxidase, teal, or DAB staining (single or double staining). Slides were digitized using an Aperio XT full-slide scanner and Aperio ImageScope software (V12.3.3.5048).

[0288] 6.1.4 StatisticsStatistical analyses were performed using GraphPad Prism (V10). Unless otherwise stated, data are presented as mean ± sem. Two-way ANOVA mixed effects followed by Dunnett's multiple comparison test was used to analyze three groups or repeated measures. Statistical details for each experiment are also provided in the legend. Power calculations were performed using historical internal data and data distributions determined by QQ plot analysis to determine sample size. For all other experiments, no statistical methods were used to predetermine sample size. Data distributions were assumed to be normal, and individual data points are presented in all plots. In the pig model, animals were assessed based on ejection fraction after MI and subsequently randomized to either the mediator group or the HVP treatment group. cMRI was analyzed in a blinded manner by two independent cardiologists. Ex vivo experiments were randomly assigned to experimental and control groups. Data collection was not blinded due to experimental conditions.

[0289] 6.2 Results .

[0290] 6.2.1 Strong efficacy signal confirmed in a 3-month GLP study in pigs with myocardial infarction In a rigorously conducted GLP study involving pigs with myocardial infarction, we observed robust efficacy signals. Our previous work demonstrated that a dose of 1 billion HVP cells resulted in successful implantation into the host myocardium in a porcine model of chronic ischemic cardiomyopathy, leading to a reduction in infarct volume and a gradual decrease in cardiac function

[26] . In this current study, we used the same model, inducing myocardial infarction by occlusion of the left anterior descending artery (LAD) in male and female pigs, with a lower dose of cells incorporated. Three different doses of HVP cells were injected into the boundary between the infarct and scar areas, along with a control group receiving the mediator. Immunosuppressant treatment was initiated 6 days prior to cell delivery and maintained throughout the 3-month study period. Subsequently, a total of 26 pigs underwent cMRI to assess left ventricular function before cell injection and at 1, 2, and 3 months post-cell transplantation. Figure 8a). Prior to cell transplantation, all groups experienced decreased left ventricular function, with a mean EF of 44.85% (mediator, 45.00%; HVP-cell-low, 43.88%; HVP-cell-medium, 45.40%; HVP-cell-high, 45.13%). Over the following 3 months, LVEF further deteriorated significantly by approximately -20% in the mediator-treated groups, while the treatment groups showed a reduction or improvement in LVEF compared to pre-cell transplantation LVEF (HVP-cell-low, 4%; HVP-cell-medium, -8%; HVP-cell-high, 0%). Notably, no significant differences were observed between the 100 million, 300 million, and 600 million cell dose groups at 3 months post-transplantation. A decrease in LVEF was observed in the administered animals during the first month, which may be attributed to transient transplant arrhythmias observed during this period (see next section). Figure 8 b). These trends in LVEF are reflected in the total longitudinal strain (GLS) in the drug-treated animals ( Figure 8 c) and stroke volume (SV) Figure 8 d) Improvement is reflected in this. Additionally, the Wall Motion Score Index (WMSI) ( Figure 8 e) and infarct volume ( Figure 8 f).

[0291] This comprehensive assessment of cardiac function and infarct volume over a 3-month period provides strong evidence for the potential therapeutic impact of HVP cell therapy in the context of chronic ischemic cardiomyopathy, thus addressing the complexity of myocardial infarction and its impact on cardiac function.

[0292] 6.2.2 Evidence of human heart grafts and the absence of teratomas or other vegetation changes in porcine myocardium. Histological examination of porcine heart tissue performed 8 days and 3 months after cell transplantation revealed a large number of human cell grafts in the porcine myocardium, which positively expressed ventricular myosin light chain (MLC2v). Figure 9 Furthermore, anti-N-cadherin immunostaining of transplanted cells provided evidence of insert disc-like structure formation and integration of human cardiomyocytes into the host porcine myocardium. For animals terminated at 3 months, myocardial infarction was induced prior to cell implantation. In these animals, human cell grafts were observed at the boundaries of the infarct tissue and in the scar area. Most pigs euthanized at 3 months showed minimal or almost no inflammatory response at the cell injection site in the heart, serving as a promising indicator of the efficacy of our immunosuppressive regimen. Figure 9Following delivery of HVP cells to any porcine tissue, no evidence of teratoma formation or other neoplastic cells was observed at 8 days or 3 months post-delivery, and in NOD mice, no evidence of tumor formation or other neoplastic cells was observed at the injection site at 6 months post-delivery. Furthermore, our analysis revealed no presence of human cells in any porcine organs other than the heart, and no indication of systemic toxicity or neoplastic effects in cardiac or non-cardiac tissues of mice or pigs (data not shown). These findings underscore the success of the HVP cell transplantation approach, demonstrating not only the integration of human cardiomyocytes into the host myocardium but also the absence of any adverse systemic effects, further supporting the safety and potential therapeutic value of our novel cell therapy strategy.

[0293] References

[0294] All publications mentioned in this specification are incorporated herein by reference in their entirety.

[0295] [1]AP Ambrosy et al., J Am Coll Cardiol 63, 1123 (2014).

[0296] [2]TA McDonagh et al., Eur Heart J 42, 3599 (2021).

[0297] [3]MA Pfeffer, AM Shah and BA Borlaug, Circ Res 124, 1598(2019).

[0298] [4] G. Savarese and LH Lund, Card Fail Rev 3, 7 (2017).

[0299] [5]JA Iwaz et al., Drugs 76, 187 (2016).

[0300] [6] KS Foo et al., Mol Ther 26, 1644 (2018).

[0301] [7] KR Chien and X. Lian, WO2016029122A1 (February 25, 2016).

[0302] [8] CY LEUNG et al., WO2017172086A1 (October 5, 2017).

[0303] [9] KR CHIEN et al., WO2018100433A1 (June 7, 2018).

[0304]

[10] KR Chien, J. Clarke, and CY LEUNG, WO2019038587A1 (February 28, 2019).

[0305]

[11] CA Cowan et al., N Engl J Med 350, 1353 (2004).

[0306]

[12] W. Zhou and CR Freed, Stem Cells 27, 2667 (2009).

[0307]

[13] D. Huangfu et al., Nat Biotechnol 26, 795 (2008).

[0308]

[14] K. Woltjen et al., Nature 458, 766 (2009).

[0309]

[15] H. Zhou et al., Cell Stem Cell 4, 381 (2009).

[0310]

[16] T. Kuroda et al., PLOS ONE 7, e37342 (2012).

[0311]

[17] S. Kusakawa et al., Sci Rep 5, 17892 (2015).

[0312]

[18] AS Lee et al., Cell Cycle 8, 2608 (2009).

[0313]

[19] WM Schopperle and WCDeWolf, STEM CELLS 25, 723 (2007).

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[20] JA Thomson et al., Science 282, 1145 (1998).

[0315]

[21] AH Kashani et al., Ophthalmol Retina 4, 264 (2020).

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[22] J. Piao et al., Cell Stem Cell 28, 217 (2021).

[0317]

[23] K. Tano et al., PLoS One 9, e110496 (2014).

[0318]

[24] K. Masumoto et al., Sci Rep 12, 10351 (2022).

[0319]

[25] M. Lemmens et al., Cytotherapy 25, 59 (2023).

[0320]

[26] C. Poch et al., Nat Cell Biol 24(5), 659-671 (2022).

Claims

1. A population of cells, wherein: a) At least 70% of the cells in the cell population express cardiac progenitor cell (CPC)-related markers, and b) 1% or less of the cells in the cell population express octamer transcription factor 4 (OCT4).

2. The cell population according to claim 1, wherein the cell population comprises, is composed of, or is substantially composed of dissociated cells.

3. The cell population according to claim 1 or 2, wherein the cell population comprises, consists of, or is substantially composed of dissociated single cells.

4. The cell population according to any of the preceding claims, wherein the cell population comprises cells in a suspension, is composed of cells in a suspension, or is substantially composed of cells in a suspension.

5. The cell population according to any of the preceding claims, wherein the cell population is characterized as a cardiac lineage, optionally wherein the cell population is characterized as a ventricular cardiac lineage.

6. The cell population according to any of the preceding claims, wherein less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3% or 0.2% of the cells in the cell population express OCT4.

7. The cell population according to any of the preceding claims, wherein about 0.1% of the cells in the cell population express OCT4.

8. The cell population according to any of the preceding claims, wherein no cells in the cell population express OCT4.

9. The cell population according to any of the preceding claims, wherein the percentage (%) of cells expressing OCT4 in the cell population is measurable by flow cytometry, single-cell RNA sequencing or immunofluorescence, or determined by flow cytometry, single-cell RNA sequencing or immunofluorescence.

10. The cell population according to any of the preceding claims, wherein 1.5% or less of the cells in the population express T cell receptor α locus 1-60 (TRA-1-60).

11. The cell population according to any of the preceding claims, wherein less than 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3% or 0.2% of the cells in the cell population express TRA-1-60.

12. The cell population according to any of the preceding claims, wherein 1.5% or less of the cells in the population express TRA-1-60.

13. The cell population according to any of the preceding claims, wherein the percentage (%) of cells expressing TRA-1-60 in the cell population is measurable by flow cytometry, single-cell RNA sequencing or immunofluorescence, or determined by flow cytometry, single-cell RNA sequencing or immunofluorescence.

14. The cell population according to any of the preceding claims, wherein, as measured by flow cytometry, microarray, RNA sequencing and / or quantitative polymerase chain reaction (PCR), the expression of OCT4 in the cell population is below the detection limit.

15. The cell population according to any of the preceding claims, wherein 1% or less of the cells in the population express NANOG.

16. The cell population according to any of the preceding claims, wherein less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, or 0.2% of the cells in the cell population express NANOG.

17. The cell population according to claim 15 or 16, wherein the percentage (%) of cells expressing NANOG in the cell population is measurable by flow cytometry, single-cell RNA sequencing or immunofluorescence, or determined by flow cytometry, single-cell RNA sequencing or immunofluorescence.

18. The cell population according to any of the preceding claims, wherein 3% or less of the cells in the population express SOX2.

19. The cell population according to any of the preceding claims, wherein less than 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, or 2.3% of the cells in the cell population express SOX2.

20. The cell population according to claim 18 or 19, wherein the percentage (%) of cells expressing SOX2 in the cell population is measurable by flow cytometry, single-cell RNA sequencing or immunofluorescence, or determined by flow cytometry, single-cell RNA sequencing or immunofluorescence.

21. The cell population according to any of the preceding claims, wherein, as measured by a high-efficiency culture (HEC) assay, the cell population produces less than one OCT4 per million cells. + Colony.

22. The cell population according to any of the preceding claims, wherein, as measured by Western blotting, the cells in the cell population do not express OCT4.

23. The cell population according to any of the preceding claims, wherein at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells in the cell population express cardiac progenitor cell (CPC) related markers.

24. The cell population according to any of the preceding claims, wherein the cardiac progenitor cell-associated marker is Islet-1 (ISL1).

25. The cell population according to any of the preceding claims, wherein the percentage of cells expressing the CPC in the cell-related markers is measurable by flow cytometry, single-cell RNA sequencing or immunofluorescence.

26. The cell population according to any of the preceding claims, wherein the cell population comprises cells expressing ventricular progenitor cell-related markers.

27. The cell population according to any of the preceding claims, said cell population comprising cells expressing ISL1, PDGFRA, TBX1, HAND1, TBX5 and / or SMARCD3.

28. The cell population according to any of the preceding claims, said cell population comprising cells expressing Jagged-1 (JAG1), Frizzled-4 (FZD4), fibroblast growth factor receptor 3 (FGFR3), leukemia inhibitory factor receptor (LIFR) and / or TNF superfamily member 9 (TNFSF9).

29. The cell population according to any of the preceding claims, wherein the cell population comprises cells expressing LIFR and ISL1.

30. The cell population according to any of the preceding claims, wherein at least 70% of the cells in the cell population express LIFR and ISL1.

31. The cell population according to any of the preceding claims, wherein at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the cells in the cell population express LIFR and ISL1.

32. The cell population of claim 30 or 31, wherein the percentage of cells expressing LIFR and ISL1 is measurable by flow cytometry, single-cell RNA sequencing or immunofluorescence, or determined by flow cytometry, single-cell RNA sequencing or immunofluorescence.

33. The cell population according to any of the preceding claims, wherein the cell population comprises or is composed of human cells.

34. The cell population according to any of the preceding claims, wherein the cell population is AZD6414.

35. The cell population according to any of the preceding claims, wherein the cell population comprises ventricular progenitor cells (VPCs).

36. The cell population according to any of the preceding claims, wherein the cell population comprises hPSC-pan-mesoderm-cardiac mesoderm-pancreatic islet ventricular progenitor cells.

37. The cell population according to any of the preceding claims, wherein ≥80%, ≥85%, ≥90%, ≥95%, or ≥99% of the cells in the cell population are VPCs.

38. The cell population according to any of the preceding claims, wherein ≤5%, ≤4%, ≤3%, ≤2%, or ≤1% of the cells in the cell population are stem cells.

39. The cell population according to any of the preceding claims, wherein ≤5%, ≤4%, ≤3%, ≤2%, or ≤1% of the cells in the cell population are pluripotent stem cells.

40. The cell population according to any of the preceding claims, wherein ≤10% of the cells in the cell population are fully differentiated cells.

41. The cell population according to any of the preceding claims, wherein the cell population is characterized as cardiac differentiation.

42. The cell population according to any of the preceding claims, wherein the cell population is characterized as cardiac ventricular differentiation.

43. The cell population according to any of the preceding claims, wherein the cell population is capable of differentiating into cardiac tissue upon delivery to the heart of a subject.

44. The cell population according to any of the preceding claims, wherein the cell population comprises cells capable of differentiating into cardiomyocytes in vitro.

45. The cell population according to any of the preceding claims, wherein the cell population comprises cells capable of differentiating into beating cardiomyocytes in vitro.

46. ​​The cell population according to any of the preceding claims, wherein at least 70% of the cells in the cell population express cardiac troponin T after at least 15 days of in vitro differentiation.

47. The cell population according to any of the preceding claims, wherein the cell population is capable of differentiating into ventricular wall tissue upon delivery to the ventricular wall of a subject.

48. The cell population according to any of the preceding claims, wherein the cell population is capable of differentiating into pulsating myocardium after delivery to the ventricular wall of the subject.

49. The cell population according to any of the preceding claims, wherein the cell population is capable of repairing damaged cardiac tissue after delivery to a subject's damaged heart.

50. The cell population according to any of the preceding claims, wherein the cell population is used to repair damaged cardiac tissue.

51. The cell population according to any of the preceding claims, wherein the cell population is used to repair damaged myocardium.

52. The cell population according to any of the preceding claims, wherein, after delivery of the cell population to the heart of a subject, the cell population is capable of forming a vascularized, electrically responsive ventricular myocardial patch that secretes extracellular matrix.

53. The cell population according to any of the preceding claims, wherein the cell population does not form a teratoma after being delivered to the ventricular wall of the subject.

54. The cell population according to any of the preceding claims, wherein the cell population comprises cells that have been engineered to be low in immunogenicity or low in allogeneity.

55. The cell population according to any of the preceding claims, wherein the cell population comprises or is composed of cells derived from stem cells.

56. The cell population of claim 55, wherein the stem cells are embryonic stem cells (ESCs).

57. The cell population according to any of the preceding claims, wherein the cell population comprises or is composed of cells derived from stem cell lines.

58. The cell population according to any of the preceding claims, wherein the cell population comprises or is composed of cells derived from induced pluripotent stem cells (iPSCs).

59. The cell population according to any of the preceding claims, wherein the cell population does not contain cells derived from pluripotent cells.

60. The cell population according to any of the preceding claims, wherein the cell population is derived from one or more cells that have been expanded in vitro.

61. The cell population according to any of the preceding claims, wherein the cell population is derived from one or more cells isolated from the subject.

62. The cell population according to any of the preceding claims, wherein the cell population is artificial or comprises artificial cells.

63. The cell population according to any of the preceding claims, wherein the cell population does not exist in nature, or comprises cells that do not exist in nature.

64. The cell population according to any of the preceding claims, wherein the cell population comprises modified cells.

65. The cell population according to any of the preceding claims, wherein the cell population is a separate cell population.

66. The cell population according to any of the preceding claims, wherein the cell population comprises engineered cells.

67. The cell population according to any of the preceding claims, wherein the cell population comprises cells that have been modified or engineered in vitro.

68. The cell population according to any of the preceding claims, wherein the cell population comprises cells that have been modified or engineered in vitro.

69. The cell population according to any of the preceding claims, wherein the cell population is isolated from the body and contained in the pharmaceutical composition.

70. A method for preparing a pharmaceutical composition, the method comprising combining a cell population according to any one of claims 1 to 69 with a pharmaceutical excipient.

71. The use of isolated cell populations in therapeutic methods, among which a) At least 70% of the cells in the population express cardiac progenitor cell (CPC)-related markers, and b) 1% or less of the cells in the population express OCT4.

72. A composition comprising a cell population according to any one of claims 1 to 69.

73. A method for producing a cell population comprising any one of claims 1 to 69, the method comprising harvesting cells from a cell culture, wherein the harvested cells are derived from stem cells cultured for 8 to 12 days under conditions suitable for cardiomyogenesis.

74. A method for producing a cell population comprising cardiac progenitor cells, the method comprising harvesting the cell population, wherein the harvested cells are derived from stem cells cultured for 8 to 12 days under conditions suitable for cardiomyogenesis.

75. The cell population according to any one of claims 1 to 69, wherein the cell population is produced by a method comprising harvesting cells from an adherent cell culture, wherein the adherent cell culture is derived from stem cells cultured for 8 to 12 days under conditions suitable for cardiomyogenesis.

76. A cell population comprising cardiac ventricular progenitor cells, wherein the cell population is produced by a method comprising harvesting cells from an adherent cell culture, wherein the adherent cell culture is cultured for 8 to 12 days under conditions suitable for cardiomyogenesis.

77. The method of claim 73 or 74 or the cell population of claim 75 or 76, wherein the conditions suitable for cardiomyogenesis include: a) On day 0, activate Wnt / Wnt / β-catenin signaling in the culture. b) From day 3 to day 5, Wnt / β-catenin signaling in the culture was inhibited to generate a population of cardiac ventricular progenitor cells (VPCs).

78. The method or cell population of claim 77, wherein Wnt / Wnt / β-catenin signaling is activated by a GSK3 inhibitor, optionally wherein the GSK3 inhibitor is CHIR-98014.

79. The method or cell population of claim 77 or 78, wherein Wnt / β-catenin signaling is inhibited by a Wnt inhibitor, optionally wherein the Wnt inhibitor is Wnt-C59.

80. The method or cell population according to any one of claims 77 to 79, wherein the method comprises culling pluripotent cells from the harvested cells to produce a purified VPC population.

81. The method or cell population of claim 80, wherein removing pluripotent cells from the harvested cells includes removing cells expressing markers of pluripotency from the harvested cells.

82. The method or cell population according to claim 80 or 81, wherein removing pluripotent cells from the harvested VPC population includes removing cell populations expressing TRA-1-60.

83. The method according to any one of claims 80 to 82, wherein removing pluripotent cells from the harvested VPC population comprises removing the TRA-1-60-expressing cell population by magnetically activated cell sorting.

84. The method or cell population according to any one of claims 77 to 82, wherein the stem cells are induced pluripotent cells or embryonic stem cells.

85. The method or cell population according to any one of claims 77 to 84, wherein: a) At least 70% of the cells in the cell population express cardiac progenitor cell (CPC)-related markers, and b) 1% or less of the cells in the cell population express OCT4.

86. A method of treating or preventing a disease in a subject, the method comprising administering to a subject in need an effective amount of a cell population according to any one of claims 1 to 69 or 75 or 76.

87. A method for treating or preventing a disease in a subject, the method comprising: a) The method according to any one of claims 73, 74 or 77 to 85 for producing cell populations in vitro, and b) The cell population is administered to the subject.

88. The method of claim 86 or 87, wherein the cell population comprises cells derived from one or more cells not isolated from the subject, consists of cells derived from one or more cells not isolated from the subject, or consists substantially of cells derived from one or more cells not isolated from the subject.

89. The method of claim 86 or 87, wherein the cell population comprises cells derived from one or more cells isolated from the subject, consists of cells derived from one or more cells isolated from the subject, or consists substantially of cells derived from one or more cells isolated from the subject.

90. The method according to any one of claims 86 to 89, wherein the disease is a disease associated with myocardial loss and / or fibrotic scarring.

91. The method according to any one of claims 86 to 90, wherein the disease is: a) Heart failure; b) Chronic ischemic cardiomyopathy; or c) Myocardial infarction.

92. The method according to any one of claims 86 to 91, wherein the subject is receiving immunosuppressive therapy.

93. A pharmaceutical composition comprising a cell population according to any one of claims 1 to 69, and a pharmaceutical excipient.

94. The pharmaceutical composition according to claim 93, wherein the pharmaceutical composition comprises a freezing medium.

95. A container comprising the pharmaceutical composition according to claim 93 or 94.

96. The container of claim 95, wherein the container is a storage vial, a frozen vial, an injection device, or a syringe.

97. A pharmaceutical composition for treating or preventing a disease in a subject in need, wherein the pharmaceutical composition comprises a cell population according to any one of claims 1 to 69, wherein the method comprises administering the pharmaceutical composition to the subject.

98. Use of the cell population according to any one of claims 1 to 69 for the manufacture of a medicament for the treatment or prevention of a disease in a subject.

99. Use of the pharmaceutical composition according to claim 97 or the cell population according to claim 98, wherein the disease is: a) Heart failure; b) Chronic ischemic cardiomyopathy; or c) Myocardial infarction.

Images