Molecular identification of off-target cardiac cell populations

Abstract

This disclosure relates generally to methods of screening and preparing high purity cardiac cell therapies and related cell preparations.

Description

[0001] MOLECULAR IDENTIFICATION OF OFF-TARGET CARDIAC CEEE POPUEATIONS

[0002] REEATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 663,044, filed June 21, 2024, entitled “MOLECULAR IDENTIFICATION OF OFF-TARGET CARDIAC CELL POPULATIONS,” the entire disclosure of which is hereby incorporated by reference herein in its entirety.

[0004] FIELD

[0005] This disclosure relates to compositions and methods related to cardiac cells assessed for high purity.

[0006] BACKGROUND

[0007] The ability of stem cells to regenerate injured tissue holds great promise for the treatment of cardiac disease and injury; however, several obstacles to their adoption persist. Though a batch of stem cells may undergo a cell-type specific differentiation protocol, heterogeneous cell populations may still arise. Methods for screening batches for off-target cells and potential off- target cells may be useful for a variety of purposes, such as research and preparing cardiac cell therapies.

[0008] SUMMARY

[0009] Cardiac cell therapies (e.g., cardiomyocyte grafts) are useful for treating subjects in need thereof, such as those having injured cardiac tissue (e.g., as the result of heart disease or failure). The success of these therapies may be influenced by the purity of certain cell population used during the differentiation, maturation, and / or preparation process. Currently available methods for identifying off-target cell populations are imperfect and are unable to yield pure cardiac populations. Surprisingly, the inventors of the present disclosure have identified methods and compositions that are useful for identifying cardiac cell populations (e.g., human cardiac cell populations), such as non-cardiomyocytes, epicardial-like cells, fibroblast-like cells, and other sub-fractions within differentiated and matured cardiac cells generated from stem cells.

[0010] Described herein, in some aspects, are compositions for cardiac cell therapy. In some aspects, a composition for cardiac cell therapy comprises: a plurality of cells in a physiologically acceptable medium suitable for administration to a patient in need thereof; wherein at least 50% of the plurality of cells comprise a molecular profile comprising: i) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80. CDC42EP5, CDH11, CDON. CFH, COL1A2, COL3A1, COL5A1, CRIME CTSH, CXCLI2, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBNI, FLRT2, FNI, GATA3, GATA6, KRT19, LIMA1, LRPR2, LRRC17, MGP, MMP2, NUPR1, OGN, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNT1, and / or UPK3B, ii) negative / low expression of one or more markers selected from: ANXA1, CCDC80, CDON, CRIM1, COL3A1, CTSH, DCN, FLRT2, KRT19, LRPR2, NPNT, NUPR1, PLAC9, PODXL, POSTN, RDH10, SBSON, S100A10, S100A11, SPARC, TMEM88, TNNT1, and / or UPK3B, iii) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, COL1A2, COL3A1, CXCL12, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBNI, FNI, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN, PTX3, S100A10, S100A11, SLIT2, SPARC, TFPI2, TGFb, and / or THY], iv) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CDH11, COL1A2, COL3A1, COL5A1, EVAIB, FBNI, LIMA1, MGP, MMP2, OGN, PLAC9, S100A10 v) negative / low expression of one or more markers selected from COL3A1, PODXL, and / or KRT19, vi) negative / low expression of one or more markers selected from KRT19 and / or PODXL, vii) negative / low expression of one or more markers selected from COL1A2 and / or FNL, and / or viii) negative / low expression of COL3A1.

[0011] In some aspects, a composition for cardiac cell therapy comprises: a plurality of cells in a physiologically acceptable medium suitable for administration to a patient in need thereof; wherein at least 50% of the plurality of cells comprise a molecular profile characteristic of cardiomyocyte identity. In some embodiments, the molecular profile characteristic of cardiomyocyte identity comprises the molecular profile characteristic of cardiomyocytes comprises one of the following: i) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CDC42EP5, CDH11, CDON, CFH, COL1A2, COL3A1, COL5A1, CRIM1, CTSH, CXCL12, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBNI, FLRT2, FNI, GATA3, GATA6, KRT19, LIMA1, LRPR2, LRRC17, MGP, MMP2, NUPRI, OGN, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNT1, and / or UPK3B, ii) negative / low expression of one or more markers selected from: ANXA1, CCDC80, CDON, CRIM1, COLS Al, CTSH, DCN, FLRT2, KRT19, LRPR2, NPNT, NUPR1, PLAC9, PODXL, POSTN, RDH10, SBSON, S100A10, S100A11, SPARC, TMEM88, TNNT1, and / or UPK3B, iii) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, COL1A2, COL3A1, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FN1, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN, PTX3, S100A10, S100A11, SLIT2, SPARC, TFP12, TGFb, and / or THY1, iv) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CDH11, COL1A2, COL3A1, COL5A1, EVA1B, FBN1, L1MA1, MGP, MMP2, OGN, PLAC9, S100A10 v) negative / low expression of one or more markers selected from COL3A1, PODXL, and / or KRT19, vi) negative / low expression of one or more markers selected from KRT19 and / or PODXL, vii) negative / low expression of one or more markers selected from COL1A2 and / or FNL, and / or viii) negative / low expression of COL3A1.

[0012] In some aspects, a composition for cardiac cell therapy comprises: a plurality of cells in a physiologically acceptable medium suitable for administration to a patient in need thereof; wherein at least 50% of the plurality of cells comprise negative / low expression of COL3A1.

[0013] In some embodiments, the plurality of cells comprises mature cardiomyocytes. In some embodiments, the mature cardiomyocytes are derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells comprise positive / high expression of one or more of the following markers: SSEA3 / 4, TRA-1-60, OCT3 / 4, NANOG, and SOX2. In some embodiments, the mature cardiomyocytes are derived from embryonic stem cells. In some embodiments, the mature cardiomyocytes comprise positive / high expression of MLC2v and / or CTNT. In some embodiments, the mature cardiomyocytes further comprises negative / low expression of MLC2a.

[0014] Described herein, in some aspects, are methods of preparing a cellular composition for cardiac cell therapy. In some aspects, the method comprises: (a) determining that a plurality of cells have a molecular profile characteristic of cardiomyocyte identity, wherein the molecular profile comprises negative / low expression of COLS Al, and

[0015] (b) preparing a cellular composition for cardiac cell therapy from the plurality of cells having a molecular profile characteristic of cardiomyocyte identity.

[0016] In some embodiments, the plurality of cells have a molecular profile characteristic of cardiomyocyte identity represents greater than 50% of all cells used to prepare the cardiac cell therapy. In some embodiments, the plurality of cells have a molecular profile characteristic of cardiomyocyte identity represents greater than 60%, 70%, 80%, or 90% of all cells used to prepare the cardiac cell therapy.

[0017] In some embodiments, the plurality of cells having a molecular profile characteristic of cardiomyocyte identity is obtained by:

[0018] (i) determining a molecular profde of a batch of cells;

[0019] (ii) selecting the plurality of cells having a molecular profile characteristic of cardiomyocyte identity from the batch of cells.

[0020] In some embodiments, the molecular profile of the batch of cells is obtained via flow cytometry. In some embodiments, the molecular profile of the batch of cells is obtained from a transcriptome of the batch of cells. In some embodiments, the transcriptome is obtained via single cell ribonucleic acid sequencing (scRNA-seq).

[0021] In some embodiments, the molecular profile characteristic of cardiomyocyte identity comprises negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CDC42EP5, CDH11, CDON, CFH, COL1A2, COLS Al, COL5A1, CR1M1, CTSH, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FLRT2, FN1, GATA3, GATA6, KRT19, L1MA1, LRPR2, LRRC17, MGP, MMP2, NUPR1, OGN, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNT1, and / or UPK3B. In some embodiments, the molecular profile characteristic of cardiomyocyte identity comprises negative / low expression of one or more markers selected from: COL3A1, PODXL, and / or KRT19.

[0022] In some embodiments, the plurality of cells having a molecular profile characteristic of cardiomyocyte identity comprises mature cardiomyocytes. In some embodiments, the mature cardiomyocytes are derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells express one or more of the following markers: SSCB3 / 4, TRA-1-60, OCT3 / 4, NANOG, and SOX2. In some embodiments, the mature cardiomyocytes are derived from embryonic stem cells. In some embodiments, the mature cardiomyocytes comprise positive / high expression of MLC2v and / or CTNT. In some embodiments, the mature cardiomyocytes comprise negative / low expression of MLC2a.

[0023] In some embodiments, preparing the cellular composition for cardiac cell therapy from the plurality of cells having a molecular profile characteristic of cardiomyocyte identity comprises contacting the plurality of cells having a molecular profile characteristic of cardiomyocyte identity with a physiologically acceptable medium.

[0024] Described herein, in some aspects, are methods of preparing a cellular composition for cardiac cell therapy. In some aspects, the method comprises:

[0025] (a) determining that a plurality of cells do not have a molecular profile characteristic of non- cardiomyocytes; and

[0026] (b) preparing the cellular composition for cardiac cell therapy from the plurality of cells.

[0027] In some embodiments, the molecular profile of the plurality of cells is obtained via flow cytometry. In some embodiments, the molecular profile of the plurality of cells is obtained from a transcriptome of the plurality of cells. In some embodiments, the transcriptome is obtained via single cell ribonucleic acid sequencing (scRNA-Seq).

[0028] In some embodiments, the molecular profile characteristic of non-cardiomyocytes is a non-cardiomyocyte molecular profile. In some embodiments, the molecular profile characteristic of non-cardiomyocytes comprises positive / high expression of one or more markers selected from: ANXA1, BGN, CCDC80, CDC42EP5, CDH11, CDON. CFH, COL1A2, COLS Al, COL5A1, CRIM1, CTSH, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FLRT2, FN1, GATA3, GATA6, KRT19, L1MA1, LRPR2, LRRC17, MGP, MMP2, NUPR1, OGN, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNT1, and / or UPK3B. In some embodiments, the molecular profile characteristic of non-cardiomyocytes comprises positive / high expression of COL3A1, PODXL, and / or KRT19.

[0029] In some embodiments, the molecular profile characteristic of non-cardiomyocytes is an epicardial-like cell (EC) molecular profile. In some embodiments, the EC molecular profile comprises positive / high expression of one or more markers selected from: ANXA1, CCDC80, CDON, CRIM1, COL3A1, CTSH, DCN, FLRT2, KRT19, LRPR2, NPNT, NUPR1, PLAC9, PODXL, POSTN, RDH10, SBSON, S100A10, S100A11, SPARC, TMEM88, TNNT1, and / or UPK3B. In some embodiments, the EC molecular profile comprises positive / high expression of KRT19 and / or PODXL.

[0030] In some embodiments, the molecular profile characteristic of non-cardiomyocytes is a fibroblast-like (FB) molecular profile. In some embodiments, the FB molecular profile comprises positive / high expression of one or more markers selected from: ACTA2, ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, C0LIA2, C0L3AI, CXCLI2, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBNI, FN1, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN, PTX3, S100A10, S100A11, SLIT2, SPARC, TFP12, TGFb, and / or THYE In some embodiments, the FB molecular profile comprises positive / high expression of COL1A2 and / or FN1.

[0031] In some embodiments, no more than 50% of the cells in the cellular composition comprise a molecular profile characteristic of non-cardiomyocytes. In some embodiments, no more than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the cells in the cellular composition comprise a molecular profile characteristic of non-cardiomyocytes.

[0032] In some embodiments, preparing the cellular composition for cardiac cell therapy from the plurality of cells comprises contacting the plurality of cells with a physiologically acceptable medium.

[0033] Described herein, in some aspects, is a method of preparing a plurality of cells for cardiac cell therapy, wherein at least 95% of the plurality cells are characterized as having a molecular profile comprising one of the following: i) negative / low expression of one or more markers selected ANXA 1, BGN, CCDC80, CDC42EP5, CDH11, CDON, CFH, COL1A2, COL3A1, COL5A1, CRIM1, CTSH, CXCL12, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBNI, FLRT2, FN1, GATA3, GATA6, KRT19, LIMA1, LRPR2, LRRC17, MGP, MMP2, NUPRI, OGN, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNTI, and / or UPK3B, ii) negative / low expression of one or more markers selected from: ANXA1, CCDC80, CDON, CRIM1, COL3A1, CTSH, DCN, FLRT2, KRT19, LRPR2, NPNT, NUPRI, PLAC9, PODXL, POSTN, RDH10, SBSON, SI00AI0, S100A11, SPARC, TMEM88, TNNTI, and / or UPK3B, iii) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, COL1A2, COL3A1, CXCL12, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBNI, FNI, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN, PTX3, S100A10, S100A11, SLIT2, SPARC, TFPI2, TGFb, and / or THYE iv) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CDH11, COL1A2, COL3A1, COL5A1, EVAIB, FBNI, LIMA1, MGP, MMP2, OGN, PLAC9, S100A10 v) negative / low expression of one or more markers selected from COL3A1 , PODXL, and / or KRT19, vi) negative / low expression of one or more markers selected from KRT19 and / or PODXL, vii) negative / low expression of one or more markers selected from COL1A2 and / or FNP, and / or viii) negative / low expression of COL3A1.

[0034] Described herein, in some aspects, is a method of preparing a plurality of mature cardiomyocytes for cardiac cell therapy, the method comprising contacting a plurality of mature cardiomyocytes that been determined to have a molecular profile characteristic of cardiomyocyte identity with a physiologically acceptable medium suitable for administration to a subject in need of cardiac cell therapy.

[0035] In some aspects, the method of preparing a batch of cells for cardiac cell therapy comprises:

[0036] (a) determining that an at least first batch of cells among a plurality of batches comprises a molecular profde characteristic of cardiomyocyte identity and;

[0037] (b) preparing the at least first batch of cells for cardiac cell therapy.

[0038] In some embodiments, preparing the mature cardiomyocytes for cardiac cell therapy comprises contacting the mature cardiomyocytes with a physiologically acceptable medium suitable for administration to a subject.

[0039] Described herein, in some aspects, is a method of determining whether a plurality of cells comprises a molecular profde characteristic of cardiomyocyte identity and; if the plurality of cells comprises a molecular profile characteristic of cardiomyocyte identity, preparing the plurality of cells for cardiac cell therapy.

[0040] Described herein, in some aspects, is a method of determining cardiomyocyte identity of a cell, the method comprising determining the level of COL3A1 in the cell.

[0041] Described herein, in some aspects, is a method of treating a subject in need of cardiac cell therapy, the method comprising:

[0042] (a) determining that no more than 50% of cardiomyocytes in a batch of cells comprise a molecular profile characteristic of a non-cardiomyocyte identity; and

[0043] (b) administering the batch of cells or a portion thereof to the subject. Described herein, in some aspects, is a method of treating a subject in need of cardiac cell therapy, the method comprising administering a plurality of cells to a subject, wherein at least 50% of cells in the plurality of cells has been determined to have a molecular profile characteristic of cardiomyocyte identity.

[0044] Described herein, in some aspects, is a method of treating a subject in need of cardiac cell therapy, the method comprising administering a plurality of cells to a subject, wherein at least 50% of cells in the plurality of cells have a molecular profile characteristic of cardiomyocyte identity.

[0045] Described herein, in some aspects, is a method of treating a subject in need of cardiac cell therapy, the method comprising administering to the subject a cardiac cell therapy described herein, a composition prepared according to a method described herein, or a batch of cells prepared according to a method described herein.

[0046] BRIEF DESCRIPTION OF DRAWINGS

[0047] FIG. 1 shows identification of non-cardiomyocyte markers using single-cell RNA-seq (scRNA-seq) data from samples of cells having undergone cardiomyocyte differentiation processes, on Day 51 post-mesoderm induction. Each plot shows uniform manifold approximation and projection (UMAP) clustering of scRNA-seq data colored by gene expression values for collagen type III alpha 1 chain (COL3A1), matrix Gia protein (MGP), cysteine-rich acidic matrix-associated protein (SPARC), S100 calcium binding protein Al 1 (S100A11), collagen type I alpha 2 chain (COL1A2), osteoglycin (OGN), fibrillin-1 (FBN1), S100 calcium binding protein A10 (S100A10), biglycan (BGN), coiled-coil domain containing 80 (CCDC80), annexin Al (ANXA1), placenta associated 9 (PLAC9), cadherin-11 (CDH11 ), sulfatase 1 (SULFI), matrix metallopeptidase 2 (MMP2), Eva-1 homolog B (EVA IB), sprouty RTK signaling antagonist 1 (SPRY / ), LIM domain and actin binding 1 (E1MA1), collagen type V alpha 1 chain (COE5A1), and seizure related 6 homolog like 2 (SEZ6E2).

[0048] FIG. 2 shows identification of fibroblast-like markers using single-cell RNA-seq (scRNA-seq) data from samples of cells having undergone cardiomyocyte differentiation processes, on Day 51 post-mesoderm induction. Each plot shows uniform manifold approximation and projection (UMAP) clustering of scRNA-seq data colored by gene expression values for collagen type I alpha 2 chain (COE1A2), biglycan (BGN), tropoelastin (ELN), C-X-C motif chemokine ligand 12 (CXCE12), leucine rich repeat containing 17 (LRRC17), osteoglycin (OGN), fibulin 5 (FBLN5). fibrillin-1 (FBNI), elastin microfibril interfacer 1 (EMILIN 1), matrix metallopeptidase 2 (MMP2). CDC42 effector protein 5 (CDC42EP5). Eva-1 homolog B (EVA1B), matrix Gia protein (MGP). S100 calcium binding protein Al l (S100A11), collagen type III alpha 1 chain (COL3AI), cysteine-rich acidic matrix- associated protein (SPARC), annexin Al (ANXA1), placenta associated 9 (PLAC9), coiled-coil domain containing 80 (CCDC80), and S100 calcium binding protein A10 (SI00A I0).

[0049] FIG. 3 shows identification of epicardial-like markers using single-cell RNA-seq (scRNA-seq) data from samples of cells having undergone cardiomyocyte differentiation processes, on Day 51 post-mesoderm induction. Each plot shows uniform manifold approximation and projection (UMAP) clustering of scRNA-seq data colored by gene expression values for keratin 19 (KRT19), somatomedin B and thrombospondin type 1 domain containing (SBSON), podocalyxin like (PODXL), troponin T1 (TNNT1), megalin (LRPR2), uroplakin 3B (UPK3B), cell adhesion associated oncogene regulated (CDON), transmembrane protein 88 (TMEM88), retinol dehydrogenase 10 (RDH10), cathepsin H (CTSH), decorin (DCN), cysteine-rich transmembrane BMP regulator 1 (CRIME), S100 calcium binding protein A10 (S100A10), coiled-coil domain containing 80 (CCDC80), fibronectin leucine rich transmembrane protein 2 (FLRT2), placenta associated 9 (PLAC9), (NUPR1), annexin Al (ANXA1), collagen type III alpha 1 chin (COL3AI), S100 calcium binding protein Al 1 (SI00AII).

[0050] FIGs. 4A-4C show representative flow cytometry scatter plots evaluating the utility of COL3A1 as a non-cardiomyocyte marker in samples of cells having undergone cardiomyocyte differentiation processes, on Day 51 post-mesoderm induction FIG. 4A shows the proportion of mature cardiomyocytes (CTNT positive / high) and non-cardiomyocytes (CD90 positive / high) in a sample of putative mature cardiomyocytes. FIG. 4B shows the proportion of mature cardiomyocytes (CTNT positive / high) and non-cardiomyocytes (COL3A1 positive / high) in a sample of putative mature cardiomyocytes. FIG. 4C shows the proportion of non- cardiomyocytes (CD90 positive / high and COL3A1 positive / high) in a sample of putative mature cardiomyocytes. Numbers in each quadrant indicate the proportion of cells belonging to each population.

[0051] FIGs. 5A-5D show representative flow cytometry scatter plots evaluating the cellular composition of samples of cells having undergone cardiomyocyte differentiation processes, on Day 51 post-mesoderm induction FIG. 5A shows the proportion of mature cardiomyocytes (CTNT positive / high) and non-cardiomyocytes (COL3A1 positive / high). FIG. 5B shows the proportion of mature ventricular cardiomyocytes (MLC2v positive / high and CTNT positive / high). FIG. 5C shows the proportion of ventricular cardiomyocytes (MLC2v positive / high) or atrial cardiomyocytes MLC2a positive / high). FIG. 5D shows the proportion of mature cardiomyocytes (CTNT positive / high) and immature / precursor cardiomyocytes K167 positive / high). Numbers in each quadrant indicate the proportion of cells belonging to each population.

[0052] FIGs. 6A-6E show representative flow cytometry scatter plots quantifying the relative proportion of cell types in samples of cells having undergone cardiomyocyte differentiation processes, on Day 51 post-mesoderm induction FIG. 6A is a representative plot showing 87.8% of cells in a sample of putative mature cardiomyocytes are mature cardiomyocytes. FIG. 6B is a representative plot showing 74.3% of cells in a sample of putative mature cardiomyocytes are ventricular cardiomyocytes. FIG. 6C is a representative plot showing 15.3% of cells in a sample of putative mature cardio myocytes are non-cardiomyocytes. FIG. 6D is a representative plot showing 80.3% of cells in a sample of putative mature cardiomyocytes are atrial cardiomyocytes. FIG. 6E is a representative plot showing 8.24% of cells in a sample of putative mature cardiomyocytes are immature or precursor cardiomyocytes.

[0053] FIGs. 7A-7C show representative flow cytometry scatter plots evaluating the composition of mature cardiomyocytes (CTAWT-FITC positive / high) and non-cardiomyocyte cells (COL3A 1 -AF594 positive / high) in samples of cells having undergone cardiomyocyte differentiation processes, on Day 51 post-mesoderm induction FIG. 7A shows a representative plot showing the proportion of mature cardiomyocytes and non-cardiomyocytes in a control, undifferentiated pluripotent stem cell sample. FIG. 7B shows a representative plot showing the proportion of mature cardiomyocytes and non-cardiomyocytes in a sample of putative immature cardiomyocytes (Day 14 post-mesoderm induction). FIG. 7C shows a representative plot showing the proportion of mature cardiomyocytes and non-cardiomyocytes in the sample of putative mature cardiomyocytes.

[0054] FIG. 8A-8B show uniform manifold approximation and projection (UMAP) clustering of scRNA-seq data from samples of cells having undergone cardiomyocyte differentiation processes, on Day 51 post-mesoderm induction FIG. 8A shows the identification of cardiomyocyte (CM) and non-cardiomyocyte (non-CM) cell clusters. FIG. 8B shows subclustering of the CM and non-cardiomyocyte clusters reveals six cardiomyocyte clusters (0, 1, 2, 4, 5, and 6) and three non-cardiomyocyte clusters (3, 7, and 8).

[0055] FIGs. 9A-9C show overlapping expression of cardiac marker genes projected onto UMAP space. FIG. 9A shows expression of heart and neural crest derivatives expressed 2 (HAND2). FIG. 9B shows expression of GATA binding protein 4 (GATA4). FIG. 9C shows expression of GATA binding protein 6 (GATA6).

[0056] FIGs. 10A-10F show overlapping expression of epicardial-like cell marker genes projected onto UMAP space. FIG. 10A shows expression of podocalyxin like (PODXL). FIG. 10B shows expression of keratin 19 (KRT19). FIG. IOC shows expression of basonuclin zinc finger protein 1 (BNC1). FIG. 10D shows expression of uroplakin 3B (UPK3B). FIG. 10E shows expression of troponin T1 (TNNT1). FIG. 10F shows expression of collagen type IX alpha 3 chain (COL9A3).

[0057] FIGs. 11A-11H show overlapping expression of fibroblast-like cell marker genes projected onto UMAP space. FIG. 11A shows expression of collagen type III alpha 1 chain (COL3A1). FIG. 11B shows expression of fibronectin 1 (FN1). FIG. 11C shows expression of Thy-1 cell surface antigen (THY1). FIG. 11D shows expression of platelet derived growth factor receptor alpha (PDGFRA). FIG. HE shows expression of periostin (POSTN). FIG. HF shows expression of actin alpha 2 smooth muscle (ACTA2). FIG. 11G shows expression of transforming growth factor beta 1 (TGFB1). FIG. 11H shows expression of tissue factor pathway inhibitor 2 (TFPI2).

[0058] FIGs. 12A-12B show exemplary flow cytometry scatter plots of samples of cells having undergone cardiomyocyte differentiation processes, on Day 51 post-mesoderm induction. FIG. 12A shows identification of a subpopulation of non-cardiomyocytes as epicardial-like cells using KRT19. Left panel is a representative plot showing that 17.5% of cells in the sample of putative cardiomyocytes are epicardial-like cells (KRT19 positive / high and COL3A1 positive / high). Middle panel is a representative plot showing that 66.7% of the cells in a sample of putative cardiomyocytes are mature cardiomyocytes (CTNT positive / high) while 7.68% of cells are epicardial-like cells (KRT19 positive / high). Right panel is a representative plot showing 33.8% of cells in a sample of putative cardiomyocytes express an epicardial marker gene, KRT19. FIG. 12B shows identification of epicardial-like cells using PODXL. The top left panel is a representative plot showing the proportion of cell populations in a sample of putative cardiomyocytes express CD 172a and / or CD36. The top middle panel from the left is a representative plot showing that 25.7% of cells in a sample of putative mature cardiomyocytes are off-target cells (CD90 positive / high). The top right panel is a representative plot showing 2.35% of cells in a sample of putative mature cardiomyocytes are epicardial-like cells (PODXL positive / high). The bottom left panel is a representative plot showing that 2.33% of cells in a sample of putative mature cardiomyocytes are epicardial-like cells (PODXL positive / high). The bottom middle panel is a representative plot showing that 7.13% of cells in a sample of putative mature cardiomyocytes are epicardial-like cells.

[0059] FIG. 13 shows mean expression of certain markers by putative mature cardiomyocytes using flow cytometry based on expression of CTNT, MLC2v, MLC2a, CD172, CD36, COL3A1, KI67, CD31, CD90, and PODXL

[0060] FIG. 14 shows representative flow cytometry scatter plots evaluating heterogeneity of mature cardiomyocytes in samples of cells having undergone cardiomyocyte differentiation processes, on Day 51 post-mesoderm induction. Two cell populations, “T-PD6-D51” (top row) and “W-PD8-D51” (bottom row), were assessed for expression of molecular profiles associated with mature cardiomyocyte identity (positive / high CTMT-FITC); fibroblast-like identity (positive / high COL3A1 -AF647); endothelial-like identity (positive / high CD3 / -BV605); neural- like identity (positive / high PAX6-PE); and myofibroblast-like non-cardiomyocyte identity (<z.S'A7A-CL594). Long-dashed boxes indicate cells are likely to be non-cardiomyocytes and short-dashed boxes indicate cells are likely to be cardiomyocytes.

[0061] FIGs. 15A-15B show expression of certain Actin-related markers in putative mature cardiomyocytes. FIG. 15A shows ACTA2 (a.k.a. aSMA) expression in a putative mature cardiomyocyte population (scRNA-seq). FIG. 15B shows expression of ACTA2 (a.k.a. aSMA), ACTA1, and ACTC1 in putative mature cardiomyocytes (top panel); ACTA2 (a.k.a. aSMA) expression is expected to decrease with maturity and ACTC1 expression is expected to increase with maturity (bottom panel).

[0062] FIG. 16 shows flow cytometric analysis of putative cardiomyocyte populations having a sub-population of ACTA2 (a.k.a. <z.S' 4)-cnrichcd cells on Day 51 (top panel) and Day 65 (bottom panel).

[0063] FIG. 17 is a schematic showing a proposed timeline for dynamic expression of CTNT, MLC2a, MLC2v, ACTA2 (a.k.a. aSMA), and cTNI.

[0064] FIG. 18 shows a PAX6-cnrichcd subpopulation in a heterogenous population primarily containing putative cardiomyocytes, as determined by flow cytometric analysis.

[0065] FIG. 19 shows flow cytometric analysis of a heterogenous population primarily containing putative cardiomyocytes.

[0066] FIG. 20 shows a PAX6-cnrichcd subpopulation in a second population of otherwise putative cardiomyocytes, as determined by flow cytometric analysis.

[0067] FIG. 21 shows expression of CTNT, MLC2v, MLC2a, COL3A1, KI67, and PAX6 in putative cardiomyocytes, as determined by flow cytometric analysis. DETAILED DESCRIPTION

[0068] Described herein, in some aspects, are methods and compositions useful for preparing cardiac cell therapies. Cardiac cell therapies generally refer to stem-cell-derived exogenous cells that are administered to the heart of a subject (e.g., in the form of a graft) in order to repair cardiac tissue in need thereof. Though cardiac cell therapies hold great promise for treating cardiac injuries, currently available differentiation protocols and their resulting cardiac cell populations still contain a considerable percentage of cell population heterogeneity. However, using clustering analysis of high dimensional genetic and molecular data and a flow cytometrybased assay, the inventors have surprisingly identified methods for identifying high purity cell populations suitable for preparation into cardiac cell therapies. Described herein, in some aspects, are compositions and methods related to identifying and / or preparing suitable cells (e.g., human cells) and / or suitable batches of cells (e.g., human cells) for cardiac cell therapies.

[0069] In some aspects, compositions disclosed herein comprise isolated cardiac cells (e.g., cardiomyocytes), wherein the molecular profile is a molecular profile characteristic of cardiomyocytes. In some embodiments, the molecular profile characteristic of cardiomyocytes is a molecular profile selected from one or more of the following: al. negative / low expression of one or more markers selected ANXA1, BGN, CCDC80, CDC42EP5, CDH11, CDON, CFH, COLIA2, COL3AI, COL5AI, CRIMI, CTSH, CXCLI2, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBN1, FLRT2, FN1, GATA3, GATA6, KRT19, LIMA1, LRPR2, LRRC17, MGP, MMP2, NUPRI, OGN, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNTI, and / or UPK3B, a2. negative / low expression of one or more markers selected from: ANXA1, CCDC80, CDON, CRIM1, COL3A1, CTSH, DCN, FLRT2, KRT19, LRPR2, NPNT, NUPRI, PLAC9, PODXL, POSTN, RDH10, SBSON, S100A10, S100A11, SPARC, TMEM88, TNNTI, and / or UPK3B, a3. negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, COL1A2, COL3A1, CXCL12, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBN1, FN1, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN, PTX3, S100A10, S100A11, SLIT2, SPARC, TFPI2, TGFb, and / or THY1, a4. negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CDH11, COL1A2, COL3A1, COL5A1, EVAIB, FBNI, LIMA1, MGP, MMP2, OGN, PLAC9, S100A10,' a5. negative / low expression of one or more markers selected from COL3A1, PODXL, and / or KRT19, a6. negative / low expression of one or more markers selected from KRT19 and / or PODXL, a7. negative / low expression of one or more markers selected from COL1A2 and / or FNI and / or a8. negative / low expression of COL3A1 In some embodiments, an isolated cardiac cell having the molecular profile of any one of al-a7 also exhibit positive / high expression of CTNT. In some embodiments, an isolated cardiac cell having the molecular profile of any one of al-a7 also exhibits positive / high expression of MLC2v. In some embodiments, an isolated cardiac cell having the molecular profile of any one of al-a7 also exhibit positive / high expression of CTNT and / or MLC2v. In some embodiments, an isolated cardiac cell having the molecular profile of any one of al-a7 also exhibits negative / low of MLC2a. In some embodiments, an isolated cardiac cell having the molecular profile of any one of al-a7 also exhibits positive / high expression of CTNT and MLC2V and negative / low expression of MLC2a.

[0070] In some aspects, compositions disclosed herein comprise isolated cardiac cells (e.g., epicardial-like cells), wherein the molecular profile is a molecular profile characteristic of epicardial-like cells. In some embodiments, the molecular profile characteristic of epicardial-like cells is a molecular profile selected from one or more of the following: bl. positive / high expression of ANXA1, CCDC80, CDON. CRIM1, COL3A1, CTSH,

[0071] DCN, FLRT2, KRT19, LRPR2, NPNT, NUPR1, PLAC9, PODXL. POSTN. RDH10, SBSON, S100A10, S100A11, SPARC, TMEM88, TNNT1, and / or UPK3B, b2. positive / high expression of ANXA1, CCDC80, CDON, COL3A1, CRIM1, CTSH,

[0072] DCN, FLRT2, KRT19, LRPR2, NUPR1, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, TMEM88, TNNT1, and / or UPK3B, b3. positive / high expression of BNC1 , COL9A3, KRT19, NPNT, PODXL, TNNT1 , and / or UPK3B„ b4. negative / low expression of CD 172a and positive / high expression of CD90 and PODXL, b5. negative / low expression of CD36 and positive / high expression of CD90 and PODXL, b6. positive / high expression of CD90 and PODXL, or b7. positive / high expression of KRT19 and / or PODXL.

[0073] In some embodiments, an isolated cardiac cell having the molecular profile of any one of bl-b7 also exhibit negative / low expression of CTNT. In some embodiments, an isolated cardiac cell having the molecular profile of any one of cl-c7 also exhibits negative / low expression of MLC2v. In some embodiments, an isolated cardiac cell having the molecular profile of any one of bl-b7 also exhibit negative / low expression of CTNT and / or MLC2v. In some embodiments, an isolated cardiac cell having the molecular profile of any one of bl-b7 also exhibits positive / high expression of MLC2a. In some embodiments, an isolated cardiac cell having the molecular profile of any one of bl-b7 also exhibits negative / low expression of CTNT and MLC2V and positive / high expression of MLC2a.

[0074] In some aspects, compositions disclosed herein comprise isolated cardiac cells (e.g., fibroblast-like cells), wherein the molecular profile is a molecular profile characteristic of fibroblast- like cells. In some embodiments, the molecular profile characteristic of fibroblast- like cells is a molecular profile selected from one or more of the following: cl. positive / high expression of ACTA2, ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, COL1A2, COLS Al, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FN1, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN, PTX3, S100A10, S100A11, SLIT2, SPARC, TFP12, TGFb, and / or THY], cl. positive / high expression of ANXA1, BGN, CCDC80, CDC42EP5, COL1A2, COLS Al, CXCL12, ELN, EMILIN 1, EVA1B, FBLN5, FBN1, LRRC17, MGP, MMP2, OGN, PLAC9, S100A10, S100A11, and / or SPARC, c3. positive / high expression of ATCA2, COLS Al , FN1 , PDGFRa, POSTN, TFP12,

[0075] TGFB \ . and / or THY; c4. negative / low expression of CD 172a and positive / high expression of CD90, c5. negative / low expression of CD36 and positive / high expression of CD90, c6. negative / low expression of PODXL and positive / high expression of CD90, or c7. positive / high expression of COL1A2 or FN1.

[0076] In some embodiments, an isolated cardiac cell having the molecular profile of any one of cl-c7 also exhibit negative / low expression of CTNT. In some embodiments, an isolated cardiac cell having the molecular profile of any one of cl-c7 also exhibits negative / low expression of MLC2v. In some embodiments, an isolated cardiac cell having the molecular profile of any one of cl-c7 also exhibit negative / low expression of CTNT and / or MLC2v. In some embodiments, an isolated cardiac cell having the molecular profile of any one of cl-c7 also exhibits positive / high expression of MLC2a. In some embodiments, an isolated cardiac cell having the molecular profile of any one of cl-c7 also exhibits negative / low expression of CTNT and MLC2V and positive / high expression of MLC2a.

[0077] Cardiac Cell Types

[0078] The present disclosure relates, in some aspects, to cardiac cell therapies (e.g., cardiac grafts) comprising a plurality of cells, wherein the plurality of cells have been determined to have a molecular profile characteristic of a particular cardiac cell type (e.g., cardiomyocytes, epicardial-like cells, and fibroblast-like cells). Cardiomyocytes

[0079] In some embodiments, a plurality of cells have been determined to have a molecular profile characteristic of cardiomyocytes (e.g., characteristic of cardiomyocyte identity). In some embodiments, cardiomyocytes are cells having cardiomyocyte lineage, including, but not limited to, ventricular cardiomyocytes, atrial cardiomyocytes, and / or cardiac smooth muscle cells. Cardiomyocytes can be understood to be cells at any stage of cardiomyocyte development without restriction, unless stated otherwise.

[0080] In some embodiments, cardiomyocytes are mature cardiomyocytes. In some embodiments, a mature cardiomyocyte is any terminally differentiated (e.g., quiescent) cardiac muscle cell. In some embodiments, a mature cardiomyocyte is derived from an embryonic stem cell. In some embodiments, a maturer cardiomyocyte is derived from a pluripotent stem cell. In some embodiments, the pluripotent stem cells comprise positive / high expression of one or more of the following markers: stage- specific embryonic antigen 3 and / or 4 (SSEA3 / 4), podocalyxin (TRA-1-60, also referred to synonymously herein as PODXLy octamer-binding transcription 3 and / or 4 (OCT3 / 4). homeobox protein NANOG (NANOG), and sex-determining region Y-box 2 (SOX2). In some embodiments, a mature cardiomyocyte is derived from an immature cardiomyocyte which has been contacted by a maturation cocktail (e.g., RPMI-1640 supplemented with b27 minus insulin).

[0081] In some embodiments, a cardiomyocyte is derived from a stem cell (e.g., a human stem cell). In some embodiments, a cardiomyocyte is or is derived from an induced pluripotent stem cell (iPSC). In some embodiments, a cardiomyocyte is or is derived from a human pluripotent stem cell (hPSC). In some embodiments, a cardiomyocyte is or is derived from an embryonic stem cell (ESC) (e.g., a human embryonic stem cell).

[0082] In some embodiments, cardiomyocytes are matured in vivo (e.g., in a subject). In some embodiments, cardiomyocytes are matured in vitro. In some embodiments, cardiomyocytes are mature ventricular cardiomyocytes. In some embodiments, cardiomyocytes are mature atrial cardiomyocytes. In some embodiments, cardiomyocytes are mature cardiac smooth muscle cells. In some embodiments, mature cardiomyocytes exhibit the following characteristics within about 51 days from mesoderm induction (Day 51): (1) cluster of differentiation 36 (CD36) positive / high and / or myosin regulatory light chain 2v (MEC2v) positive / high, and / or downregulated myosin regulatory light chain 2a (MLC2a') (relative to immature cardiomyocytes), e.g., using flow cytometric analysis; and (2) capable of contraction with application of an external stimulus (e.g., electrical stimulus). In some embodiments, a cell having cardiomyocyte identity is a cell expressing one or more markers characteristic of cardiomyocytes (e.g., having a molecular profile characteristic of cardiomyocytes). In some embodiments, cardiomyocytes have positive / high expression of cardiac muscle troponin T (CTNT, also referred to synonymously herein as TNNT2). In some embodiments, a cell having cardiomyocyte identity has positive / high expression of one or more positive / high genetic or molecular markers provided in Table 1 and has negative / low expression of one or more negative genetic or molecular markers provided in Table 1.

[0083] Table 1. Non-limiting characteristics of cardiomyocytes throughout development

[0084] Non-limiting examples of cardiomyocyte differentiation and maturation procedures can be found in PCT Publication No.: WO 2014 / 200339, PCT Publication No.: WO 2017 / 039445, PCT Publication No.: WO 2020 / 227232, U.S. Publication No.: US 2020 / 0407687, each of which are incorporated herein by reference. Epicardial-like Cells

[0085] In some embodiments, a plurality of cells have been determined to have a molecular profile characteristic of epicardial-like cells (e.g., characteristic of epicardial cell identity). An epicardial-like cell is an epicardial cell or a cell that exhibits characteristics similar to those cells of the epicardium, but may not be a true epicardial cell. The epicardium refers to the outermost protective layer of the heart, comprising a single layer of mesothelial cells. During development, the epicardium supports embryonic heart formation by, for example, harboring progenitor cells which may differentiate into various nonmyocyte cardiac lineages (e.g., vascular smooth muscle cells, pericytes, and fibroblasts). In healthy adults, the epicardium becomes dormant; however, cardiac injury may reactivate epicardial cells and contribute to tissue repair (Quijada, P., el al. (2020). Circulation research, 126(3), 377-394.). In some embodiments, epicardial-like cells are epicardial cells. In some embodiments, epicardial-like cells are epicardial-derived progenitor cells (EPDCs). In some embodiments, epicardial-like cells are epicardioids.

[0086] In some embodiments, an epicardial-like cell is derived from a stem cell (e.g., a human stem cell). In some embodiments, an epicardial-like cell is or is derived from an induced pluripotent stem cell (iPSC). In some embodiments, an epicardial-like cell is or is derived from a human pluripotent stem cell (hPSC). In some embodiments, an epicardial-like cell is or is derived from an embryonic stem cell (ESC) (e.g., a human embryonic stem cell).In some embodiments, a cell having epicardial-like cell (e.g., epicardial cell) identity is a cell expressing one or more markers characteristic of epicardial-like cells (e.g., having a molecular profile characteristic of epicardial-like cells). In some embodiments, an epicardial-like cell comprises one or more markers related to expression (e.g., transcript, protein) of epicardial or epicardiallike cell markers. Non-limiting examples of epicardial or epicardial-like cell markers include keratin 19 (KRT19), podocalyxin-like (PODXL), uroplakin 3B (UPK3B), retinoic acid receptor responder protein 2 (RARRES2), basonuclin 1 (BNC1), collagen type IX alpha 3 chain (COL9A3), decorin (DCN). twist family bHLH transcription factor 1 (TWIST1), periostin (POSTN). receptor (calcitonin) activity modifying protein 1 (RAMP1), neuropilin 2 (NRP2), secreted protein acidic and rich in cysteine (SPARC), lysine hydroxylase 6 (EYH6), troponin T1 slow skeletal type (TNNT1), intelectin 1 (ITEN1), calbindin 2 (CAEB2), sphingomyelin phosphodiesterase 3 (SMPD3), Somatomedin b and thrombospondin type 1 domain containing (SBSON), low density lipoprotein receptor-related protein 2 (LRPR2), cell adhesion associated, oncogene regulated (CDON), transmembrane protein 88 (TMEM88), retinol dehydrogenase 10 (RDH10), cathepsin H (CTSH), cysteine-rich transmembrane BMP regulator 1 (CRIME), S100 calcium-binding protein A10 (S100A10) , S100 calcium-binding protein Al l (S100A11), coiled- coil domain containing 80 ( CCDC80), fibronectin leucine rich transmembrane protein 2 (FLRT2), placenta- specific protein 9 (PLAC9), nuclear protein- 1 (NUPRI ), annexin Al (ANXA1), collagen type III, alpha I (COL3A I). and nephronectin (NPNT).

[0087] Fibroblast-like Cells

[0088] In some embodiments, a plurality of cells have been determined to have a molecular profile characteristic of fibroblast- like cells (e.g., having fibroblast- like cell identity). A fibroblast-like cell is a fibroblast or a cell that exhibits characteristics similar to fibroblasts, but may not be true fibroblast. Cardiac fibroblasts are cells which produce connective tissue (e.g., periostin, vimentin, fibronectin, and collagen types I, III, V, and VI) in the heart. During development, fibroblasts and fibroblast-like cells proliferate in the heart to deposit and / or degrade the extracellular matrix. In healthy adults, fibroblasts enter a “resting” phase during which the extracellular matrix is maintained, with limited to no proliferation; however, cardiac injury may result in proliferation of fibroblasts (e.g., activated fibroblasts) which may mediate changes to the extracellular matrix (Ivey, M. J., & Tallquist, M. D. (2016). Circulation Journal, 80(11), 2269-2276.). Fibroblast-like cells often share morphological features (e.g., an elongated, spindle-shaped appearance) and / or functional properties (e.g., involved in synthesizing and maintaining the extracellular matrix) as fibroblasts. In some embodiments, a fibroblast- like cell is a fibroblast. In some embodiments, a fibroblast-like cell is a fibrocyte. In some embodiments, a fibroblast-like cell is a telocyte. In some embodiments, a fibroblast-like cell is a myofibroblast. In some embodiments, a fibroblast-like cell is a protomyofibroblast. In some embodiments, a fibroblast-like cell is a mesenchymal cell. In some embodiments, a fibroblast-like cell is a stromal cell. In some embodiments, a fibroblast- like cell is derived from a stem cell (e.g., a human stem cell). In some embodiments, a fibroblast- like cell is or is derived from an induced pluripotent stem cell (iPSC). In some embodiments, an fibroblast-like cell is or is derived from a human pluripotent stem cell (hPSC). In some embodiments, an fibroblast-like cell is or is derived from an embryonic stem cell (ESC) (e.g., a human embryonic stem cell).

[0089] In some embodiments, a cell having fibroblast-like cell (e.g., fibroblast cell) identity is a cell expressing one or more markers characteristic of fibroblast-like cells (e.g., having a molecular profile characteristic of fibroblast-like cells).

[0090] In some embodiments, a fibroblast-like cell molecular profile comprises one or more markers related to expression (e.g., transcript, protein) of fibroblast or fibroblast-like cell markers. Non-limiting examples of fibroblast or fibroblast-like cell markers include collagen type III alpha 1 chain (COL3A1 collagen type I alpha 2 chain (COL1A2), thy-1 cell surface antigen (THYF), transforming growth factor beta (TGFB), actin alpha 2 smooth muscle aorta (ACTA2), fibronectin 1 (FN1), decorin (DCN), fibrillin 1 (FBN1), periostin (POSTN), neuropilin 2 (NPN2), slit guidance ligand 2 (SLIT2), secreted protein acidic and rich in cysteine (SPARC), platelet-derived growth factor receptor alpha (PDGFRA), complement factor H (CFEL), pentraxin 3 (PTX3), oncostatin M receptor (OSMR), fibulin 2 (FBLN2), biglycan (BGN), elastin (ELN), C-X-C motif chemokine 12 (CXCL12’, also known as stromal cell-derived factor 1 (SDF1)), leucine-rich repeat-containing protein 17 (LRRC17), osteoglycin (OGN), fibulin 5 (FBLN5; also known as developmental arteries and neural crest epidermal growth factor (DANCE)), elastin microfibril interfacer 1 (EM1L1N1), matrix metalloproteinase-2 (MMP-2), cell division control protein 42 effector protein 5 (CDC42EP5), Eva-1 homolog B (EVA IB), matrix Gia protein (MGP), S100 calcium-binding protein Al l (S100A11), annexin Al (ANXA1), placenta- specific protein 9 (PLAC9), coiled-coil domain containing 80, S100 calcium-binding protein A10 (S100A10), heart- and neural crest derivatives-expressed protein 2 (HAND2), GATA binding protein 4 (GATA4), GATA binding protein 6 (GATA6), and myristoylated alanine-rich C- kinase substrate (MARCKS).

[0091] Identifying Cells Suitable for Cardiac Cell Therapy

[0092] The present disclosure relates, in some aspects, to identification of cells (e.g., a cell, a batch of cells) that are suitable (e.g., are “on target”) or unsuitable (e.g., are “off target”) for a particular composition or method.

[0093] In some embodiments, cells are identified as having a molecular profile characteristic of a particular cell type. In some embodiments, cells are identified as having a molecular profile which excludes a particular cell type. Cells (e.g., cardiomyocytes) can be understood to comprise a molecular profile. A cell’s molecular profile encompasses a signature comprising expression levels of one or more markers (e.g., nucleic acids, peptide / protein). The term “marker” is used widely in the art and can be commonly understood to refer to a biological molecule and / or a detectable fragment thereof. In some embodiments, markers comprise nucleic acids, peptides / proteins, and detectable fragments thereof. Nucleic acid markers may comprise nuclear RNA, pre-mRNA, mRNA, copy DNA, and fragments thereof. Peptides / proteins may comprise any polymer comprising amino acid residues linked by peptide bonds, including, but not limited to, native proteins, recombinant proteins, synthetic proteins, proteins having one or more co- or post-expression modifications (e.g., glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, etc.), and any fragments thereof. In some embodiments, the method comprises identifying cells as having a molecular profile characteristic of cardiomyocyte identity. In some embodiments, the method comprises identifying cells as having a molecular profile characteristic of epicardial-like cell identity. In some embodiments, the method comprises identifying cells as having a molecular profile characteristic of fibroblast-like cell identity. In some embodiments, the method comprises identifying cells as having a molecular profile characteristic of non-cardiomyocyte identity (e.g., a molecular profile which excludes cardiomyocytes).

[0094] Expression of a marker by a cell can be measured in any number of ways, but generally refers to quantification of the presence (or absence) of a distinct signal corresponding to the marker in or on the cell, compared to a control and / or baseline. In some instances, expression of a marker is measured as an absolute quantity (e.g., weight, molar amount, concentration). In some instances, expression of a marker is expressed as a relative quantity (e.g., fold-increase, fold-decrease, percent). In some instances, expression of a marker in a cell is measured in a semi-quantitative manner (e.g., “high” or “low” fluorescence, as used in flow cytometry).

[0095] Cells can be understood to be “positive” or have “high expression” of one or more markers if they express the marker at or above a baseline level. For example, a cell may be understood to be positive (+) for a marker which is detected in the cell. A cell may be understood to have “high” expression for a marker when the quantity (e.g., absolute, relative, semi-quantitative) of the marker in the cell is above some baseline or threshold (e.g., population average, control cell). Cells can be understood to be “negative” or have “low expression” of one or more markers if they do not express the marker at or above a baseline level. For example, a cell may be understood to be negative (-) for a marker which is not detected in the cell. A cell may be understood to have “low” expression for a marker when the quantity (e.g., absolute, relative, semi-quantitative) of the marker in the cell is below some baseline or threshold (e.g., population average, control cell).

[0096] In some embodiments, methods of identifying a cell (e.g., cardiomyocyte) comprise identifying expression of one or more cell markers by the cell. In some embodiments, one or more of the cell markers is a cell type-specific marker. Methods of identifying cell type-specific markers are known to those of skill in the art and are dependent on the marker of interest. In a non-limiting example, cell surface proteins or cell type-specific genes may be used to identify or define specific cell populations or sub-populations (i.e., subsets) using flow cytometry, immunohistochemistry, immunofluorescence, polymerase chain reaction (e.g., reverse transcription PCR), or fluorescence-activated cell sorting (FACS). The markers of interest may be determined by a number of techniques, including microarrays and ribonucleic acid (RNA) sequencing, such as single cell RNA sequencing (scRNA-seq).

[0097] In some instances, quantification of a signal for a marker in a test cell is determined by detecting the presence of a marker (e.g., a nucleic acid or protein derived therefrom). In some instances, quantification of a signal for a marker in a test cell is compared to quantification of a signal for the same marker in a negative control cell (e.g., a cell known to not highly express the marker) or a positive control cell (e.g., a cell known to highly express the marker). For example, in some embodiments, a test cell determined to be “positive” for the marker or have “high” expression of the marker expresses the marker at an increased average level compared a negative control cell, for example, 1.5-fold increase, 2-fold increase, 3-fold increase, 4-fold increase, 5- fold increase, 10-fold increase, 20-fold increase, 30-fold increase, 40-fold increase, 50-fold increase, or higher. In another example, a test cell determined to be “positive” for the marker or have “high” expression of the marker expresses the marker at the same or an increased average level compared to a positive control cell. In another example, a test cell determined to be “negative” for the marker or have “low” expression of the marker expresses the marker at a reduced average level compared to a positive control cell, for example, 1.5-fold decrease, 2-fold decrease, 3-fold decrease, 4-fold decrease, 5-fold decrease, 10-fold decrease, 20-fold decrease, 30-fold decrease, 40-fold decrease, 50-fold decrease, or less. In another example, a test cell determined to be “negative” for the marker or have “low” expression of the marker expresses the marker at the same or a reduced average level compared to a negative control cell.

[0098] When used in the context of expression of one or more markers by a cell, the terms “positive / high” and “negative / low” can be understood to mean “positive or high” and “negative or low” expression of the one or more markers, respectively. One of ordinary skill in the art will appreciate that the term “positive / high” and “negative / low” encompasses different methods of determining presence of and / or an expression level of a marker; for example, a cell population may be determined to be “positive” for a marker when analyzed using scRNA-seq (e.g., based on an absolute level of an RNA transcript) or “high” for the same marker when analyzed using flow cytometric analysis (e.g., based on assessment of a level relative to a control cell).

[0099] In some instances, quantification of a signal for a marker in a first test cell is compared to quantification of a signal for the same marker in a second test cell, such that expression of the marker in the first cell is described relative to the second cell. In some embodiments, a first test cell is deemed to comprise positive / high expression of one or more markers relative to expression of the same marker by a second cell. In some embodiments, a first test cell is identified as comprising an off-target molecular profile because of positive / high expression of one or more markers (e.g., markers in Table 2) relative to a second cell. In some embodiments, a first test cell is identified as comprising an off-target molecular profile because of negative / low expression of one or more markers (e.g., markers in Table 2) relative to a second cell. In some embodiments, a first test cell and a second test cell are identified as comprising a target molecular profile because of positive / high expression of one or more markers (e.g., markers in Table 2). In some embodiments, a first test cell and a second test cell are identified as comprising a target molecular profile because of negative / low expression of one or more markers (e.g., markers in Table 2).

[0100] In some embodiments, a cell is identified as being of a particular cell type (e.g., identified as cardiomyocyte) or at a certain stage of development (e.g., identified as a mature cardiomyocyte) using cell-type labeling. Cell-type labeling, as it relates to RNA sequencing, refers to the process of identifying cell-type- specific canonical marker genes that are differentially expressed and labeling the cells based on the canonical marker genes. In some embodiments, cells are identified as being cardiac cells using cell-type labeling. In some embodiments, cells are identified as being cardiomyocytes, endothelial cells, and / or fibroblasts using cell-type labeling. In some embodiments, cells are analyzed to determine their cell-type. In some embodiments, cells known to have specific cell-type (e.g., identified as such by a method described herein) are used as control cells to determine the cell-type of unidentified test cells. In some embodiments, cells are identified as being at a particular stage of maturity (e.g., precursor cells, immature cells, mature cells). In some embodiments, cells are identified as mature cells (e.g., mature cardiomyocytes). In some embodiments, cells are analyzed to determine their stage of maturity. In some embodiments, cells known to be at a certain stage of maturity (e.g., identified as such by a method described herein) are used as control cells to determine the stage of maturity of unidentified test cells.

[0101] In some embodiments, cells of a first type are differentiated from cells of a second type using differential gene analysis. Differential gene expression analysis, as it relates to RNA sequencing, identifies genes that are differentially expressed (e.g., more highly or lowly expressed) between two or more states (e.g., cell types, bioprocess) that may be a molecular marker that differentiates the two or more states. Methods of differential gene expression analysis are known to those of skill in the art and may employ Bayesian statistics, linear models, negative binomial distributions, t-tests, or machine learning algorithms (e.g., random forest, support vector machine). In some embodiments, cells in a first developmental stage are differentiated from cells in a second developmental stage using differential gene analysis. In some embodiments, a batch of on-target cells (e.g., mature cardiomyocytes) is differentiated from a batch of off-target cells (e.g., fibroblast-like cells, epicardial-like cells) using differential gene analysis.

[0102] In some embodiments, a cell (e.g., a batch of cells) is identified as expressing one or more cell markers using flow cytometry. In some embodiments, a cell is identified as expressing one or more cell markers relative to a control cell using flow cytometry. In some embodiments, a control cell is a fluorescence minus one (FMO) control cell, an unstained cell, a single-color stained cell, or a sample known to express certain markers (e.g., a viability-only sample, a sample of cells previously identified as having a particular molecular profile). In some embodiments, a fluorescence minus one (FMO) cell population is used as a control cell population, e.g., to improve gating accuracy of negative fractions of a test cell population; for example, by including all markers in a panel except a specific marker of interest. In some embodiments, a viability-only cell population (e.g., a population of cells stained with a viability dye) is used a control cell population. In some embodiments, cells are fixed (e.g., when detecting intracellular markers). In some embodiments, cells are live (e.g., when detecting extracellular markers).

[0103] In some embodiments, a cell (e.g., a batch of cells) is identified as expressing one or more cell markers using RNA sequencing (RNA-seq). Non-limiting examples of RNA-seq include bulk RNA sequencing, single-cell RNA sequencing, spatial RNA sequencing, mRNA sequencing, total RNA-seq, strand- specific RNA-seq, and long read RNA-seq. Those of skill in the art will recognize that methods of RNA-seq may vary depending on the RNA of interest, sequencing platform, and the intended application, but generally require 1) reverse transcription of RNA into DNA; 2) preparation of a sequencing library from the DNA; 3) sequencing; and, 4) data processing, which may include batch correction, clustering, cell-type labeling, and differential gene expression analysis.

[0104] In some embodiments, cells having molecular profiles associated with certain cardiac cell populations are identified using clustering (e.g., RNA-seq clustering). Clustering, as it relates to RNA-seq, refers to the process of grouping individual cells based on their gene expression profiles to identify cell populations within a heterogenous sample. Since gene expression profiles are typically high-dimensional, clustering usually requires dimensionality reduction prior to clustering to facilitate analysis. Methods of dimensionality reduction are known to those of skill in the art and can include principal component analysis (PCA), nonnegative matrix factorization (NMF), linear discriminant analysis (LDA), T-distributed Stochastic Neighbor Embedding (t-SNE), and uniform manifold approximation and projection (UMAP). Each method of dimensionality reduction is known to those of skill in the art to differ in their ability to preserve global and local structure. Other algorithms for dimensionality reduction are contemplated. Clustering algorithms can then be applied to the reduced gene expression profile to group and visualize cells that have similar gene expression profiles. Nonlimiting clustering algorithms include k-means clustering, hierarchical clustering, and expectation-maximization. Visualized clusters e.g., on a t-SNE or UMAP plot) can be used by those of skill in the art to explore heterogeneity within cell populations and relationships between cell populations. In some embodiments, clustering analysis is used to identify molecular profiles associated with one or more desirable characteristics.

[0105] In some embodiments, a cell, or a plurality of cells (e.g., a batch of cells) is identified as being “on target.” A cell identified as being “on target” is any cell having one or more desirable characteristics. In some embodiments, a cell identified as being “on target” has a molecular profile comprising one or more desired markers. In some embodiments, a cell identified as being “on-target” has a molecular profile associated with a certain cardiac cell population. In some embodiments, a cell identified as being “on-target” has a molecular profile associated with epicardial-like cells. In some embodiments, a cell identified as being “on-target” has a molecular profile associated with fibroblast-like cells. In some embodiments, cells identified as being “on- target” have a molecular profile which excludes a certain cell type. Identifying certain cells as “on-target” may be useful, for example, for preparing a cardiac graft by contacting a batch identified as having a high percentage of “on target” cells with a physiologically acceptable medium. In some embodiments, at least 50% of cells in a plurality of cells are “on target.” In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% of cells in a plurality of cells are “on target.” In a preferred embodiment, at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100%) of cells in a plurality of cells are “on target.”

[0106] In some embodiments, cells are identified as being “off target.” A cell identified as being “off target” is any cell having a molecular profile comprising one or more undesired markers. In some embodiments, cells identified as being “off-target” have a molecular profile associated with a certain cardiac cell population. In some embodiments, cells identified as being “off- target” have a molecular profile associated with epicardial-like cells. In some embodiments, cells identified as being “off-target” have a molecular profile associated with fibroblast-like cells. In some embodiments, cells identified as being “off-target” have a molecular profile which excludes a certain cell type. Identifying certain cells as “off-target” may be useful, for example, for preparing a cardiac graft by contacting a batch of cells identified as having a low percentage of “off target” cells with a physiologically acceptable medium. In some embodiments, no more than 50% of cells in a plurality of cells are “off target.” In some embodiments, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, no more than 0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2%, no more than 0.1%, or 0% of cells in a plurality of cells are “off target.” In a preferred embodiment, no more than 10% (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0%) of cells in a plurality of cells are “off target.”

[0107] Distinguishing Markers

[0108] Several markers have been identified as potentially useful for determining whether cells are “on target” or “off target” based on expression patterns of the same. For example, in some embodiments, a molecular profile for a certain cardiac cell population comprises one or more of ACSL1, ACTA2 (a.k.a. a-SMA), ACTC1 , ACTN2, ADAMTSI9, ALDOC, ALPK3, ANXA1, AQP1, BGN, BNCI, CACNAID, CALB2, CAVI, CCDC80, CCNB1, CD 172a, CD36, CD90, CDC42EP5. CDH11, CDK1, CDKN2A. CDON. CFH, CKMT2, COLIA2, COL3AI, COL5AI, COL9A3. CRIME CSRP3, CTNT, CTSH, CXCLI2, DANCE, DCN, DES, ELN, EMILIN 1, ESAM, EVA1B, FABP3, FBLN2, FBLN5, FBN1, FHL2, FLRT2, FN1, FOXA2, FOXD3-AS1, G0S2, GAL, GATA3, GATA4, GATA6, GSN, HAND1, HAND2, HCN4, HEY2, HIF1A, HOPX, IGF2, IGFBP7, IRX4, ITGA7, ITLN1, KCNJ2, KCNJ3, KCTD12, KRT19, L1TD1, LEF1, LIMA1, LINC00678, LMOD1, LMOD2, LRPR2, LRRC17, LYH6, MAN1C1, MARCKS, MEF2C, MGP, MKI67, MLC2a, MLC2v, MMP2, MT-ND2, MYBPC3, MYH10, MYH11, MYH6, MYH7, MYL3, MYL4, MYL9, MYOZ2, NANOG, NKK2-5, NKX2-5, NPNT, NPPA, NR2F2, NUPRI, OCT3 / 4, OGN, OSMR, PAX6, PDGFRa, PDLIM1, PECAM1, PGAM2, PGC1A, PKP2, PLAC9, PLVAP, PODXL, POSTN, POU5F1, PPP1R3A, PTX3, RAMP1, RARRES2, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SHOX2, SLC25A4, SLC27A6, SLC2A1, SLIT2, SMPD3, SMTN, SOX2, SPARC, SPRY1, SSCB3 / 4, SSCC3 / 4, SSEA3 / 4, SULF1, SYNPO2L, TBX2, TBX5, TCAP, TFPI2, TGFb, TGM2, THY1, TMEM88, TNI, TNNCI, TNNI3, TNNTI, TPMI, TRA-1-60, TTN, TWIST 1, UPK3B, VCAM1, and / or VCAN.

[0109] ACSL1 is a gene encoding long-chain-fatty-acid— CoA ligase 1 (also known as ACS1, LACS, FACL1, FACL2, LACS1, and LACS2), which is involved in lipid biosynthesis and fatty acid degradation. In some embodiments, expression of ACSL1 by a cell is determined by measuring a level of an ACSL1 RNA (e.g., mRNA transcribed from ACSL1). Non-limiting examples of ribonucleic acid sequences of human ACSL1 can be found, for example, at GenBank Accession Nos. NM_001286708.2, NM_001286710.2, and UniProt Accession No. P33121.

[0110] ACTA2 is a gene encoding actin, aortic smooth muscle protein (also known as a-SMA, ACTSA, SMDYS, and ACTVS), which is involved in blood pressure homeostasis and vascular contractility. In some embodiments, expression of ACTA2 by a cell is determined by measuring a level of ACTA2 RNA (e.g., mRNA transcribed from ACTA2). Exemplary nucleic acid sequences of human ACTA2 can be found, for example, at GenBank Accession Nos. NG_011541.1, NM_001141945.3, and UniProt Accession No. P62736.

[0111] ACTG2 is a gene encoding actin gamma 2 smooth muscle (also known as ACT, ACTA3, ACTE, ACTL3, ACTSG, MMIHS5, VSCM, VSCM1), which is involved in cell motility of smooth muscle in enteric tissues. In some embodiments, expression of ACTG2 by a cell is determined by measuring a level of ACTG2 RNA (e.g., mRNA transcribed from ACTG2). Exemplary nucleic acid sequences of human ACTG2 can be found, for example, at GenBank Accession Nos. NG_034140.1, NM_001199893.2, and UniProt Accession No. B4DW52.

[0112] ACTC1 is a gene encoding actin, alpha cardiac muscle 1 (also known as ACTC, ASD5, CMD1R, CMH11, and LVNC4), which is involved in the cellular motility of cardiac muscle. In some embodiments, expression of ACTC1 by a cell is determined by measuring a level of an ACTC1 RNA (e.g., mRNA transcribed from ACTC1). Non-limiting examples of ribonucleic acid sequences of human ACTC1 can be found, for example, at GenBank Accession Nos.

[0113] NM_001406482.1, NM_001406483.1, and UniProt Accession No. P68032.

[0114] ACTN2 is a gene encoding alpha-actinin-2 (also known as MPD6, CMH23, CMY08, CMYP8, CMD1AA, and MYOCOZ), which is involved in the motility of cardiac and skeletal muscle. In some embodiments, expression of ACTN2 by a cell is determined by measuring a level of an ACTN2 RNA (e.g., mRNA transcribed from ACTN2). Non-limiting examples of ribonucleic acid sequences of human ACTN2 can be found, for example, at GenBank Accession Nos. NM_001103.4, NM_001278343.2, and UniProt Accession No. P35609.

[0115] ADAMTS19 is a gene encoding A disintegrin and metalloproteinase with thrombospondin motifs 19 (also known as CVDP2), which is involved in aortic valve morphogenesis. In some embodiments, expression of ADAMTS19 by a cell is determined by measuring a level of an ADAMTS19 RNA (e.g., mRNA transcribed from ADAMTS19). Non-limiting examples of ribonucleic acid sequences of human ADAMTS19 can be found, for example, at GenBank Accession No. NM_133638.6, and UniProt Accession No. Q8TE59. ALDOC is a gene encoding fructose-bisphosphate aldolase C (also known as ALDC), which is a glycolytic enzyme expressed in Purkinje cells and hippocampal cells in the brain. In some embodiments, expression of ALDOC by a cell is determined by measuring a level of an ALDOC RNA (e.g., mRNA transcribed from ALDOC). Non-limiting examples of ribonucleic acid sequences of human ALDOC can be found, for example, at GenBank Accession No. NM_005165.3, and UniProt Accession No. P09972.

[0116] ALPK3 is a gene encoding alpha-protein kinase 3 (also known as MAK, CMH27, and MIDORI), which is involved in cardiac muscle cell development. In some embodiments, expression of ALPK3 by a cell is determined by measuring a level of an ALPK3 RNA (e.g., mRNA transcribed from ALPK3). Non-limiting examples of ribonucleic acid sequences of human ALPK3 can be found, for example, at GenBank Accession No. NM_020778.5, and UniProt Accession No. Q96L96.

[0117] ANXA1 is a gene encoding annexin Al protein (also known as ANX1, and LPC1), which is involved in inhibition of phospholipase A2 and anti-inflammatory activities. In some embodiments, expression of ANXA1 by a cell is determined by measuring a level of ANXA1 RNA (e.g., mRNA transcribed from ANXA1). Exemplary nucleic acid sequences of human ANXA1 can be found, for example, at GenBank Accession Nos. NM_000700.3, XM_017014657.2, and UniProt Accession No. P04083.

[0118] AQP1 is a gene encoding aquaporin-1 (also known as CO, CHIP28, and AQP-CHIP), which is involved in the passive transport of water along an osmotic gradient into a cell. In some embodiments, expression of AQP1 by a cell is determined by measuring a level of an AQP1 RNA (e.g., mRNA transcribed from AQP1). Non-limiting examples of ribonucleic acid sequences of human AQP1 can be found, for example, at GenBank Accession Nos.

[0119] NM_00 1329872.2, NM_198098.4, and UniProt Accession No. Q5R819.

[0120] AS1 is a gene encoding prostaglandin D2 receptor (also known as PTGDR, DP, DPI, ASRT1, and PTGDR1), which is a receptor for prostaglandin D2, a mediator of allergic inflammation. In some embodiments, expression of AS1 by a cell is determined by measuring a level of an AS1 RNA (e.g., mRNA transcribed from AS1). Non-limiting examples of ribonucleic acid sequences of human AS1 can be found, for example, at GenBank Accession Nos. NM_000953.3, NM_001281469.2, and UniProt Accession No. Q13258.

[0121] BGN is a gene encoding biglycan protein (also known as PGI, MRLS, DSPG1, PG-S1, SEMDX, and SLRR1A), which is involved in bone growth, collagen fibril assembly, and muscle development / regeneration. In some embodiments, expression of BGN by a cell is determined by measuring a level of BGN RNA (e.g., mRNA transcribed from BGN). Exemplary nucleic acid sequences of human BGN can be found, for example, at GenBank Accession Nos. NG_015961.1, NM_001711.6, and UniProt Accession No. P21810.

[0122] BNC1 is a gene encoding zinc finger protein basonuclin-1 protein (also known as BNC, bnl, BSN1, POF16, and HsT19447), which is involved in keratinocyte proliferation and regulation for rRNA transcription. In some embodiments, expression of BNC1 by a cell is determined by measuring a level of BNC1 RNA (e.g., mRNA transcribed from BNC1). Exemplary nucleic acid sequences of human BNC1 can be found, for example, at GenBank Accession Nos. NM_001301206.2, NM_001717.4, and UniProt Accession No. Q01954.

[0123] CACNA1D is a gene encoding voltage-dependent L-type calcium channel subunit alpha- 1D (also known as CACH3, CACN4, CACNL1A2, CCHL1A2, CAV1.3, PASNA, and SANDD), which is a voltage gated calcium channel involved in muscle contraction, neurotransmitter release, and other calcium-dependent processes. In some embodiments, expression of CACNA1D by a cell is determined by measuring a level of an CACNA1D RNA (e.g., mRNA transcribed from CACNA I D). Non-limiting examples of ribonucleic acid sequences of human CACNA1D can be found, for example, at GenBank Accession Nos. NM_000720.4, NM_001128839.3, and UniProt Accession No. Q01668.

[0124] CALB2 is a gene encoding calretinin protein (also known as CR, CAL2, and CAB29), which is involved in message targeting, intracellular calcium buffering, and modulation of neuronal excitability. In some embodiments, expression of CALB2 by a cell is determined by measuring a level of CALB2 RNA (e.g., mRNA transcribed from CALB2). Exemplary nucleic acid sequences of human CALB2 can be found, for example, at GenBank Accession Nos. NM_001740.5, NM_007088.4, and UniProt Accession No. P22676.

[0125] CAV1 is a gene encoding caveolin-1 (also known as CGL3, PPH3, BSCL3, LCCNS, VIP21, and MSTP085), which is a scaffolding protein involved in caveolae plasma membranes. In some embodiments, expression of CAV1 by a cell is determined by measuring a level of an CAV1 RNA (e.g., mRNA transcribed from CAVI). Non-limiting examples of ribonucleic acid sequences of human CAVI can be found, for example, at GenBank Accession Nos. NM_001172895.1, NM_001172896.2, and UniProt Accession No. Q03135.

[0126] CCDC80 is a gene encoding coiled-coil domain-containing protein 80 protein (also known as CL2, URB, DRO1, SSG1, okuribin, and LINC01279), which is involved in fibronectin and heparin binding activity, extracellular matrix organization, and positive regulation of cell-substrate adhesion. In some embodiments, expression of CCDC80 by a cell is determined by measuring a level of CCDC80 RNA (e.g., mRNA transcribed from CCDC80). Exemplary nucleic acid sequences of human CCDC80 can be found, for example, at GenBank Accession Nos. NM_199511.3, NM_199512.3, and UniProt Accession No. Q76M96.

[0127] CCNBI is a gene encoding G2 / mitotic- specific cyclin-Bl (also known as CCNB), which is involved in the transition between the G2 and M phases in mitosis. In some embodiments, expression of CCNBI by a cell is determined by measuring a level of an CCNBI RNA (e.g., mRNA transcribed from CCNBI). Non-limiting examples of ribonucleic acid sequences of human CCNBI can be found, for example, at GenBank Accession Nos. NM_001354844.2, NM_001354845.2, and UniProt Accession No. P14635.

[0128] CD 172a is a gene encoding tyrosine-protein phosphatase non-receptor type substrate 1 protein (also known as SIRPA, BIT, MFR, P84, SIRP, MYD-1, SHPS1, and PTPNS1), which is involved in negative regulation of receptor tyrosine kinase-coupled signaling processes. In some embodiments, expression of CD 172a by a cell is determined by measuring a level of CD 172a RNA (e.g., mRNA transcribed from CD] 72a). Exemplary nucleic acid sequences of human CD172a can be found, for example, at GenBank Accession Nos. NM_001040022.1, NM_001040023.2, and UniProt Accession No. P78324.

[0129] CD36 is a gene encoding platelet glycoprotein 4 protein (also known as FAT, GP4, GP3B, GPIV, CHDS7, PASIV, SCARB3, and BDPLT10), which is a receptor for thrombospondin and is involved in cytoadherence of Plasmodium falciparum parasitized erythrocytes and regulation of fatty acid transport. In some embodiments, expression of CD36 by a cell is determined by measuring a level of CD36 RNA (e.g., mRNA transcribed from CD36). Exemplary nucleic acid sequences of human CD36 can be found, for example, at GenBank Accession Nos. NG_008192.1, NM_000072.3, and UniProt Accession No. P16671.

[0130] CD90 is a gene encoding Thy- 1 membrane glycoprotein protein (also known as THY1 and CDw90) which is a member of the immunoglobulin superfamily of proteins and is involved in cell communication and cell adhesion. In some embodiments, expression of CD90 by a cell is determined by measuring a level of CD90 RNA (e.g., mRNA transcribed from CD90). Exemplary nucleic acid sequences of human CD90 can be found, for example, at GenBank Accession Nos. NM_001311160.2, NM_001311162.2, and UniProt Accession No. P04216.

[0131] CDC42EP5 is a gene encoding Cdc42 effector protein 5 protein (also known as CEP5 and Borg3), which is involved in regulating the formation of F-actin-containing structures and negative regulation of Cdc42. In some embodiments, expression of CDC42EP5 by a cell is determined by measuring a level of CDC42EP5 RNA (e.g., mRNA transcribed from CDC42EP5). Exemplary nucleic acid sequences of human CDC42EP5 can be found, for example, at GenBank Accession No. NM_145057.4, and UniProt Accession No. Q6NZY7. CDH11 is a gene encoding cadherin-11 protein (also known as OB, ESWS, CAD11, CDHOB, OSF-4, TBHS2), which is involved in mediation of calcium-dependent cell-cell adhesion. In some embodiments, expression of CDH11 by a cell is determined by measuring a level of CDH11 RNA (e.g., mRNA transcribed from CDH17). Exemplary nucleic acid sequences of human CDH11 can be found, for example, at GenBank Accession Nos. NG_029491.2, NM_001308392.2, and UniProt Accession No. P55287.

[0132] CDK1 is a gene encoding cyclin-dependent kinase 1 (also known as CDC2, CDC28A, and P34CDC2), which is involved in cell cycle control. In some embodiments, expression of CDK1 by a cell is determined by measuring a level of an CDK1 RNA (e.g., mRNA transcribed from CDK1). Non-limiting examples of ribonucleic acid sequences of human CDK1 can be found, for example, at GenBank Accession Nos. NM_001170406.1, NM_001170407.2, and UniProt Accession No. P06493.

[0133] CDKN2A is a gene encoding CDKN2A -interacting protein (also known as ARF, CAI2, CDK4I, CDKN2, CMM2, INK4, INK4A, MLM, MTS-1, MTS1, P14, P14ARF, P16, P16- INK4A, P16INK4, P16INK4A, P19, P19ARF, and TP16), which is involved in stabilizing the tumor suppressor gene p53, as well as cell cycle G1 control. In some embodiments, expression of CDKN2A by a cell is determined by measuring a level of an CDKN2A RNA (e.g., mRNA transcribed from CDKN2A). Non-limiting examples of ribonucleic acid sequences of human CDKN2A can be found, for example, at GenBank Accession Nos. NM_000077.5, NM_001195132.2, and UniProt Accession No. Q9NXV6.

[0134] CDON is a gene encoding cell adhesion molecule-related / down-regulated by oncogenes protein (also known as CDO, Ihog, CDON1, HPE11, and ORCAM), which is a member of the immunoglobulin superfamily and is involved in mediation of cell-cell interactions between muscle precursor cells and positive regulation of myogenesis. In some embodiments, expression of CDON by a cell is determined by measuring a level of CDON RNA (e.g., mRNA transcribed from CDON). Exemplary nucleic acid sequences of human CDON can be found, for example, at GenBank Accession Nos. NG_029776.1, NM_001243597.3, and UniProt Accession No. Q4KMG0.

[0135] CFH is a gene encoding complement factor H protein (also known as FH, HF, HF1, HF2, HUS, FHE1, AHUS1, AMBP1, ARMD4, ARMS1, CFHE3), which is involved in regulating complement activation and restricting this innate defense mechanism to microbial infections. In some embodiments, expression of CF77 by a cell is determined by measuring a level of CFH RNA (e.g., mRNA transcribed from CFH). Exemplary nucleic acid sequences of human CFH can be found, for example, at GenBank Accession Nos. NG_007259.1, NM_000186.4, and UniProt Accession No. P08603.

[0136] CKMT2 is a gene encoding creatine kinase S-type, mitochondrial (also known as SMTCK), which is involved in the transfer of high-energy phosphate from mitochondria to creatine. In some embodiments, expression of CKMT2 by a cell is determined by measuring a level of an CKMT2 RNA (e.g., mRNA transcribed from CKMT2). Non-limiting examples of ribonucleic acid sequences of human CKMT2 can be found, for example, at GenBank Accession Nos. NM_001099735.2, NM_001099736.2, and UniProt Accession No. P17540.

[0137] COL1A2 is a gene encoding collagen alpha-2(l) chain protein (also known as 014, EDSCV, and EDSARTH2), which is a component of fibril-forming collagen and is involved in skeletal system and blood vessel development, Rho protein signal transduction, and blood pressure regulation. In some embodiments, expression of C0L1A2 by a cell is determined by measuring a level of C0L1A2 RNA (e.g., mRNA transcribed from C0L1A2). Exemplary nucleic acid sequences of human C0L1A2 can be found, for example, at GenBank Accession Nos. NG_007405.1, NM_000089.4, and UniProt Accession No. P08123.

[0138] C0L3A1 is a gene encoding collagen alpha- 1 (III) chain protein (also known as EDS4A, EDSVASC, and PMGEDSV), which is a component of a fibrillar collagen found in most soft connective tissues and involved in regulation of cortical development and tissue homeostasis. In some embodiments, expression of C0L3A1 by a cell is determined by measuring a level of C0L3A1 (e.g., mRNA transcribed from COLA A l). Exemplary nucleic acid sequences of human C0L3A1 can be found, for example, at GenBank Accession Nos. NG_007404.1, NM_000090.4, and UniProt Accession No. P02461.

[0139] COL5A1 is a gene encoding collagen alpha- 1(V) chain protein (also known as EDSC, FMDMG, and EDSCL1), which is involved in a component of fibrillar collagen and is involved in blood vessel development, cell adhesion and migration, and heart morphogenesis. In some embodiments, expression of COL5A1 by a cell is determined by measuring a level of COL5A1 RNA (e.g., mRNA transcribed from COL5A1). Exemplary nucleic acid sequences of human COL5A1 can be found, for example, at GenBank Accession Nos. NG_008030.1, NM_000093.5, and UniProt Accession No. P20908.

[0140] COL9A3 is a gene encoding collagen alpha-3(IX) chain protein (also known as IDD, MED, EDM3, STL6, and DJ885L7.4.1) which is a major component of hyaline collagen and is involved in extracellular matrix organization. In some embodiments, expression of COL9A3 by a cell is determined by measuring a level of COL9A3 RNA (e.g., mRNA transcribed from COL9A3). Exemplary nucleic acid sequences of human COL9A3 can be found, for example, at GenBank Accession Nos. NG_016353.1, NM_001853.4, and UniProt Accession No. Q14050.

[0141] CR1M1 is a gene encoding cysteine-rich motor neuron 1 protein (also known as S52 and CRIM-1), which is involved in tissue development through interactions with members of the transforming growth factor beta family. In some embodiments, expression of CR1M1 by a cell is determined by measuring a level of CR1M1 RNA (e.g., mRNA transcribed from CRIMP). Exemplary nucleic acid sequences of human CR1M1 can be found, for example, at GenBank Accession Nos. NM_016441.3, XM_011532899.4, and UniProt Accession No. Q9NZV1.

[0142] CSRP3 is a gene encoding cysteine and glycine -rich protein 3 (also known as CLP, MLP, CRP3, LM04, CMD1M, and CMH12), which is involved in gene regulation, cell growth, and somatic differentiation. In some embodiments, expression of CSRP3 by a cell is determined by measuring a level of an CSRP3 RNA (e.g., mRNA transcribed from CSRP3). Non-limiting examples of ribonucleic acid sequences of human CSRP3 can be found, for example, at GenBank Accession Nos. NM_001369404.1, NM_003476.5, and UniProt Accession No. P50461.

[0143] CTNT is a gene encoding troponin T, cardiac muscle protein (also referred to synonymously herein as TNNT2, also known as CMH2, RCM3, TnTC, CMD1D, CMPD2, and LVNC6), which is a component of the troponin complex and is involved in regulation of muscle contraction in response to alterations in intracellular calcium ion concentration. In some embodiments, expression of CTNT by a cell is determined by measuring a level of CTNT NA (e.g., mRNA transcribed from CTNT). Exemplary nucleic acid sequences of human CTNT can be found, for example, at GenBank Accession Nos. NG_007556.1, NM000364.4, and UniProt Accession No. P45379.

[0144] CTSH is a gene encoding pro-cathepsin H protein (also known as ACC4, ACC5, CPSB, ACC-4, and ACC-5), which is involved in degradation of lysosomal proteins. In some embodiments, expression of CTSH by a cell is determined by measuring a level of CTSH RNA (e.g., mRNA transcribed from CTSH). Exemplary nucleic acid sequences of human CTSH can be found, for example, at GenBank Accession Nos. NG_009614.2, NM_001319137.2, and UniProt Accession No. P09668.

[0145] CXCL12 is a gene encoding stromal cell-derived factor 1 protein (also known as IRH, PBSF, SDF1, TLSF, TPAR1, and SCYB12), which is involved in embryogenesis, immune surveillance, inflammation response, tumor growth and metastasis, and tissue homeostasis. In some embodiments, expression of CXCL12 by a cell is determined by measuring a level of CXCL12 RNA (e.g., mRNA transcribed from CXCL12). Exemplary nucleic acid sequences of human CXCL12 can be found, for example, at GenBank Accession Nos. NG_016861.2, NM_000609.7, and UniProt Accession No. P48061.

[0146] DCN is a gene encoding decorin protein (also known as CSCD, PG40, PGII, PGS2, DSPG2, and SLRR1B), which is involved in collagen fibril assembly and tumor suppression. In some embodiments, expression of DCN by a cell is determined by measuring a level of DCN RNA (e.g., mRNA transcribed from DCN). Exemplary nucleic acid sequences of human DCN can be found, for example, at GenBank Accession Nos. NG_011672.2, NM_001920.5, and UniProt Accession No. P07585.

[0147] DES is a gene encoding desmin (also known as CSM1, CSM2, CDCD3, LGMD1D, LGMD1E, and LGMD2R), which is involved in connecting myofibrils to each other and to the plasma membrane. In some embodiments, expression of DES by a cell is determined by measuring a level of a DES RNA (e.g., mRNA transcribed from DES). Non-limiting examples of ribonucleic acid sequences of human DES can be found, for example, at GenBank Accession Nos. NM_001382708.1, NM_001382709.1, and UniProt Accession No. P17661.

[0148] ELN is a gene encoding elastin (also known as WS, WBS, SVAS, and ADCL1), which is a component of elastin fibers and it gin involved in aortic valve morphogenesis, blood circulation, respiratory gaseous exchange by the respiratory system, and skeletal muscle tissue development. In some embodiments, expression of ELN by a cell is determined by measuring a level of ELN RNA (e.g., mRNA transcribed from ELN). Exemplary nucleic acid sequences of human ELN can be found, for example, at GenBank Accession Nos. NG_009261.1, NM_000501.4, and UniProt Accession No. P15502.

[0149] EMILIN 1 is a gene encoding elastin microfibril interface 1 protein (also known as EMI, ATBFS, HMN10, gpll5, EMILIN, and HMND10), which is involved in development of elastic tissues including heart, lung, skin, and large blood vessels. In some embodiments, expression of EMILIN1 by a cell is determined by measuring a level of EMILIN1 RNA (e.g., mRNA transcribed from EMILIN 1). Exemplary nucleic acid sequences of human EMILIN 1 can be found, for example, at GenBank Accession Nos. NG_046849.1, NM_007046.4, and UniProt Accession No. Q9Y6C2.

[0150] ESAM is a gene encoding endothelial cell-selective adhesion molecule (also known as NEDIHSS and W117m), which is involved in cell-cell adhesion and regulation of actin filament polymerization. In some embodiments, expression of ESAM by a cell is determined by measuring a level of an ESAM RNA (e.g., mRNA transcribed from ESAM). Non-limiting examples of ribonucleic acid sequences of human ESAM can be found, for example, at GenBank Accession No. NM_138961.3, and UniProt Accession No. Q96AP7. EVA1B is a gene encoding eva-1 homolog B protein (also known as Clorf78 and FAM176B), which is likely located in the membrane and is a paralog of EVA1A which acts upstream within several processes. In some embodiments, expression of EVA1B by a cell is determined by measuring a level of EVA1B RNA (e.g., mRNA transcribed from EVA IB). Exemplary nucleic acid sequences on human EVA1B can be found, for example, at GenBank Accession Nos. NM_001304762.2, NM_018166.3, and UniProt Accession No. Q9NVM1.

[0151] FABP3 is a gene encoding fatty acid-binding protein, heart (also known as MDGI, FABP11, H-FABP, M-FABP, and O-FABP), which is involved in intracellular metabolism and transport of long-chain fatty acids. In some embodiments, expression of FABP3 by a cell is determined by measuring a level of an FABP3 RNA (e.g., mRNA transcribed from FABP3). Non-limiting examples of ribonucleic acid sequences of human FABP3 can be found, for example, at GenBank Accession Nos. NM_001320996.2, NM_004102.5, and UniProt Accession No. P07483.

[0152] FBFN2 is a gene encoding fibulin-2 protein (also known as FIBL-2), which is involved in organ development and differentiation of neuronal, skeletal, and heart structures. In some embodiments, expression of FBFN2 by a cell is determined by measuring a level of FBFN2 RNA (e.g., mRNA transcribed from FBLN2). Exemplary nucleic acid sequences of human FBFN2 can be found, for example, at GenBank Accession Nos. NM_001004019.2, NM_001165035.3, and UniProt Accession No. P98095.

[0153] FBFN5 is a gene encoding fibulin-5 protein (also known as EVEC, UP50, ADCL2, ARMD3, CMT1H, DANCE, ARCL1A, FIBL-5, HNARMD), which is involved in promoting endothelial cell adhesion and vascular development and remodeling. In some embodiments, expression of FBFN5 by a cell is determined by measuring a level of FBFN5 RNA (e.g., mRNA transcribed from FBLN5). Exemplary nucleic acid sequences of human FBFN5 can be found, for example, at GenBank Accession Nos. NG_008254.1, NM_001384158.1, and UniProt Accession No. Q9UBX5.

[0154] FBN1 is a gene encoding fibrillin- 1 protein (also known as FBN, SGS, WMS, MASS, MFLS, MFS1, OCTD, SSKS, WMS2, ACMICD, ECTOL1, and GPHYSD2), which is involved in providing force-bearing structural support in elastic and nonelastic connective tissues. In some embodiments, expression of FBN1 by a cell is determined by measuring a level of FBN1 RNA (e.g., mRNA transcribed from FBNI). Exemplary nucleic acid sequences of human FBN1 can be found, for example, at GenBank Accession Nos. NG_008805.2, NM_000138.5, and UniProt Accession No. P35555. FHL2 is a gene encoding four and a half LIM domains protein 2 (also known as DRAL, AAG11, FHL-2, SMIL3, and SLIM-3), which is involved in the assembly of extracellular membranes. In some embodiments, expression of FHL2 by a cell is determined by measuring a level of an FHL2 RNA (e.g., mRNA transcribed from FHL2). Non-limiting examples of ribonucleic acid sequences of human FHL2 can be found, for example, at GenBank Accession Nos. NM_001039492.3, NM_001318894.1, and UniProt Accession No. Q14192.

[0155] FLRT2 is a gene encoding fibronectin leucine-rich repeat transmembrane protein 2, which is involved in regulation of embryonic neural and vascular development. In some embodiments, expression of FLRT2 by a cell is determined by measuring a level of FLRT2 RNA (e.g., mRNA transcribed from FLRT2). Exemplary nucleic acid sequences of human FLRT2 can be found, for example, at GenBank Accession Nos. NM_001346143.2, NM_001346144.2, and UniProt Accession No. 043155.

[0156] FN1 is a gene encoding fibronectin protein (also known as FN, CIG, FNZ, MSF, ED-B, FINC, GFND, LETS, GFND2, and SMDCF), which is involved in cell adhesion and migration processes such as metastasis, embryogenesis, blood coagulation, host defense, and wound healing. In some embodiments, expression of FN1 by a cell is determined by measuring a level of FN1 RNA (e.g., mRNA transcribed from FNI ). Exemplary nucleic acid sequences of human FN1 can be found, for example, at GenBank Accession Nos. NG_012196.2, NM_001306129.2, and UniProt Accession No. P02751.

[0157] F0XA2 is a gene encoding hepatocyte nuclear factor 3-beta (also known as HNF-3-beta, HNF3B, and TCF3B), which is involved in the regulation of metabolism and differentiation and the liver and pancreas. In some embodiments, expression of F0XA2 by a cell is determined by measuring a level of an F0XA2 RNA (e.g., mRNA transcribed from F0XA2). Non-limiting examples of ribonucleic acid sequences of human F0XA2 can be found, for example, at GenBank Accession Nos. NM_021784.5, NM_153675.3, and UniProt Accession No. Q9Y261.

[0158] F0XD3 is a gene encoding forkhead box protein D3 (also known as AIS1, Genesis, HFH2, and VAMAS2), which is involved in development of neural crest cells and maintenance of pluripotency for stem cells. In some embodiments, expression of F0XD3 by a cell is determined by measuring a level of an F0XD3 RNA (e.g., mRNA transcribed from F0XD3). Non-limiting examples of ribonucleic acid sequences of human F0XD3 can be found, for example, at GenBank Accession No. NM_012183.3, and UniProt Accession No. Q9UJU5.

[0159] G0S2 is a gene encoding G0 / G1 switch protein 2, which is involved in positive regulation of the extrinsic apoptotic signaling pathway. In some embodiments, expression of G0S2 by a cell is determined by measuring a level of an G0S2 RNA (e.g., mRNA transcribed from G0S2). Non-limiting examples of ribonucleic acid sequences of human G0S2 can be found, for example, at GenBank Accession No. NM_015714.4, and UniProt Accession No. P27469.

[0160] GAL is a gene encoding galanin peptides (also known as ETL8, GAL-GMAP, GALN, GLNN, and GMAP), which is a neuroendocrine peptide involved in nociception, food and energy homeostasis, osmotic regulation, and water balance. In some embodiments, expression of GAL by a cell is determined by measuring a level of an GAL RNA (e.g., mRNA transcribed from GAL). Non-limiting examples of ribonucleic acid sequences of human GAL can be found, for example, at GenBank Accession No. NM_015973.5, and UniProt Accession No. P22466.

[0161] GATA3 is a gene encoding trans-acting T-cell-specific transcription factor GATA binding protein 3(also known as HDR and HDRS), which is involved in regulating T-cell development and endothelial cell biology. In some embodiments, expression of GATA3 by a cell is determined by measuring a level of GATA3 RNA (e.g., mRNA transcribed from GATA3). Exemplary nucleic acid sequences of human GATA3 can be found, for example, at GenBank Accession Nos. NG_015859.2, NM_001002295.2, and UniProt Accession No. P23771.

[0162] GATA4 is a gene encoding transcription factor GATA binding protein 4 (also known as TOF, ASD2, VSD1, and TACHD), which is involved in regulation of embryogenesis genes and in myocardial differentiation and function. In some embodiments, expression of GATA4 by a cell is determined by measuring a level of GATA4 RNA (e.g., mRNA transcribed from GATA4). Exemplary nucleic acid sequences of human GATA4 can be found, for example, at GenBank Accession Nos. NG_008177.2, NM_001308093.3, and UniProt Accession Number P43694.

[0163] GATA6 is a gene encoding transcription factor GATA binding protein 6, which is involved in cellular differentiation and organogenesis during development. In some embodiments, expression of GATA6 by a cell is determined by measuring a level of GATA6 RNA (e.g., mRNA transcribed from GATA6). Exemplary nucleic acid sequences of human GATA6 can be found, for example, at GenBank Accession Nos. NG_032677.2, NM_005257.6, and UniProt Accession No. Q92908.

[0164] GSN is a gene encoding gelsolin (also known as ADF, AGEL, GELS, and AMYLD4), which is involved in the assembly and disassembly of actin filaments. In some embodiments, expression of GSN by a cell is determined by measuring a level of an GSN RNA (e.g., mRNA transcribed from GSN). Non-limiting examples of ribonucleic acid sequences of human G can be found, for example, at GenBank Accession Nos. NM_000177.5, NM_001127662.2, and UniProt Accession No. P06396.

[0165] HAND1 is a gene encoding heart- and neural crest derivatives-expressed protein 1 (also known as Hxt, Thing 1, bHLHa27, and eHand), which is involved in cardiac morphogenesis. In some embodiments, expression of HAND1 by a cell is determined by measuring a level of an HAND1 RNA (e.g., mRNA transcribed from HAND]). Non-limiting examples of ribonucleic acid sequences of human HAND1 can be found, for example, at GenBank Accession No. NM_004821.3, and UniProt Accession No. 096004.

[0166] HAND2 is a gene encoding heart and neural crest derivatives-expressed protein 2 (also known as Hed, dHand, DHAND2, Thing2, bHLHa26), which is involved in cardiac morphogenesis. In some embodiments, expression of HAND2 by a cell is determined by measuring a level of HAND2 RNA (e.g., mRNA transcribed from HAND2). Exemplary nucleic acid sequences of human HAND2 can be found, for example, at GenBank Accession Nos. NG_046954.1, NM_021973.3, and UniProt Accession No. P61296.

[0167] HCN4 is a gene encoding potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (also known as SSS2, EIG18, and BRGDA8), which is a hyperpolarization-activated nucleotide-gated potassium channel involved in maintaining optimal cardiac rhythm. In some embodiments, expression of HCN4 by a cell is determined by measuring a level of an HCN4 RNA (e.g., mRNA transcribed from HCN4'). Non-limiting examples of ribonucleic acid sequences of human HCN4 can be found, for example, at GenBank Accession No. NM_005477.3, and UniProt Accession No. Q9Y3Q4.

[0168] HEY2 is a gene encoding hairy / enhancer-of-split related with YRPW motif protein 2 (also known as GRL, CHF1, HRT2, HERP1, HESR2, bHLHb32, and GRIDLOCK), which is involved in embryonic cardiovascular development. In some embodiments, expression of HEY2 by a cell is determined by measuring a level of an HEY2 RNA (e.g., mRNA transcribed from HEY2). Non-limiting examples of ribonucleic acid sequences of human HEY2 can be found, for example, at GenBank Accession No. NM_012259.3, and UniProt Accession No. Q9UBP5.

[0169] H1F1A is a gene encoding hypoxia-inducible factor 1-alpha (also known as HIF-l-alpha, HIF-1A, HIF-lalpha, HIF1, HIF1-ALPHA, M0P1, PASD8, and bHLHe78), which is involved in the homeostatic reaction to hypoxia. In some embodiments, expression of H1F1A by a cell is determined by measuring a level of an H1F1A RNA (e.g., mRNA transcribed from HIF1A). Non-limiting examples of ribonucleic acid sequences of human H1F1A can be found, for example, at GenBank Accession Nos. NM_001243084.2, NM_001530.4, and UniProt Accession No. Q309Z6.

[0170] HOPX is a gene encoding homeodomain-only protein (also known as CAMEO, HOD, HOP, LAGY, NECC1, OBI, SMAP31, and TOTO), which is involved in cardiac- specific gene expression and development. In some embodiments, expression of HOPX\yy a cell is determined by measuring a level of an HOPX RNA (e.g., mRNA transcribed from HOPX). Non-limiting examples of ribonucleic acid sequences of human HOPX can be found, for example, at GenBank Accession Nos. NM_001145459.2, NM_001145460.2, and UniProt Accession No. Q9BPY8.

[0171] 1GF2 is a gene encoding insulin-like growth factor 2 (also known as Cllorf43, GRDF, IGF-II, PP9974, and SRS3), which is involved in tissue differentiation and glucose metabolism in adipose, muscle, and liver tissue. In some embodiments, expression of 1GF2 by a cell is determined by measuring a level of an 1GF2 RNA (e.g., mRNA transcribed from IGF2). Nonlimiting examples of ribonucleic acid sequences of human 1GF2 can be found, for example, at GenBank Accession Nos. NM_000612.6, NM_001007139.6, and UniProt Accession No. P01344.

[0172] 1GFBP7 is a gene encoding insulin-like growth factor-binding protein 7 (also known as AGM, FSTL2, IBP-7, IGFBP-7, IGFBP-7v, IGFBPRP1, MAC25, PSF, RAMSVPS, and TAF), which is involved in the stimulation of prostacyclin production and cell adhesion. In some embodiments, expression of 1GFBP7 by a cell is determined by measuring a level of an 1GFBP7 RNA (e.g., mRNA transcribed from IGFBP7). Non-limiting examples of ribonucleic acid sequences of human 1GFBP7 can be found, for example, at GenBank Accession Nos. NM_001253835.2, NM_001553.3, and UniProt Accession No. Q16270.

[0173] 1RX4 is a gene encoding Iroquois-class homeodomain protein IRX-4 (also known as IRXA3), which is involved in ventricular differentiation during cardiac development. In some embodiments, expression of 1RX4 by a cell is determined by measuring a level of an 1RX4 RNA (e.g., mRNA transcribed from IRX4). Non-limiting examples of ribonucleic acid sequences of human 1RX4 can be found, for example, at GenBank Accession Nos. NM_001278632.1, NM_001278633.1, and UniProt Accession No. P78413.

[0174] 1TGA7 is a gene encoding integrin alpha-7, which is involved in cellular migration, differentiation, and metastasis of skeletal and cardiac muscles. In some embodiments, expression of 1TGA7 by a cell is determined by measuring a level of an 1TGA7 RNA (e.g., mRNA transcribed from ITGA7). Non-limiting examples of ribonucleic acid sequences of human 1TGA7 can be found, for example, at GenBank Accession Nos. NM_001144996.2, NM_001144997.2, and UniProt Accession No. Q13683.

[0175] 1TLN1 is a gene encoding intelectin-1 protein (also known as HL1, LFR, HL-1, INTL, ITLN, hlntL, and omentin), which is involved in calcium ion binding activity, positive regulation of D-glucose import, protein homotrimerization, and positive regulation of protein phosphorylation. In some embodiments, expression of 1TLN1 by a cell is determined by measuring a level of 1TLN1 RNA (e.g., mRNA transcribed from ITLNP). Exemplary nucleic acid sequences of human 1TLN1 can be found, for example, at GenBank Accession No. NM_017625.3, and UniProt Accession No. Q8WWA0.

[0176] KCNJ2 is a gene encoding inward rectifier potassium channel 2 (also known as IRK1, LQT7, SQT3, ATFB9, HHIRK1, KIR2.1, and HHBIRK1), which is a potassium channel involved in the establishment of the action potential in muscle tissues. In some embodiments, expression of KCNJ2 by a cell is determined by measuring a level of an KCNJ2 RNA (e.g., mRNA transcribed from KCNJ2). Non-limiting examples of ribonucleic acid sequences of human KCNJ2 can be found, for example, at GenBank Accession No. NM_000891.3 and UniProt Accession No. P63252.

[0177] KCNJ3 is a gene encoding G protein-activated inward rectifier potassium channel 1 (also known as GIRK1, KG A, and KIR3.1), which is a potassium channel involved in regulating heartbeat. In some embodiments, expression of KCNJ3 by a cell is determined by measuring a level of an KCNJ3 RNA (e.g., mRNA transcribed from KCNJ3). Non-limiting examples of ribonucleic acid sequences of human KCNJ3 can be found, for example, at GenBank Accession Nos. NM_001260508.2, NM_001260509.2, and UniProt Accession No. P48549.

[0178] KCTD12 is a gene encoding BTB / POZ domain-containing protein KCTD12 (also known as C13orf2, PFET1, and PFETIN which is involved in the regulation of the G protein-couples receptor signaling pathway. In some embodiments, expression of KCTD12 by a cell is determined by measuring a level of an KCTD12 RNA (e.g., mRNA transcribed from KCTD12). Non-limiting examples of ribonucleic acid sequences of human KCTD12 can be found, for example, at GenBank Accession No. NM_138444.4, and UniProt Accession No. Q96CX2.

[0179] KRT19 is a gene encoding keratin, type I cytoskeletal 19 protein (also known as K19, CK19, and K1CS), which is involved in maintaining structural integrity of epithelial cells. In some embodiments, expression of KRT19 by a cell is determined by measuring a level of KRT19 RNA (e.g., mRNA transcribed from KRT1 ). Exemplary nucleic acid sequences of human KRT19 can be found, for example, at GenBank Accession Nos. NG_012285.1, NM_002276.2, and UniProt Accession No. P08727.

[0180] L1TD1 is a gene encoding LINE-1 type transposase domain-containing protein 1 (also known as ECAT11), which is involved in retrotransposition and single- stranded RNA binding activity. In some embodiments, expression of L1TD1 by a cell is determined by measuring a level of an L1TD1 RNA (e.g., mRNA transcribed from L1TD1). Non-limiting examples of ribonucleic acid sequences of human L1TD1 can be found, for example, at GenBank Accession Nos. NM_001164835.2, NM_019079.5, and UniProt Accession No. Q5T7N2. LEF1 is a gene encoding lymphoid enhancer-binding factor 1 (also known as ECTD1, ECTD17, LEF-1, TCF10, TCF1ALPHA, and TCF7L3), which is involved in T-cell receptor alpha enhancer function. In some embodiments, expression of LEF1 by a cell is determined by measuring a level of an EEF1 RNA (e.g., mRNA transcribed from LEF1). Non-limiting examples of ribonucleic acid sequences of human EEF1 can be found, for example, at GenBank Accession Nos. NM_001130713.3, NM_001130714.3, and UniProt Accession No. Q9UJU2.

[0181] E1MA1 is a gene encoding LIM domain and actin-binding protein 1 (also known as EPLIN, LDLCQ8, and SREBP3), which is involved in inhibition of actin filament depolymerization and cross-links filaments in bundles. In some embodiments, expression of E1MA1 by a cell is determined by measuring a level of E1MA1 RNA (e.g., mRNA transcribed from LIMA ]). Exemplary nucleic acid sequences of human LIMA1 can be found, for example, at GenBank Accession Nos. NG_029859.1, NM_001113546.2, and UniProt Accession No. Q9UHB6.

[0182] LINC00678 is a gene encoding long intergenic non-protein coding RNA 678, which is predicted to be involved in body weight. In some embodiments, expression of LINC00678 by a cell is determined by measuring a level of an LINC00678 RNA (e.g., mRNA transcribed from LLNC00678). Non-limiting examples of ribonucleic acid sequences of human LINC00678 can be found, for example, at GenBank Accession No. NR_102708.1.

[0183] LMOD1 is a gene encoding leiomodin-1 (also known as ID, DI, 64Kd, MMIHS3, SMLMOD, and SM-LMOD), which is involved in the contractility of smooth muscle cells. In some embodiments, expression of LMOD1 by a cell is determined by measuring a level of an LMOD1 RNA (e.g., mRNA transcribed from LMOD1). Non-limiting examples of ribonucleic acid sequences of human LM0D1 can be found, for example, at GenBank Accession No. NM_012134.3 and UniProt Accession No. P29536.

[0184] LMOD2 is a gene encoding leiomodin-2 (also known as CLMOD, CMD2G, and C- LMOD), which is involved in actin polymerization and sarcomere organization in the heart. In some embodiments, expression of LMOD2 by a cell is determined by measuring a level of an LMOD2 RNA (e.g., mRNA transcribed from LMOD2). Non-limiting examples of ribonucleic acid sequences of human LM0D2 can be found, for example, at GenBank Accession No. NM_207163.3 and UniProt Accession No. Q6P5Q4.

[0185] LOX is a gene encoding lysyl oxidase (also known as AAT10 and protein-lysine 6- oxidase), which is involved in crosslinking of collagens and elastin, e.g., related to angiogenesis. In some embodiments, expression of LOX by a cell is determined by measuring a level of an LOX RNA (e.g., mRNA transcribed from LOX). Non-limiting examples of ribonucleic acid sequences of human LOX can be found, for example, at GenBank Accession No. NG_008722.1, NM_001178102.2, and UniProt Accession No. P28300.

[0186] LRPR2 is a gene encoding low-density lipoprotein receptor-related protein 2 (also known as DBS, GP330, LRP-2), which is involved in cell-signaling and in reuptake of sterols, hormones, lipoproteins, and vitamin-binding proteins. In some embodiments, expression of LRP2 by a cell is determined by measuring a level of LRP2 RNA (e.g., mRNA transcribed from LRP2). Exemplary nucleic acid sequences of human LRP2 can be found, for example, at GenBank Accession Nos. NG_012634.1, NM_004525.3, and UniProt Accession No. P98164.

[0187] LRRC17 is a gene encoding leucine-rich repeat-containing protein 17 (also known as P37NB), which is involved in bone marrow development and negative regulation of osteoclast differentiation. In some embodiments, expression of LRRC17 by a cell is determined by measuring a level of LRRC17 RNA (e.g., mRNA transcribed from LRRC17). Exemplary nucleic acid sequences of human LRRC17 can be found, for example, at GenBank Accession Nos. NM_001031692.3, NM_005824.3, and UniProt Accession No. Q8N6Y2.

[0188] LYH6 is a gene encoding lymphocyte antigen 6H protein (also known as NMLY6), which is involved in the acetylcholine receptor signaling pathway. In some embodiments, expression of LYH6 by a cell is determined by measuring a level of LYH6 RNA (e.g., mRNA transcribed from LYH6). Exemplary nucleic acid sequences of human LYH6 can be found, for example, at GenBank Accession Nos. NM_001130478.2, NM_001135655.2, and UniProt Accession No. 094772.

[0189] MAN1C1 is a gene encoding mannosyl-oligosaccharide 1,2-alpha-mannosidase IC (also known as HMIC, MANI A3, MANIC, and pp6318), which is involved in the ERAD pathway of extracellular exosomes. In some embodiments, expression of MAN1C1 by a cell is determined by measuring a level of an MAN1C1 RNA (e.g., mRNA transcribed from MAN1C1 ). Nonlimiting examples of ribonucleic acid sequences of human MAN1C1 can be found, for example, at GenBank Accession Nos. NM_001289010.2, NM_001385182.1, and UniProt Accession No. Q9NR34.

[0190] MARCKS is a gene encoding myristoylated alanine-rich C-kinase substrate (also known as MACS, 80K-L, PKCSL, and PRKCSL), which is involved in cell motility, membrane trafficking, and phagocytosis. In some embodiments, expression of MARCKS by a cell is determined by measuring a level of MARCKS RNA (e.g., mRNA transcribed from MARCKS). Exemplary nucleic acid sequences of human MARCKS can be found, for example, at GenBank Accession No. NM_002356.7, and UniProt Accession No. P29966. MEF2C is a gene encoding myocyte- specific enhancer factor 2C (also known as C5DELql4.3, DEL5ql4.3, and NEDHSIL), which is involved in myogenesis and maintaining the differentiated state of muscle cells. In some embodiments, expression of MEF2C by a cell is determined by measuring a level of an MEF2C RNA (e.g., mRNA transcribed from MEF2C). Non-limiting examples of ribonucleic acid sequences of human MEF2C can be found, for example, at GenBank Accession Nos. NM_001131005.2, NM_001193347.1, and UniProt Accession No. Q06413.

[0191] MGP is a gene encoding matrix gla protein (also known as NTI, GIG36, and MGLAP), which is involved in inhibiting ectopic tissue calcification. In some embodiments, expression of MGP by a cell is determined by measuring a level of MGP RNA (e.g., mRNA transcribed from MGP). Exemplary nucleic acid sequences of human MGP can be found, for example, at GenBank Accession Nos. NG_023331.2, NM_000900.5, and UniProt Accession No. P08493.

[0192] MK167 is a gene encoding proliferation marker protein Ki-67 (also known as KIA, MIB-, MIB-1, and PPP1R105), which is involved in chromosome segregation and mitotic nuclear division. In some embodiments, expression of MKI67 \y a cell is determined by measuring a level of an MK167 RNA (e.g., mRNA transcribed from MKI67). Non-limiting examples of ribonucleic acid sequences of human MK167 can be found, for example, at GenBank Accession Nos. NM_001145966.2, NM_002417.5, and UniProt Accession No. P46013.

[0193] MEC2v is a gene encoding myosin regulatory light chain 2, ventricular / cardiac muscle isoform protein (also known as MLC2, CMH10, MFM12, MLC-2, MYL2, and MLC-2s / v), which is involved in embryonic heart muscle structure and function, and in cardiac myosin cycling kinetics. In some embodiments, expression of MEC2v by a cell is determined by measuring a level of MEC2v RNA (e.g., mRNA transcribed from MLC2v). Exemplary nucleic acid sequences of human MEC2v can be found, for example, at GenBank Accession Nos. NG_007554.1, NM_000432.4, and UniProt Accession No. P10916.

[0194] MMP2 is a gene encoding matrix metalloproteinase-2 protein (also known as CLG4, MONA, CLG4A, MMP-2, TBE-1, and MMP-II), which is involved in endometrial menstrual breakdown, regulation of vascularization, and metastasis. In some embodiments, expression of MMP2 by a cell is determined by measuring a level of MMP2 RNA (e.g., mRNA transcribed from MMP2). Exemplary nucleic acid sequences of human MMP2 can be found, for example, at GenBank Accession Nos. NG_008989.1, NM_001127891.3, and UniProt Accession No. P08253.

[0195] MT-ND2 is a gene encoding NADH-ubiquinone oxidoreductase chain 2 (also known as MTND2 and ND2), which is involved in electron transport in mitochondria. In some embodiments, expression of MT-ND2 by a cell is determined by measuring a level of an MT- ND2 RNA (e.g., mRNA transcribed from MT-ND2). Non-limiting examples of ribonucleic acid sequences of human MT-ND2can be found, for example, at UniProt Accession No. P03891.

[0196] MYBPC3 is a gene encoding myosin binding protein C, cardiac type protein (also known as cMYBP-C, CMD1MM, CMH4, FHC, LVNC10, and MYBP-C), which is involved in the regulation of positioning of myosin and actin in cardiomyocytes. In some embodiments, expression of MYBPC3 by a cell is determined by measuring a level of an MYBPC3 RNA (e.g., mRNA transcribed from MYBPC3). Non-limiting examples of ribonucleic acid sequences of human MYBPC3 can be found, for example, at GenBank Accession No. NM_000256.3, and UniProt Accession No. Q14896.

[0197] MYH10 is a gene encoding myosin-10 (also known as NMMHCB and NMMHC-IIB), which is involved in muscle formation and contractility. In some embodiments, expression of MYH10 by a cell is determined by measuring a level of an MYH10 RNA (e.g., mRNA transcribed from MYHIO). Non-limiting examples of ribonucleic acid sequences of human MYH10 can be found, for example, at GenBank Accession Nos. NM_001256012.3, NM_001256095.2, and UniProt Accession No. P35580.

[0198] MYH11 is a gene encoding myosin- 11 (also known as AAT4, FAA4, SMHC, SMMHC, SMMS-1, and VSCM2), which is involved in contraction of smooth muscle. In some embodiments, expression of MYH11 by a cell is determined by measuring a level of an MYH11 RNA (e.g., mRNA transcribed from MYH1P). Non-limiting examples of ribonucleic acid sequences of human MYH11 can be found, for example, at GenBank Accession Nos. NM_001040113.2, NM_001040114.2, and UniProt Accession No. P35749.

[0199] MYH6 is a gene encoding myosin-6 (also known as ASD3, MYHC, SSS3, CMH14, MYHCA, CMD1EE, and alpha-MHC), which is an integral subunit of cardiac myosin. In some embodiments, expression of MYH6 by a cell is determined by measuring a level of an MYH6 RNA (e.g., mRNA transcribed from MYH6). Non-limiting examples of ribonucleic acid sequences of human MYH6 can be found, for example, at GenBank Accession No. NM_002471.4, and UniProt Accession No. P13533.

[0200] MYH7 is a gene encoding myosin-7 (also known as CMH1, MPD1, SPMD, SPMM, CMD1S, MYHCB, CMY07A, CMY07B, CMYP7A, and CMYP7B), which is an integral component of cardiac myosin. In some embodiments, expression of MYH7 by a cell is determined by measuring a level of an MYH7 RNA (e.g., mRNA transcribed from MYH7). Nonlimiting examples of ribonucleic acid sequences of human MYH7 can be found, for example, at GenBank Accession Nos. NM_000257.4, NM_001407004.1, and UniProt Accession No. P12883.

[0201] MYL3 is a gene encoding myosin light chain 3 (also known as CMH8, VLC1, VLCI, MLC1V, MLC1SB, and MLC-IV / sb), which is an integral subunit of myosin implicated in heart function. In some embodiments, expression of MYL3 by a cell is determined by measuring a level of an MYL3 RNA (e.g., mRNA transcribed from MYL3). Non-limiting examples of ribonucleic acid sequences of human MYL3 can be found, for example, at GenBank Accession Nos. NM_000258.3, NM_001406937.1, and UniProt Accession No. P08590.

[0202] MYL4 is a gene encoding myosin light chain 4 (also known as ALC1, AMLC, GT1, and PRO1957), which is involved in embryonic cardiac muscle and adult atria. In some embodiments, expression of MYL4 by a cell is determined by measuring a level of an MYL4 RNA (e.g., mRNA transcribed from MYL4). Non-limiting examples of ribonucleic acid sequences of human MYL4 can be found, for example, at GenBank Accession Nos. NM_001002841.2, NM_002476.2, and UniProt Accession No. P12829.

[0203] MLC2A is a gene encoding myosin regulatory light chain 2, atrial isoform (also known as MYL2N and MYL7 which is involved in cardiac muscle development and heart contraction. In some embodiments, expression of MLC2A by a cell is determined by measuring a level of an MLC2A RNA (e.g., mRNA transcribed from MLC2A). Non-limiting examples of ribonucleic acid sequences of human MLC2A can be found, for example, at GenBank Accession No. NM_021223.3, and UniProt Accession No. Q01449.

[0204] MYL9 is a gene encoding myosin regulatory light polypeptide 9 (also known as LC20, MLC-2C, MLC2, MMIHS4, MRLC1, and MYRL2), which is involved in the regulation of muscle contraction. In some embodiments, expression of MYL9 by a cell is determined by measuring a level of an MYL9 RNA (e.g., mRNA transcribed from MYL9). Non-limiting examples of ribonucleic acid sequences of human MYL9 can be found, for example, at GenBank Accession Nos. NM_006097.5, NM_181526.3, and UniProt Accession No. P24844.

[0205] MYOZ2 is a gene encoding myozenin-2 (also known as CS-1, CMH16, C4orf5, and FATZ-2), which is involved in calcium-dependent signal transduction in cardiac and skeletal muscle cells. In some embodiments, expression of MYOZ2 by a cell is determined by measuring a level of an MYOZ2 RNA (e.g., mRNA transcribed from MY0Z2). Non-limiting examples of ribonucleic acid sequences of human MYOZ2 can be found, for example, at GenBank Accession Nos. NM_001440645.1, NM_001440646.1, and UniProt Accession No. Q9NPC6.

[0206] NANOG is a gene encoding homeobox protein NANOG, which is involved in embryonic stem cell renewal, proliferation, and pluripotency. In some embodiments, expression of NANOG by a cell is determined by measuring a level of NANOG RNA (e.g., mRNA transcribed from NANOG). Exemplary nucleic acid sequences of human NANOG can be found, for example, at GenBank Accession Nos. NM_001297698.2, NM_024865.4, and UniProt Accession No. Q9H9S0.

[0207] NKX2-5 is a gene encoding Homeobox protein Nkx-2.5 (also known as NK2 Homeobox 5, NKX2.5, NKX4-1, NKX2E, CSX1, CSX, Homeobox Protein NK-2 Homolog E, Homeobox protein Nkx-2.5, Homeobox Protein CSX, VSD3, CHNG5, and HLHS2), which is involved in heart formation and development. In some embodiments, expression of NKX2-5 by a cell is determined by measuring a level of an NKX2-5 RNA (e.g., mRNA transcribed from NKX2-5). Non-limiting examples of ribonucleic acid sequences of human NKX2-5 transcripts can be found, for example, at GenBank Accession Nos. NM_001166175.2, NM_001166176.2, NM_004387.4, and UniProt Accession No. P52952.

[0208] NPNT is a gene encoding nephronectin protein (also known as POEM and EGFL6L), which is involved in integrin binding activity and positive regulation of the ERK1 and ERK2 cascade. In some embodiments, expression of NPNT by a cell is determined by measuring a level of A WTRNA (e.g., mRNA transcribed from NPNT). Exemplary nucleic acid sequences of human NPNT can be found, for example, at GenBank Accession Nos. NM_001033047.3, NM_001184690.2, and UniProt Accession No. Q6UXI9.

[0209] NPPA is a gene encoding natriuretic peptides A (also known as ANF, ANP, ATFB6, ATRST2, CDD, CDD-ANF, CDP, and PND), which are involved in extracellular fluid volume regulation and electrolyte homeostasis. In some embodiments, expression of NPPA by a cell is determined by measuring a level of an NPPA RNA (e.g., mRNA transcribed from NPPA). Nonlimiting examples of ribonucleic acid sequences of human NPPA can be found, for example, at GenBank Accession No. NM_006172.4, and UniProt Accession No. P01160.

[0210] NR2F2 is a gene encoding nuclear receptor subfamily 2 group F member 6 (also known as ARP-1, ARP1, CHTD4, COUPTF2, COUPTFB, COUPTFII, NF-E3, SRXX5, SVP40, and TFCOUP2), which is involved in modulation of hormonal responses. In some embodiments, expression of NR2F2 by a cell is determined by measuring a level of an NR2F2 RNA (e.g., mRNA transcribed from NR2F2). Non-limiting examples of ribonucleic acid sequences of human NR2F2 can be found, for example, at GenBank Accession Nos. NM_001145155.2, NM_001145156.1, and UniProt Accession No. P 10588.

[0211] NUPR1 is a gene encoding nuclear protein 1 (also known as P8 and COMI), which is involved in positive regulation of oxidative phosphorylation, regulation of catabolic processes, and negative regulation of programmed cell death. In some embodiments, expression of NUPR1 by a cell is determined by measuring a level of NUPR1 RNA (e.g., mRNA transcribed from NUPRI). Exemplary nucleic acid sequences of human NUPR1 can be found, for example, at GenBank Accession Nos. NM_001042483.2, NM_012385.3, and UniProt Accession No. 060356.

[0212] OGN is a gene encoding Mimecan protein (also known as OG, OIF, SLRR3A), which is involved in ectopic bone formation and osteoblast differentiation. In some embodiments, expression of OGN by a cell is determined by measuring a level of OGN RNA (e.g., mRNA transcribed from OGN). Exemplary nucleic acid sequences of human OGN can be found, for example, at GenBank Accession Nos. NM_014057.5, NM_024416.4, and UniProt Accession No. P20774.

[0213] OSMR is a gene encoding oncostatin-M-specific receptor subunit beta protein (also known as OSMRB, PLCA1, IL-31RB, OSMRbeta, and IL-31R-beta), which is involved in positive regulation of cell population proliferation and cytokine response. In some embodiments, expression of OSMR by a cell is determined by measuring a level of OSMR RNA (e.g., mRNA transcribed from OSMR). Exemplary nucleic acid sequences of human OSMR can be found, for example, at GenBank Accession Nos. NG_016236.2, NM_001168355.3, and UniProt Accession No. Q99650.

[0214] PAX6 is a gene encoding paired box 6 (also known as AN, AN2, D11S812E, FVH1, MGDA, WAGR, ASGD5), which is a transcription factor involved in development of neural tissues (e.g., the eye). In some embodiments, expression of PAX6 by a cell is determined by measuring a level of PAX6 RNA (e.g., mRNA transcribed from PAX6). Exemplary nucleic acid sequences of human PAX6 can be found, for example, at GenBank Accession Nos. NM_000280.6, NM_001127612.3, and UniProt Accession No. P26367.

[0215] PDGFRa is a gene encoding platelet-derived growth factor receptor alpha (also known as CD140A, PDGFR2, and PDGFR-2), which is involved in organ development, tumor progression, and wound healing. In some embodiments, expression of PDGFRa by a cell is determined by measuring a level of PDGFRa RNA (e.g., mRNA transcribed from PDGFRa). Exemplary nucleic acid sequences of human PDGFRa can be found, for example, at GenBank Accession Nos. NG_009250.1, NM_001347827.2, and UniProt Accession No. P16234.

[0216] PDL1M1 is a gene encoding PDZ and LIM domain protein 1 (also known as CLIM1, CLP36, CLP-36, hCLIMl, and HEL-S-112), which is involved in the assembly, disassembly, and directioning of stress fibers in fibroblasts. In some embodiments, expression of PDL1M1 by a cell is determined by measuring a level of an PDL1M1 RNA (e.g., mRNA transcribed from PDL1M1). Non-limiting examples of ribonucleic acid sequences of human PDL1M1 can be found, for example, at GenBank Accession No. NM_020992.4, and UniProt Accession No. 000151.

[0217] PECAM1 is a gene encoding platelet endothelial cell adhesion molecule (also known as CD31, CD31 / EndoCAM, GPIIA', PECA1, PECAM-1, and endoCAM), which is involved in endothelial cell intercellular junctions. In some embodiments, expression of PECAM1 by a cell is determined by measuring a level of an PECAM1 RNA (e.g., mRNA transcribed from PEC AMI). Non-limiting examples of ribonucleic acid sequences of human PECAM1 can be found, for example, at GenBank Accession No. NM_000442.5, and UniProt Accession No. P16284.

[0218] PGAM2 is a gene encoding phosphoglycerate mutase 2 (also known as GSD10, PGAM- M, and PGAMM), which is involved in the glycolytic pathway in muscle. In some embodiments, expression of PGAM2 by a cell is determined by measuring a level of an PGAM2 RNA (e.g., mRNA transcribed from PGAM2). Non-limiting examples of ribonucleic acid sequences of human PGAM2 can be found, for example, at GenBank Accession No. NM_000290.4, and UniProt Accession No. P15259.

[0219] PGC1A is a gene encoding peroxisome proliferator-activated receptor gamma coactivator 1-alpha (also known as LEM6, PGC1, PGC1A, PGC-lv, PPARGC1, PGC-lalpha, and PGC-1 (alpha)), which is involved in energy metabolism, mitochondrial biogenesis, and muscle fiber type determination. In some embodiments, expression of PGC1A by a cell is determined by measuring a level of an PGC1A RNA (e.g., mRNA transcribed from PGC1A). Non-limiting examples of ribonucleic acid sequences of human PGC1A can be found, for example, at GenBank Accession Nos. NM_001330751.2, NM_001330752.2, and UniProt Accession No. Q865B7.

[0220] PKP2 is a gene encoding plakophilin-2 (also known as ARVD9), which is involved in cytoskeletal structure and intracellular calcium in the heart. In some embodiments, expression of PKP2 by a cell is determined by measuring a level of an PKP2 RNA (e.g., mRNA transcribed from PKP2). Non-limiting examples of ribonucleic acid sequences of human PKP2 can be found, for example, at GenBank Accession Nos. NM_001005242.3, NM_001407155.1, and UniProt Accession No. Q99959.

[0221] PEAC9 is a gene encoding placenta- specific protein 9, which is involved in inhibition of cell proliferation and stimulation of motility. In some embodiments, expression of PEAC9 by a cell is determined by measuring a level of PEAC9 RNA (e.g., mRNA transcribed from PLAC9). Exemplary nucleic acid sequences of human PEAC9 can be found, for example, at GenBank Accession Nos. NM_001012973.3, NM_001331125.2, and UniProt Accession No. Q5JTB6. PLVAP is a gene encoding plasmalemma vesicle-associated protein (also known as DIAR10, FELS, PV-1, PV1, and gp68), which is involved in microvascular permeability, embryonic development, and the MAPK cascade. In some embodiments, expression of PLVAP by a cell is determined by measuring a level of an PLVAP RNA (e.g., mRNA transcribed from PLVAP). Non-limiting examples of ribonucleic acid sequences of human PLVAP can be found, for example, at GenBank Accession No. NM_031310.3, and UniProt Accession No. Q9BX97.

[0222] PODXL is a gene encoding podocalyxin protein (also known as PC, PDX, PCLP, Gp200, gpl35, PCLP-1, PODXL1, and TRA-1-60), which is involved in hematopoietic cell differentiation, regulation of cell adhesion and morphology, and cancer progression. In some embodiments, expression of PODXL by a cell is determined by measuring a level of PODXL RNA (e.g., mRNA transcribed from PODXL). Exemplary nucleic acid sequences of human PODXL can be found, for example, at GenBank Accession Nos. NG_042104.1, NM_001018111.3, and UniProt Accession No. 000592.

[0223] POSTN is a gene encoding periostin protein (also known as PN, 0SF2, OSF-2, and PDLPOSTN), which is involved in tissue development, wound healing, and ventricular remodeling following myocardial infarction. In some embodiments, expression of POSTN by a cell is determined by measuring a level of POSTN RNA (e.g., mRNA transcribed from POSTN). Exemplary nucleic acid sequences of human POSTN can be found, for example, at GenBank Accession Nos. NM_001135934.2, NM_001135935.2, and UniProt Accession No. Q15063.

[0224] OCT3 / 4 is a gene encoding POU domain, class 5, transcription factor 1 (also known as OCT3, OCT4, OCT4Borfl, OTF-3, OTF3, OTF4, Oct-3, Oct-4, and POU5F1), which is involved in embryonic development and stem cell pluripotency. In some embodiments, expression of OCT3 / 4 by a cell is determined by measuring a level of an OCT3 / 4 RNA (e.g., mRNA transcribed from OCT3 / 4). Non-limiting examples of ribonucleic acid sequences of human OCT3 / 4 can be found, for example, at GenBank Accession Nos. NM_001173531.3, NM_001285986.2, and UniProt Accession No. Q01860.

[0225] PPP1R3A is a gene encoding protein phosphatase 1 regulatory subunit 3A (also known as GM, PP1G, and PPP1R3), which is involved in the binding of glycogen within muscle tissue. In some embodiments, expression of PPP1R3A by a cell is determined by measuring a level of an PPP1R3A RNA (e.g., mRNA transcribed from PPP1R3A). Non-limiting examples of ribonucleic acid sequences of human PPP1R3A can be found, for example, at GenBank Accession No. NM_002711.4, and UniProt Accession No. Q16821.

[0226] PTX3 is a gene encoding pentraxin-related protein 3 (also known as TSG-14 and TFNAIP5), which is involved in fibrocyte differentiation and inflammatory response. In some embodiments, expression of PTX3 by a cell is determined by measuring a level of PTX3 RNA (e.g., mRNA transcribed from PTX3). Exemplary nucleic acid sequences of human PTX3 can be found, for example, at GenBank Accession Nos. NG_051000.1, NM_002852.4, and UniProt Accession No. P26022.

[0227] RAMP1 is a gene encoding receptor activity-modifying protein 1, which is involved in calcitonin-receptor-like receptor transport, terminal glycosylation, maturation, and presentation of the CGRP receptor to the cell surface. In some embodiments, expression of RAMP1 by a cell is determined by measuring a level of RAMP1 RNA (e.g., mRNA transcribed from RAM Pl). Exemplary nucleic acid sequences of human RAMP1 can be found, for example, at GenBank Accession Nos. NM_001308353.2, NM_005855.4, and UniProt Accession No. 060894.

[0228] RARRES2 is a gene encoding retinoic acid receptor responder protein 2 (also known as TIG2 and HP10433), which is involved in chemotaxis, adipogenesis, and inflammatory response. In some embodiments, expression of RARRES2 by a cell is determined by measuring a level of RARRES2 RNA (e.g., mRNA transcribed from RARRES2). Exemplary nucleic acid sequences of human RARRES2 can be found, for example, at GenBank Accession No. NM_002889.4, and UniProt Accession No. Q99969.

[0229] RDH10 is a gene encoding retinol dehydrogenase 10 protein (also known as SDR16C4), which is involved in synthesis of embryonic retinoic acid and required for organ development. In some embodiments, expression of RDH10 by a cell is determined by measuring a level of RDH10 RNA (e.g., mRNA transcribed from RDH10). Exemplary nucleic acid sequences of human RDH10 can be found, for example, at GenBank Accession No. NM_172037.5, and UniProt Accession No. Q8IZV5.

[0230] S100A10 is a gene encoding S100 calcium binding protein A10 (also known as 42C, Pll, plO, GPU, ANX2L, CAL IL, CLP11, Ca[l], and ANX2LG). which is involved in regulation of cell cycle progression and differentiation. In some embodiments, expression of S100A10 by a cell is determined by measuring a level of S100A10 RNA (e.g., mRNA transcribed from S100A10). Exemplary nucleic acid sequences of human S100A10 can be found, for example, at GenBank Accession No. NM_002966.3, and UniProt Accession No. P60903.

[0231] SBSON is a gene encoding somatomedin-B and thrombospondin type-1 domain containing protein (also known as RPESP and C8orf84), which is involved in the structure of the extracellular matrix. In some embodiments, expression of SBSON by a cell is determined by measuring a level of SBSON RNA (e.g., mRNA transcribed from SBSON). Exemplary nucleic acid sequences of human SBSON can be found, for example, at GenBank Accession No. NM_153225.4, and UniProt Accession No. Q8IVN8. SCC3 is a gene encoding cohesion subunit SA-3 (also known as SA3, STAG3, and SPGF61), which is involved in sister chromatid cohesion and synaptonemal complex assembly. In some embodiments, expression of SCC3 by a cell is determined by measuring a level of SCC3 RNA (e.g., mRNA transcribed from SCC3). Exemplary nucleic acid sequences of human SCC3 can be found, for example, at GenBank Accession Nos. NG_034114.2, NM_001282716.1, and UniProt Accession No. Q9UJ98.

[0232] SCC4 is a gene encoding MAU2 chromatid cohesion factor homolog (also known as MAU2, mau-2, and KIAA0892), which is involved in sister chromatid cohesion and chromosome segregation. In some embodiments, expression of SCC4 by a cell is determined by measuring a level of SCC4 RNA (e.g., mRNA transcribed from SCC4). Exemplary nucleic acid sequences of human SCC4 can be found, for example, at GenBank Accession No. NM_015329.4, and UniProt Accession No. Q9Y6X3.

[0233] SEZ6E2 is a gene encoding seizure 6-like protein 2 (also known as BSRPA and PSK-1), which is involved in synapse maturation and regulation of protein kinase C signaling. In some embodiments, expression of SEZ6E2 by a cell is determined by measuring a level of SEZ6L2RNA (e.g., mRNA transcribed from SEZ6L2). Exemplary nucleic acid sequences of human SEZ6E2 can be found, for example, at GenBank Accession Nos. NG_029737.2, NM_001114099.3, and UniProt Accession No. Q6UXD5.

[0234] SEI 0X2 is a gene encoding short stature homeobox protein 2 (also known as OG12, OG12X, and SHOT), which is involved in regulating normal stature. In some embodiments, expression of SHOX2 by a cell is determined by measuring a level of an SHOX2 RNA (e.g., mRNA transcribed from SHOX2). Non-limiting examples of ribonucleic acid sequences of human SHOX2 can be found, for example, at GenBank Accession Nos. NM_001163678.2, NM_003030.4, and UniProt Accession No. 060902.

[0235] SEC25A4 is a gene encoding ADP / ATP translocase 1 (also known as AAC1, ANT, ANT 1, ANTI, MTDPS12, MTDPS12A, PEO2, PEO3, PEOA2, and Tl), which is involved in the translocation of ADP and ATP between the cytoplasm to the mitochondrial matrix. In some embodiments, expression of SEC25A4 by a cell is determined by measuring a level of an SEC25A4 RNA (e.g., mRNA transcribed from SLC25A4). Non-limiting examples of ribonucleic acid sequences of human SEC25A4 can be found, for example, at GenBank Accession No. NM_001151.4, and UniProt Accession No. P12235.

[0236] SLC27A6 is a gene encoding long-chain fatty acid transport protein 6 (also known as ACSVL2, FACVL2, FATP6, and VLCS-H1), which is involved in the uptake of long-chain fatty acids. In some embodiments, expression of SLC27A6 by a cell is determined by measuring a level of an SLC27A6 RNA (e.g., mRNA transcribed from SLC27A6). Non-limiting examples of ribonucleic acid sequences of human SLC27A6 can be found, for example, at GenBank Accession Nos. NM_001017372.3, NM_001317984.2, and UniProt Accession No. Q9Y2P4.

[0237] SLC2A1 is a gene encoding solute carrier family 2, facilitated glucose transporter member 1 (also known as CSE, DYT17, DYT18, DYT9, EIG12, GLUT, GLUT-1, GLUT1, GLUT1DS, HTLVR, PED, and SDCHCN), which is involved in the transportation of glucose across the blood-brain barrier. In some embodiments, expression of SLC2A1 by a cell is determined by measuring a level of an SLC2A1 RNA (e.g., mRNA transcribed from SLC2A1). Non-limiting examples of ribonucleic acid sequences of human SLC2A1 can be found, for example, at GenBank Accession No. NM_006516.4, and UniProt Accession No. Pl 1166.

[0238] SL1T2 is a gene encoding slit homolog 2 protein (also known as SLIL3 and Slit-2), which is involved in aortic valve morphogenesis, cell migration involved in sprouting angiogenesis, and negative regulation of leukocyte chemotaxis. In some embodiments, expression of SL1T2 by a cell is determined by measuring a level of SL1T2 RNA (e.g., mRNA transcribed from SLIT2). Exemplary nucleic acid sequences of human SL1T2 can be found, for example, at GenBank Accession Nos. NG_047105.1, NM_001289135.3, and UniProt Accession No. 094813.

[0239] SMPD3 is a gene encoding sphingomyelin phosphodiesterase 3 (also known as NSMASE2), which is involved in endochondral ossification and sphingomyelin catabolic processes. In some embodiments, expression of SMPD3 by a cell is determined by measuring a level of SMPD3 RNA (e.g., mRNA transcribed from SMPD3). Exemplary nucleic acid sequences of human SMPD3 can be found, for example, at GenBank Accession No. NM_018667.4 and UniProt Accession No. Q9NY59.

[0240] SMTN is a gene encoding smoothelin, which is a structural protein found in contractile muscle cell. In some embodiments, expression of SMTN by a cell is determined by measuring a level of SMTN RNA (e.g., mRNA transcribed from SMTN). Exemplary nucleic acid sequences of human SMTN can be found, for example, at GenBank Accession Nos. NG_029838.2, NM_001207017.1, and UniProt Accession No. A0A087X1R1.

[0241] S0X2 is a gene encoding SRY-Box transcription factor 2 protein (also known as AN0P3 and MC0PS3), which is involved in embryonic development regulation and determination of cell fate. In some embodiments, expression of S0X2 by a cell is determined by measuring a level of S0X2 RNA (e.g., mRNA transcribed from S0X2). Exemplary nucleic acid sequences of human S0X2 can be found, for example, at GenBank Accession Nos. NG_009080.1, NM_003106.4, and UniProt Accession No. P48431. SPARC is a gene encoding secreted protein acidic and cysteine rich protein (also known as ON, ONT, 0117, BM-40), which is involved in regulation of cell morphogenesis and negative regulation of angiogenesis. In some embodiments, expression of SPARC by a cell is determined by measuring a level of SPARC RNA (e.g., mRNA transcribed from SPARC). Exemplary nucleic acid sequences of human SPARC can be found, for example, at GenBank Accession Nos. NG_042174.1, NM_00130944.3, and UniProt Accession No. P09486.

[0242] SPRY1 is a gene encoding sprout RTK signaling antagonist 1 protein (also known as hSPRYl), which is involved in negative regulation of fibroblast growth factor receptor signaling pathway. In some embodiments, expression of SPRY1 by a cell is determined by measuring a level of SPRY1 RNA (e.g., mRNA transcribed from SPRY / ). Exemplary nucleic acid sequences of human SPRY1 can be found, for example, at GenBank Accession Nos. NG_042174.1, NM_00130944.3, and UniProt Accession No. P09486.

[0243] SSBP3 is a gene encoding single stranded DNA binding protein 3 (also known as CSDP, SSDP, SSDP1), which is involved in hematopoietic progenitor cell differentiation and prechordal plate formation. In some embodiments, expression of SSBP3 by a cell is determined by measuring a level of SSBP3 RNA (e.g., mRNA transcribed from SSBP3). Exemplary nucleic acid sequences of human SSBP3 can be found, for example, at GenBank Accession Nos. NM_001009955.4, NM_001394360.1, and UniProt Accession No. Q9BWW4.

[0244] SSBP4 is a gene encoding single stranded DNA binding protein 3, which is involved in positive regulation of transcription by RNA polymerase II. In some embodiments, expression of SSBP4 by a cell is determined by measuring a level of SSBP4 RNA (e.g., mRNA transcribed from SSBP4). Exemplary nucleic acid sequences of human SSBP4 can be found, for example, at GenBank Accession Nos. NM_001009998.4, NM_032627.5, and UniProt Accession No. Q9BWG4.

[0245] SULF1 is a gene encoding extracellular sulfatase 1 protein (also known as SULF-1), which is involved in apoptotic processes, chondrocyte development, and kidney development. In some embodiments, expression of SULF1 by a cell is determined by measuring a level of SULF1 RNA (e.g., mRNA transcribed from SULFI). Exemplary nucleic acid sequences of human SULFI can be found, for example, at GenBank Accession Nos. NG_042849, NM_001128204.2, and UniProt Accession No. Q8IWU6.

[0246] SYNPO2L is a gene encoding synaptopodin 2-like protein, which is involved in actin binding activity, stress fiber assembly, and sarcomere organization. In some embodiments, expression of SYNPO2L by a cell is determined by measuring a level of an SYNPO2L RNA (e.g., mRNA transcribed from SYNPO2L). Non-limiting examples of ribonucleic acid sequences of human SYNP02L can be found, for example, at GenBank Accession Nos. NM_001114133.3, NM_024875.5, and UniProt Accession No. Q9H987.

[0247] TBX2 is a gene encoding T-box transcription factor TBX2 (also known as VETD), which is involved in cardiac atrioventricular canal formation and tumorigenesis. In some embodiments, expression of TBX2 by a cell is determined by measuring a level of an TBX2 RNA (e.g., mRNA transcribed from TBX2). Non-limiting examples of ribonucleic acid sequences of human TBX2 can be found, for example, at GenBank Accession No. NM_005994.4, and UniProt Accession No. Q13207.

[0248] TBX5 is a gene encoding T-box transcription factor TBX5 (also known as HOS), which is involved in cardiac development and the specification of limb identity. In some embodiments, expression of TBX5 by a cell is determined by measuring a level of an TBX5 RNA (e.g., mRNA transcribed from TBX5). Non-limiting examples of ribonucleic acid sequences of human TBX5 can be found, for example, at GenBank Accession Nos. NM_000192.3, NM_080717.4, and UniProt Accession No. Q99593.

[0249] TCAP is a gene encoding telethonin (also known as TELE, CMD1N, CMH25, T-cap, LGM2GD, LGMDR7, and telethonin), which is involved in sarcomere assembly in striated and cardiac muscle. In some embodiments, expression of TCAP by a cell is determined by measuring a level of an TCAP RNA (e.g., mRNA transcribed from TCAP). Non-limiting examples of ribonucleic acid sequences of human TCAP can be found, for example, at GenBank Accession No. NM_003673.4, and UniProt Accession No. 015273.

[0250] TFP12 is a gene encoding tissue factor pathway inhibitor 2 protein (also known as PP5, REFI, and TFPI-2), which is involved in blood coagulation and cellular response to fluid shear stress. In some embodiments, expression of TFP12 by a cell is determined by measuring a level of TFP12 RNA (e.g., mRNA transcribed from TFPI2). Exemplary nucleic acid sequences of human TFP12 can be found, for example, at GenBank Accession Nos. NG_032914.1, NM_001271003.2, and UniProt Accession No. P48307.

[0251] TGFb is a gene encoding transforming growth factor beta-1 proprotein (also known as CED, LAP, DPD1, TGFB, IBDIMDE, TGFbeta, and TGF-betal), which is involved in cell proliferation, differentiation and growth. In some embodiments, expression of TGFb by a cell is determined by measuring a level of TGFb RNA (e.g., mRNA transcribed from TGFb). Exemplary nucleic acid sequences of human TGFb can be found, for example, at GenBank Accession Nos. NG_013364.1, NM_000660.7, and UniProt Accession No. P01137.

[0252] TGM2 is a gene encoding protein-glutamine gamma-glutamyl transferase 2 (also known as G(h), TG(C), TGC, hTG2, tTG), which is involved in apoptosis and implicated in celiac disease. In some embodiments, expression of TGM2 by a cell is determined by measuring a level of an TGM2 RNA (e.g., mRNA transcribed from TGM2). Non-limiting examples of ribonucleic acid sequences of human TGM2 can be found, for example, at GenBank Accession Nos.

[0253] NM_001323316.2, NM_001323317.2, and UniProt Accession No. Q9WVJ6.

[0254] THY1 is a gene encoding thy- 1 cell surface antigen protein (also known as CD90 and CDw90), which is involved in cell communication and cell adhesion. In some embodiments, expression of THY1 by a cell is determined by measuring a level of THY1 RNA (e.g., mRNA transcribed from THYE). Exemplary nucleic acid sequences of human THY1 can be found, for example, at GenBank Accession Nos. NM_001311160.2, NM_001311162.2, and UniProt Accession No. P04216.

[0255] TMEM88 is a gene encoding transmembrane protein 88, which is involved in the Wnt signaling pathway, protein stabilization, and protein plasma membrane localization. In some embodiments, expression of TMEM88 by a cell is determined by measuring a level of TMEM88 RNA (e.g., mRNA transcribed from TMEM88). Exemplary nucleic acid sequences of human TMEM88 can be found, for example, at GenBank Accession Nos. NM_001319941.1, NM_203411.2, and UniProt Accession No. P04216.

[0256] TNNC1 is a gene encoding troponin C, slow skeletal and cardiac muscles (also known as TNC, TN-C, TNNC, CMD1Z, and CMH13), which is involved in regulation of striated muscle contraction. In some embodiments, expression of TNNC1 by a cell is determined by measuring a level of an TNNC1 RNA (e.g., mRNA transcribed from TNNCI ). Non-limiting examples of ribonucleic acid sequences of human TNNC1 can be found, for example, at GenBank Accession No. NM_003280.3 and UniProt Accession No. P63316.

[0257] TNNI3 is a gene encoding troponin I, cardiac muscle (also known as CMH7, RCM1, cTNI, CMD2A, TNNC1, and CMD1FF), which is involved in mediating relaxation of striated muscle, exclusively in cardiac muscle tissues. In some embodiments, expression of TNNI3 by a cell is determined by measuring a level of an TNNI3 RNA (e.g., mRNA transcribed from ENNIS'). Non-limiting examples of ribonucleic acid sequences of human TNNI3 can be found, for example, at GenBank Accession No. NM_000363.5, and UniProt Accession No. P19429.

[0258] TNNT1 is a gene encoding troponin T, slow skeletal muscle protein (also known as ANM, TNT, NEM5, STNT, and TNTS), which is involved in sarcomere organization and skeletal muscle contraction. In some embodiments, expression of TNNT1 by a cell is determined by measuring a level of TNNT1 RNA (e.g., mRNA transcribed from TNNTI). Exemplary nucleic acid sequences of human TNNTI can be found, for example, at GenBank Accession Nos. NG_011829.2, NM_001126132.2, and UniProt Accession No. P13805. TPM1 is a gene encoding TPM1 tropomyosin alpha- 1 chain (also known as CMH3, TMSA, CMD1Y, LVNC9, C15ordl3, HEL-S-265, and HTM-alpha) which is involved in the contraction of striated and smooth muscles, as well as the cytoskeleton of non-muscle cells. In some embodiments, expression of TPM1 by a cell is determined by measuring a level of an TPM1 RNA (e.g., mRNA transcribed from TPMT). Non-limiting examples of ribonucleic acid sequences of human TPM1 can be found, for example, at GenBank Accession Nos. NM_000366.6, NM_001018004.2, and UniProt Accession No. P09493.

[0259] TTN is a gene encoding titin (also known as TMD, CMH9, CMPD4, CMY05, EOMFC, HMERF, MYLK5, SALMY, LGMD2J, and LGMDR10), which is an important structural protein in striated muscle, as well as a base for contractile machinery. In some embodiments, expression of TTN by a cell is determined by measuring a level of an TTN RNA (e.g., mRNA transcribed from TTN). Non-limiting examples of ribonucleic acid sequences of human TTN can be found, for example, at GenBank Accession Nos. NM_001256850.1, NM_001267550.2, and UniProt Accession No. Q8WZ42.

[0260] TWIST1 is a gene encoding twist family bHLH transcription factor 1 protein (also known as CRS, CSO, SCS, ACS3, CRS1, BPES2, BPES3, SWCOS, TWIST, and bHLHa38), which is involved in brown fat metabolism, neural tube closure, and limb development. In some embodiments, expression of TWIST1 by a cell is determined by measuring a level of TWIST1 RNA (e.g., mRNA transcribed from TWIST I). Exemplary nucleic acid sequences of human TWIST 1 can be found, for example, at GenBank Accession Nos. NG_008114.2, NM_000474.4, and UniProt Accession No. Q15672.

[0261] UPK3B is a gene encoding uroplakin-3b protein (also known as P35, UP3B, and UPIIIB), which is involved in negative regulation of gene expression. In some embodiments, expression of UPK3B by a cell is determined by measuring a level of UPK3B RNA (e.g., mRNA transcribed from UPK3B). Exemplary nucleic acid sequences of human UPK3B can be found, for example, at GenBank Accession Nos. NM_001347684.2, NM_030570.3, and UniProt Accession No. Q9BT76.

[0262] VCAM1 is a gene encoding vascular cell adhesion protein 1 (also known as CD 106 and INCAM- 100), which is involved in endothelial cell adhesion and signal transduction. In some embodiments, expression of VCAM1 by a cell is determined by measuring a level of an VCAM1 RNA (e.g., mRNA transcribed from VCAM1). Non-limiting examples of ribonucleic acid sequences of human VCAM1 can be found, for example, at GenBank Accession Nos. NM_001078.4, NM_001199834.2, and UniProt Accession No. P19320. VCAN is a gene encoding versican core protein (also known as CSPG2, ERVR, GHAP, PG-M, WGN, and WGN1), which is involved in cell adhesion, proliferation, migration, and angiogenesis. In some embodiments, expression of VCAN by a cell is determined by measuring a level of an VCAN RNA (e.g., mRNA transcribed from VCAN). Non-limiting examples of ribonucleic acid sequences of human VCAN can be found, for example, at GenBank Accession Nos. NM_001126336.3, NM_001164097.2, and UniProt Accession No. P13611.

[0263] Illustrative Molecular Profiles

[0264] Non-limiting examples of molecular profiles for certain cardiac cell populations are provided below in Table 2.

[0265] Table 2. Non-limiting molecular markers associated with specific cell populations. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes comprises negative / low expression of ANXA1, BGN, CCDC80, CDC42EP5, CDH11, CDON. CFH, COLIA2, COL3AI, COL5AI, CRIME CTSH, CXCLI2, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBN1, FLRT2, FN1, GATA3, GATA6, KRT19, LIMA1, LRPR2, LRRC17, MGP, MMP2, NUPRI, OGN, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNTI, UPK3B, lor PAX6. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes comprises negative / low expression of one or more of: ANXA1, CCDC80, CDON, CRIM1, COL3A1, CTSH, DCN, FLRT2, KRT19, LRPR2, NPNT, NUPRI, PLAC9, PODXL, POSTN, RDH10, SBSON, SI00AI0, S100A11, SPARC, TMEM88, TNNTI, and / or UPK3B. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes comprises negative / low expression of one or more of: ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, COL1A2, COL3A1, CXCL12, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBN1, FN1, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN, PTX3, S100A10, S100A11, SLIT2, SPARC, TFPI2, TGFb, and / or THY1. In some embodiments, a cell comprising a molecular profile characteristic of cardiomyocytes comprises negative / low expression of one or more of: ANXA1, BGN, CCDC80, CDH11, COL1A2, COL3A1, COL5A1, EVAIB, FBNI, LIMA1, MGP, MMP2, OGN, PLAC9, S100A10, S100A11, SEZ6L2, SPARC, SPRY1, and / or SULF1. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes comprises negative / low expression of one or more of: COL3A1, PODXL, and / or KRT19. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes comprises positive / high expression of one or more of SMAD2, TGFB, or POSTN, and negative / low expression of one or more of SMTN, MYLK, ACTA2, ACTAG2, and MYH11. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes comprises negative / low expression of one or more of: KRT19 and / or PODXL. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes comprises negative / low expression of one or more of COL1A2 and / or FN1. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes comprises negative / low expression of COL3A1. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes comprises negative / low expression of COL3A1 and positive / high expression of ACTA2. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes further comprise positive / high expression of TNNT2. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes comprises negative / low expression of COL3A1, and positive / high expression of CTNT. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes comprises negative / low expression of PAX6 and COL3A1, and positive / high expression of CTNT. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes is further identified as mature cardiomyocyte. In some embodiments, a mature cardiomyocyte (e.g., a batch thereof) comprises a molecular profile characteristic of cardiomyocytes and further comprises positive / high expression of MLC2v and / or CTNT. In some embodiments, a mature cardiomyocyte (e.g., a batch thereof) comprises a molecular profile characteristic of cardiomyocytes and further comprise negative / low expression of MLC2a.

[0266] In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of non-cardiomyocytes comprises positive / high expression of one or more of: ANXA1, BGN, CCDC80, CDC42EP5, CDH11, CDON, CFH, COL1A2, COL3A1, COL5A1, CRIM). CTSH, CXCL12, DCN. ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FLRT2, FN1, GATA3, GATA6, KRT19, LIMA1, LRPR2, LRRC17, MGP, MMP2, NUPR1, OGN, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNT1, UPK3B, and / or PAX6. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of non-cardiomyocytes comprises positive / high expression of one or more of: ANXA1, CCDC80, CDON, CRIM1, COL3A1, CTSH, DCN, FLRT2, KRT19, LRPR2, NPNT, NUPR1, PLAC9, PODXL, POSTN, RDH10, SBSON, S100A10, S100A11, SPARC, TMEM88, TNNT1, and / or UPK3B. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of non-cardiomyocytes comprises positive / high expression of one or more of: ACTA2, ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, COL1A2, COL3A1, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FN1, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN, PTX3, S100A10, S100A11, SLIT2, SPARC, TFP12, TGFb, and / or THY I. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of non- cardiomyocytes comprises positive / high expression of one or more of: ANXA1, BGN, CCDC80, CDH11, COL1A2, COL3A1, COL5A1, EVA1B, FBN1, LIMA1, MGP, MMP2, OGN, PLAC9, S100A10, S100A11, SEZ6L2, SPARC, SPRY1, and / or SULF1. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of non-cardiomyocytes comprises positive / high expression of one or more of: COL3A1, PODXL, and / or KRT19. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of non- cardiomyocytes comprises positive / high expression of PAX6. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of cardiomyocytes comprises positive / high expression of PAX6 and COL3AP, and negative / low expression of CTNT. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of non- cardiomyocytes comprises positive / high expression of one or more of: KRT19 and / or PODXL. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of non-cardiomyocytes comprises positive / high expression of one or more of: COL1A2 and / or FN1. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of non-cardiomyocytes comprises positive / high expression of COL3A1. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of non- cardiomyocytes comprises negative / low expression of one or more of SMAD2, TGFB, or POSTN, and positive / high expression of one or more of SMTN, MYLK, ACTA2, ACTAG2, and MYH11. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of non-cardiomyocytes comprises negative / low expression of TNNT2. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of non- cardiomyocytes comprises positive / high expression of COL3A1 and positive / high expression of ACTA2. In some embodiments, a cell (e.g., a batch of cells) comprising positive / high expression of COL3A1 and positive / high expression of ACTA2 is identified as a myofibroblast-like cell.

[0267] In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of epicardial-like cells comprises positive / high expression of one or more of: ANXA1, CCDC80, CDON. CRIM1, COL3A1, CTSH, DCN, FLRT2, KRT19, LRPR2, NPNT, NUPR1, PLAC9, PODXL, POSTN, RDH10, SBSON, S100A10, S100A11, SPARC, TMEM88, TNNT1, and / or UPK3B. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of epicardial-like cells comprises positive / high expression of ANXA1, CCDC80, CDON, COL3A1, CRIM1, CTSH, DCN, FLRT2, KRT19, LRPR2, NUPR1, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, TMEM88, TNNT1, and / or UPK3B. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of epicardial-like cells comprises positive / high expression of one or more of: BNC1, COL9A3, KRT19, NPNT, PODXL, TNNT1, and / or UPK3B. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of epicardial-like cells comprises negative / low expression of CD 172a and positive / high expression of CD90 and PODXL. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of epicardial-like cells comprises positive / high expression of KRT19 and / or PODXL. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of epicardial-like cells comprises negative / low expression of CD36 and / or CD172a.

[0268] In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of fibroblast-like cells comprises positive / high expression of ACTA2, ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, COL1A2, COLS Al, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FN1, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN, PTX3, S100A10, S100A11, SLIT2, SPARC, TFPI2, TGFb, and / or THY1. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of fibroblast-like cells comprises positive / high expression of ANXA1, BGN, CCDC80, CDC42EP5, COL1A2, COL3A1, CXCL12, ELN, EMILIN 1, EVA1B, FBLN5, FBN1, LRRC17, MGP, MMP2, OGN, PLAC9, S100A10, S100A11, and / or SPARC. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of fibroblast- like cells comprises negative / low expression of ATCA2, COL3A1, FN1, PDGFRa, POSTN, TFPI2, TGFB \ . and / or THY. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of fibroblast-like cells comprises negative / low expression of CD 172a, CD36, and / or PODXL. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of fibroblast- like cells comprises negative / low expression of CD 172a and positive / high expression of CD90. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of fibroblast-like cells comprises negative / low expression of CD36 and positive / high expression of CD90. In some embodiments, a cell (e.g., a batch of cells) comprising a molecular profile characteristic of fibroblast-like cells comprises negative / low expression of PODXL and positive / high expression of CD90.

[0269] Compositions and Methods of Use Thereof

[0270] As used herein, a “batch” of cells (e.g., cardiomyocytes) comprises a plurality of cells belonging to a same treatment group. In some embodiments, a batch comprises cells having about the same chronological age (e.g., days since contact with a differentiation or maturation agent). In some embodiments, a batch comprises cells having about the same developmental age (e.g., expressing cell markers within a particular range). In some embodiments, a batch comprises cells derived from the same ancestral cells. In some embodiments, a batch comprises cells derived from the same cell line. In some embodiments, a batch comprises cells from two or more different cell lines. In some embodiments, a batch of cells comprises at least 1 billion cells. In some embodiments, a batch of cells comprises about 2 billion cells. In some embodiments, a batch of cells comprises about 3 billion cells. In some embodiments, a batch of cells comprises about 4 billion cells. In some embodiments, a batch of cells comprises about 5 billion cells. In some embodiments, a batch of cells comprises about 10 billion cells. In some embodiments, a batch of cells comprises about 20 billion cells. In some embodiments, a batch of cells comprises about 1 billion cells to about 25 billion cells. In some embodiments, a batch of cells comprises about 10 billion to about 20 billion cells. In some embodiments, a batch of cells comprises about 1 billion, about 2 billion, about 3 billion, about 4 billion, about 5 billion, about 6 billion, about 7 billion, about 8 billion, about 9 billion, about 10 billion, about 11 billion, about 12 billion, about 13 billion, about 14 billion, about 15 billion, about 16 billion, about 17 billion, about 18 billion, about 19 billion, about 20 billion cells.

[0271] A sample is a portion of a batch and comprises one or more cells of the batch. In some embodiments, a sample comprises lxl0'9% to 1% of a batch. In some embodiments, properties of a batch are determined from three or more samples of the batch. Preferably, a sample is representative of a batch.

[0272] In some embodiments, a batch is heterogenous and comprises cells having two or more different characteristics, including, but not limited to, cell types (e.g., cardiomyocytes, endothelial cells, fibroblasts), ages, and / or molecular profiles. In some embodiments, a batch is homogenous and consists of cells having shared characteristics, including, but not limited to cell type (e.g., cardiomyocytes), age (e.g., chronological, developmental), and / or molecular profile.

[0273] In some embodiments, a heterogenous batch is made homogenous by isolating cells having one or more desired characteristics (e.g., molecular profile identified as being associated with cardiomyocytes) for further preparation. In some embodiments, a heterogenous batch is made homogenous by isolating cells having one or more undesired characteristics (e.g., molecular profile identified as being associated with off-target cells) for removal from the batch. For example, a heterogenous batch may comprise a first subset of cells having a molecular profile identified as being associated with cardiomyocytes and a second subset of cells having a molecular profile identified as being associated with off-target cells (e.g., non-cardiomyocytes, such as fibroblast-like cells and / or epicardial-like cells). Either subset of cells may be removed from the batch by using any number of methods, for example, immunofluorescent or immunomagnetic labeling of select surface markers (e.g., markers from molecular profiles associated with off-target cells), coupled with corresponding FACS gating strategy or immunomagnetic separation, respectively.

[0274] Cardiac Cell Therapies

[0275] Cells identified by a method disclosed herein may be used for cardiac cell therapy. The term “cardiac cell therapies,” as used herein, refers to cellular compositions comprising a plurality of cardiac cells and a physiologically acceptable medium. In some embodiments, a cardiac cell therapy comprises cardiomyocytes. In some embodiments, a cardiac cell therapy comprises epicardial-like cells. In some embodiments, a cardiac cell therapy comprises fibroblast- like cells. In a preferred embodiment, a cardiac cell therapy comprises mature cardiomyocytes.

[0276] Cardiac cell therapies comprising cardiomyocytes may comprise one or more types of cardiomyocytes (e.g., ventricular cardiomyocytes, atrial cardiomyocytes, and / or smooth muscle cells) at one or more stages of development (e.g., mature cardiomyocytes and / or immature cardiomyocytes). In some embodiments, the cardiomyocytes are ventricular cardiomyocytes. In some embodiments, cardiomyocytes are immune evading or hypoimmune. In some embodiments, cardiac cell therapies (e.g., cardiac grafts) further comprise vascular cells and / or cardiac cells. In some embodiments, cardiac cell therapies (e.g., cardiac grafts) comprising cardiomyocytes further comprise endothelial cells, conduction cells, pacemaker cells, and / or fibroblasts.

[0277] Cardiac cell therapies may comprise any physiologically acceptable medium helpful for administering, adhering, growing, and / or maintaining the cardiac cell therapy (e.g., cardiac graft) in a subject. Physiologically acceptable media for administration of tissues are known in the art.

[0278] In embodiments, cardiac cell therapies are administered to a subject having experienced cardiac injury, including, but not limited to, damage to cells and tissue of the heart, including cardiomyocytes. Cardiac injury may be caused by a number of factors, including, but not limited to, heart disease (e.g., coronary heart disease, cardiomyopathy, endocarditis, congenital cardiovascular defects, congestive heart failure), medications, and non-cardiac diseases (e.g., high blood pressure, diabetes, viruses). In some embodiments, cardiac injury is caused by heart failure (e.g., HFrEF). In some embodiments, cardiac injury is caused by myocardial infarction. In some embodiments, cardiac injury is caused by ischemia. In some embodiments, cardiac injury comprises an injured ventricle (e.g., left ventricle). In some embodiments, a cardiac cell therapy (e.g., a cardiac graft) is administered to a subject in need thereof, such as a subject having experienced cardiac injury. A subject may be any mammal, including, but not limited to, mice, rats, guinea pigs, hamsters, pigs, cows, sheep, goats, horses, and primates, including nonhuman primates and humans.

[0279] Cardiac cell therapies (e.g., cardiac grafts) are typically administered directly to the heart of a subject and may be administered to a subject through any suitable method; non-limiting examples include open surgical approaches (e.g., direct administration to the heart during open heart surgery), minimally invasive approaches (e.g., administration via a cardiac catheter or needle), or percutaneous / intravascular approaches.

[0280] In some embodiments, a cardiac cell therapy is a cardiac graft. In some embodiments, a cardiac graft comprises about 100 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 200 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 300 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 400 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 500 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 600 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 700 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 800 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 900 million to about 1 billion cardiomyocytes.

[0281] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) is a cellular composition comprising a plurality of cells wherein at least 50% of the cells are on-target cells (e.g., cardiomyocytes). In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% of the cells are on target cells. In a preferred embodiment, the on-target cells are cardiomyocytes. In a preferred embodiment, the on-target cells comprise negative / low expression of COL3A1. In another preferred embodiment, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein at least 50% of the cells have a molecular profile characteristic of cardiomyocytes. In a preferred embodiment, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein at least 50% of the cells have negative / low expression of COL3A1.

[0282] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein at least 50% of the cells have a molecular profile characteristic of non- cardiomyocytes. In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein at least 50% of the cells have a molecular profile characteristic of epicardial-like cells. In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein at least 50% of the cells have a molecular profile characteristic of fibroblast- like cells. In some embodiments, a cardiac cell therapy (e.g., cardiac graft) is a cellular composition comprising a plurality of cells wherein no more than 50% of the cells are off-target cells (e.g., fibroblast-like cells, epicardial-like cells). In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more 4%, no more than 3%, no more than 2%, no more than 1%, no more than 0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2%, or no more than 0.1% of the cells are off-target cells. In a preferred embodiment, the off-target cells are non- cardiomyocytes. In a preferred embodiment, the off-target cells comprise positive / high expression of COL3A1.

[0283] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein no more than 50% of the cells have a molecular profile characteristic of epicardiallike cells. In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein no more than 50% of the cells have a molecular profile characteristic of fibroblast-like cells. In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein no more than 50% of the cells have a molecular profile characteristic of cardiomyocytes.

[0284] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein 95% or more of the cells are on-target cells (e.g., cardiomyocytes). In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein 95.0% or more, 95.1% or more, 95.2% or more, 95.3% or more, 95.4% or more, 95.5% or more,

[0285] 95.6% or more, 95.7% or more, 95.8% or more, 95.9% or more, 96.0% or more, 96.1% or more,

[0286] 96.2% or more, 96.3% or more, 96.4% or more, 96.5% or more, 96.6% or more, 96.7% or more,

[0287] 96.8% or more, 96.9% or more, 97.0% or more, 97.1% or more, 97.2% or more, 97.3% or more,

[0288] 97.4% or more, 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more,

[0289] 98.0% or more, 98.1% or more, 98.2% or more, 98.3% or more, 98.4% or more, 98.5% or more,

[0290] 98.6% or more, 98.7% or more, 98.8% or more, 98.9% or more, 99.0% or more, 99.1% or more,

[0291] 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more,

[0292] 99.8% or more, or 99.9% or more of the cells are on-target cells.

[0293] In a preferred embodiment, the on-target cells are cardiomyocytes. In a preferred embodiment, the on-target cells comprise negative / low expression of COL3A1. In another preferred embodiment, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein at least 90% of the cells have a molecular profile characteristic of cardiomyocytes. In a preferred embodiment, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein at least 90% of the cells have negative / low expression of COL3A1.

[0294] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein at least 90% of the cells have a molecular profile characteristic of non- cardiomyocytes. In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein at least 90% of the cells have a molecular profile characteristic of epicardial-like cells. In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein at least 90% of the cells have a molecular profile characteristic of fibroblast- like cells.

[0295] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein 10% or fewer of the cells are off-target cells (e.g., fibroblast-like cells, epicardiallike cells). In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4.9%, no more than 4.8%, no more than 4.7%, no more than 4.6%, no more than 4.5%, no more than 4.4%, no more than 4.3%, no more than 4.2%, no more than 4.1%, no more than 4.0%, no more than 3.9%, no more than 3.8%, no more than 3.7%, no more than 3.6%, no more than 3.5%, no more than 3.4%, no more than 3.3%, no more than 3.2%, no more than 3.1%, no more than 3.0%, no more than 2.9%, no more than 2.8%, no more than 2.7%, no more than 2.6%, no more than 2.5%, no more than 2.4%, no more than 2.3%, no more than 2.2%, no more than 2.1%, no more than 2.0%, no more than 1.9%, no more than 1.8%, no more than 1.7%, no more than 1.6%, no more than 1.5%, no more than 1.4%, no more than 1.3%, no more than 1.2%, no more than 1.1%, no more than 1.0%, no more than 0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2%, no more than or 0.1% or 0% of the cells are off-target cells.

[0296] In a preferred embodiment, the off-target cells are non-cardiomyocytes. In a preferred embodiment, the on-target cells comprise positive / high expression of COL3A1.

[0297] In another preferred embodiment, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein 10% or fewer of the cells have a molecular profile characteristic of non-cardiomyocytes. In a preferred embodiment, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells wherein 10% or fewer of the cells have positive / high expression of COL3A1.

[0298] In some embodiments, a cardiac cell therapy (e.g., cardiac graft comprises a plurality of cells wherein no more than 10% of the cells have a molecular profile characteristic of epicardial- like cells. In some embodiments, a cardiac cell therapy (e.g., cardiac graft comprises a plurality of cells wherein no more than 10% of the cells have a molecular profile characteristic of fibroblast-like cells. In some embodiments, a cardiac cell therapy (e.g., cardiac graft comprises a plurality of cells wherein no more than 10% of the cells have a molecular profile characteristic of cardiomyocytes.

[0299] In some embodiments, a cardiac graft comprises cardiac cells produced from immature cardiac cells. In some embodiments, a cardiac graft comprises mature cardiomyocytes produced from a batch of immature cardiomyocytes. In some embodiments, a batch of immature cardiomyocytes is contacted with a maturation cocktail under conditions that promote cardiomyocyte maturation. Standard methods for maturing cardiomyocytes are described in Guo et al., Circulation Research, 2020, 126, 1086-1106, incorporated herein by reference in its entirety. In some embodiments, mature cardiomyocytes are made by culturing immature cardiomyocyte on a substrate (e.g., adherent culture) for at least 2 weeks (e.g., between 2 and 3, between 3 and 4, between 4 and 5, between 5 and 6, between 6 and 8, between 8 and 10, or between 10 and 12 weeks, inclusive, or more than 12 weeks) in culture media comprising a maturation cocktail. In some embodiments, mature cardiomyocytes are made by culturing immature cardiomyocyte on a substrate (e.g., adherent culture) for at least 2 weeks (e.g., between 2 and 3, between 3 and 4, between 4 and 5, between 5 and 6, between 6 and 8, between 8 and 10, or between 10 and 12 weeks, inclusive, or more than 12 weeks) in culture media that promotes oxidative phosphorylation.

[0300] EXAMPLES

[0301] Example 1. Molecular Identification of Off-Target Cardiac Cell Populations

[0302] Advancements in cardiac cell therapy have highlighted the potential of cardiomyocyte grafts to mitigate the effects of heart disease and failure. The success of such therapies depends, in part, on the preparation of highly pure cardiac cell populations. However, currently available strategies for purification still result in the retention of a significant portion of off-target cells. scRNA-seq analysis identifies markers associated with off-target cardiac cell populations

[0303] To identify off-target cardiac cell populations, batches of day 51 cardiomyocyte samples from various bioprocesses were characterized by single-cell RNA sequencing for patterns of gene expression among cells that were not cardiac troponin T2 ( TNNT2) positive. This strategy ensured that the off-target markers would be broad enough to capture a variety of potential failure modes. Batch corrected single-cell transcriptomic data was visualized using Uniform Manifold Approximation and Projection (UMAP) to ensure the removal of batch effects due to the use of different bioprocesses. Clustering of gene expression data revealed twenty genes associated with off-target (non-cardiomyocyte) cells (e.g., having high expression in fibroblastlike cells and epicardial-like cells, but not cardiomyocytes) (FIG. 1). Average gene expression values for these twenty genes are shown in Table 3.

[0304] Table 3. Average gene expression of off-target (non-cardiomyocyte) marker genes.

[0305] OT= off-target (non-cardiomyocyte) cells, including fibroblast-like (FB) cells and epicardial-like (EC) cells; CM = cardiomyocytes; OT-CM = difference between off-target and cardiomyocyte cells.

[0306] Differential gene expression analysis was additionally performed on each off-target population, revealing fibroblast-like-specific markers (FIG. 2 and Table 4) and epicardial-like- specific makers (FIG. 3 and Table 5).

[0307] Table 4. Average gene of fibrob ast-like marker

[0308] 0T= off-target (non-cardiomyocyte) cells, including fibroblast-like (FB) cells and epicardial-like (EC) cells; CM = cardiomyocytes; FB-EC = difference between fibroblast-like cells and epicardial-like cells.

[0309] Table 5. Average gene expression of epicardial-like marker genes.

[0310] 0T= off-target (non-cardiomyocyte) cells, including fibroblast-like (FB) cells and epicardial-like (EC) cells; CM = cardiomyocytes; EC-FB = difference between epicardial-like cells and fibroblast-like cells.

[0311] Validation of off-target (non-cardiomyocyte) cell marker C0L3A1 by flow cytometry

[0312] The off-target (non-cardiomyocyte) cell markers were then validated by flow cytometry for their use in identifying shifts in sub-population fractions for relevant deviations of bioprocesses designed to produce mature cardiomyocytes. Of the non-cardiomyocyte markers identified in Table 3, COL3A1 demonstrated the greatest difference in average gene expression value in off-target cells versus cardiomyocytes. Thus, the utility of C0L3A1 as a marker for off- target cell identification was evaluated and its ability to separate off-target markers was compared to the current gold standard marker CD90.

[0313] Cells were labelled with fluorescently conjugated antibodies targeting a combination of cardiac troponin (CTNT), Thy-1 cell surface antigen (Thy-1, also referred to as CD90), or collagen type III alpha 1 chain (C0L3A1). Analysis of flow cytometry results revealed that gating cells based on their fluorescence intensity for CTNT and CD90 resulted in approximately 7.35% of cells that were not able to be classified as either a cardiomyocyte cell or an off-target cell (FIG. 4A). In comparison, gating cells based on their fluorescence intensity for CTNT and C0L3A1 increased the percentage of classified cells (FIG. 4B). Gating cells based in their fluorescence intensity for CD90 and C0L3A1 revealed a significant portion of off-target cells that expressed C0L3A1, but not CD90, suggesting that C0L3A1 provided more complete and accurate identification of off-target cells than CD90 (FIG. 4C).

[0314] The ability of C0L3A1 to accurately identify non-cardiomyocyte cells was verified by sum-to-100 calculation. Normalized data of triplicates of three batches of flow cytometry data showed that all cells could be classified using CTNT and C0L3A1. An average of less than 5% of cells were classified as CTNT COL3A 1+, suggesting that C0L3A1 is a specific marker for non-cardiomyocyte cells (Table 6). Moreover, the utility of C0L3A1 as a non-cardiomyocyte marker was validated by comparison with normalized data from three scRNA-seq runs, which showed similar results using multiple gene panels (Table 7).

[0315] T able 6. Sum-to-100 calculation of flow cytometry runs.

[0316] >100% C0L3A1 +CTNT+ cells are double counted Table 7. Sum-to-100 calculation of scRNA-seq runs.

[0317] EC = epicardial-like cell markers; FB = fibroblast-like cell markers; non-CM = non-cardiomyocyte cell markers;

[0318] CM = cardiomyocyte cell markers

[0319] Batch test analysis demonstrates repeatable identification of off-target and mature cardiomyocytes

[0320] Using COL3A1 as a marker for off-target cells, the composition of day 14 and day 51 cardiac cell populations was characterized by flow cytometry. Mature cardiomyocytes were identified using CTNT, and further identified as ventricular cardiac muscle cells using MLC2v, atrial cardiac muscle cells using MLC2a, and actively proliferating cells (immature / precursor cells) using K167 (FIGs. 5A-5D). The results demonstrate that across a given timepoint differentiated cell populations were found to exhibit similar cell compositions (FIGs. 6A-7C and Table 8A).

[0321] Table 8A. Average cell composition in day 14 (D14) and day 51 (D51) cell populations, compared to undifferentiated pluripotent stem cells (PSC).

[0322] %CV = coefficient of variation; N = number of replicates

[0323] Identification of cells using these markers was further validated in additional mature (D51) cell populations. As shown in FIG. 13, populations identified as putative mature cardiomyocytes exhibit positive / high expression of CTNT, MLC2v, MLC2a, CD 172, and CD36 (top panel) and negative / low expression of COL3A1, KI67, CD31, CD90, and PODXL (bottom panel). Molecular profiles of individual cell populations shown in FIG. 13 are provided in Table 8B below.

[0324] Table 8B. Molecular profiles of individual D51 cell populations shown in FIG. 13

[0325] Incorporation of epicardial-like markers PODXL and KRT19 to sub-divide off-target cells

[0326] Finally, sub-populations of off-target cells were identified by clustering of gene expression data. Nine clusters were identified, three of which corresponded to off-target cells (FIGs. 8A-8B). The top differentially expressed genes in each cluster are listed in Table 9. These three off-target clusters were identified via C0L3A1 and showed overlapping expression with cardiac genes HAND2, GATA4, and GATA6 (FIGs. 9A-9C).

[0327] Table 9. Top differentially expressed genes based on clustering of gene expression data. Clusters 3, 7, and 8 correspond to off-target cells.

[0328] Further analysis of cluster 8 revealed overlapping expression of C0L13A1 with epicardial genes PODXL, KRT19, BNC1, UPK3B, TNNT1, and COL9A3 (FIGs. 10A-10F). Additionally, gene expression of off-target fibroblast-like cells were found to be similar to ECM secreting activated cardiac fibroblasts in the human heart, as demonstrated by high expression of COL3AS1, FN1, THY1, PDGFRa, P0STN, ACTA2, TGFB1, and TFPI2 (FIGs. 11A-11H).

[0329] The ability to separate sub-populations of off-target cells using the above-identified marker genes was validated by flow cytometry. Using various gating strategies, the expression of KRT19 and PODXL was successfully used to identify epicardial-like cells (FIGs. 12A-12B). Example 2. Identification of Additional Distinguishing Markers

[0330] To identify “off-target” sub-populations of cells in heterogenous putative cardiomyocyte populations, cell populations were assessed for expression of ACTA2 (also known as aSMA), a marker of smooth muscle cells, and PAX6, a neural progenitor marker.

[0331] ACTA2 (a.k.a. aSMA) As a Marker of Myofibroblast-like Sub-populations of Putative Cardiomyocytes

[0332] Heterogeneity was observed in two cell populations, W-PD8-D51 and T-PD6-D51, identified as containing primarily putative cardiomyocytes based on flow cytometric analysis. As shown in FIG. 14, both populations had similar molecular profiles for CTNT vs COL3A1 expression, CTNT vs PAX6 expression, and CD9 vs PAX6 expression. Notably, while the majority of W-PD8-D51 cells and T-PD6-D51 cells exhibited negative / low expression of COL3A1 and negative / low expression of ACTA2 (a.k.a. aSMA), a distinct cluster of T-PD6-D51 cells exhibited positive / high expression of COL3A1 and positive / high expression of ACTA2 (a.k.a. aSMA).

[0333] To better identify the lineage of ACTA2 (a.k.a. <z.S' 4)-cnrichcd sub-populations, scRNA-seq and clustering analysis, as described in Example 1, was performed on these cell populations. As shown in FIG. 15A, scRNA-seq analysis of the heterogenous cell populations identified both typical putative cardiomyocytes (“ON-target”) and putative non-cardiomyocyte sub-populations (“off-target”) that expressed relative higher levels of ACTA2 (a.k.a. aSMA). Further differential analysis (not shown) determined that the ACTA2 (a.k.a. <z.S' 4)-cnrichcd putative non-cardiomyocytes exhibit negative / low expression of ACTG2, SMTN, MYHI1 , and MYLK, which are associated with smooth muscle cells, and positive / high expression of LOX, SMAD2, TGFb, and POSTN, which are associated with myofibroblasts. These findings suggest that, while ACTA2 (a.k.a. aSMA) is expressed in both smooth muscle and myofibroblasts, the ACTA2 (a.k.a. <z.S' 4)-cnrichcd sub-population of cells has a myofibroblast lineage.

[0334] Moreover, previous studies have suggested that actin filaments in the heart undergo isoform switching during development, with a gradual transition from ACTA2 (a.k.a. aSMA) (also known as ACTA2) expression in the fetal heart to ACTA1 expression in the neonatal heart, and finally to ACTC1 expression in the adult heart. Further scRNA-seq analysis of the heterogenous cell populations suggests that while PSC-derived putative cardiomyocytes are developing along this timeline, PSC-derived ACTA2 (a.k.a. aSMA) -enriched sub-populations are not (FIG. 15B). This may indicate that these sub-populations of ACTA2 (a.k.a. aSMA) -enriched cells are of a related lineage, but at an earlier developmental stage compared to the putative cardiomyocytes. Indeed, additional flow cytometric analysis of these cells with an extended maturation period suggests that ACTA2 (a.k.a. aSMA) enrichment coincides with increasingly enriched expression of cTNl (also referred to herein as TNNI3) (FIG. 16). Without wishing to be bound by theory, it is believed that heterogeneity of cardiomyocyte populations may be attributed to differences in maturation progression, with ACTA2 (a.k.a. <z.S' 4)-cnrichcd sub-populations of cells representing cardiomyocyte cells at a different stage of maturation. A proposed timeline of dynamic marker expression relative to maturation age is shown in FIG. 17.

[0335] PAX6 As a Marker of Neural-Lineage Sub-populations of Putative Cardiomyocytes

[0336] Further flow cytometric analysis of a heterogenous cell population identified as primarily putative cardiomyocytes revealed a distinct AXd-cxprcssing sub-population. As shown in FIG. 18, while the majority of cells exhibited positive / high expression for CTNT and negative / low expression for COL3A1 (and thus were deemed putative cardiomyocytes), a distinct subpopulation of the cells exhibited relatively negative / low expression of CTNT and positive / high expression of PAX6 compared to the putative cardiomyocytes (top right panel, bottom right quadrant; bottom panel, bottom right quadrant). Analysis of a separate putative cardiomyocyte population similarly identified a sub-population of cells having relatively negative / low expression of CTNT and negative / low expression of COL3A1 (FIG. 19; bottom right quadrant of each panel). These cells were analyzed for PAX6 expression and, similarly to the cells shown in FIG. 18, a sub-population were found to exhibit positive / high expression of PAX6 (FIG. 20; bottom right quadrant of each panel). However, despite accounting for about 85% of off-target cells in putative cardiomyocyte pools, AXd-cnrichcd cells comprises only a minor percentage of each tested pool as shown in FIG. 21 and Table 10. Overall, these experiments provide additional markers for further refinement of putative cardiomyocyte cell molecular profiles described herein (e.g., positive / high CTNT and negative / low COL3A ]).

[0337] EQUIVALENTS AND SCOPE

[0338] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes aspects in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes aspects in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0339] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, certain aspects of the invention or aspects of the invention consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those aspects have not been specifically set forth in haec verba herein.

[0340] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one aspect, to A only (optionally including elements other than B); in another aspect, to B only (optionally including elements other than A); in yet another aspect, to both A and B (optionally including other elements); etc.

[0341] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0342] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one aspect, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another aspect, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another aspect, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0343] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0344] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that aspects described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative aspects, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the application describes “a composition comprising A and B,” the application also contemplates the alternative aspects “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

[0345] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different aspects of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0346] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular aspect of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such aspects are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular aspect of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0347] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific aspects described herein. The scope of the present aspects described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

[0348] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an aspect for a variable herein includes that aspect as any single aspect or in combination with any other aspects or portions thereof. The recitation of an aspect herein includes that aspect as any single aspect or in combination with any other aspects or portions thereof.

Claims

CLAIMSWhat is claimed is:

1. A composition for cardiac cell therapy, the composition comprising: a plurality of cells in a physiologically acceptable medium suitable for administration to a patient in need thereof; wherein at least 50% of the plurality of cells comprise negative / low expression of COLS Al.

2. The composition of claim 1, wherein the plurality of cells comprises mature cardiomyocytes.

3. The composition of claim 2, wherein the mature cardiomyocytes are derived from pluripotent stem cells.

4. The composition of claim 3, wherein the pluripotent stem cells comprise positive / high expression of one or more of the following markers: SSEA3 / 4, TRA-1-60, OCT3 / 4, NANOG, and SOX2.

5. The composition of claim 2, wherein the mature cardiomyocytes are derived from embryonic stem cells.

6. The composition of any one of claims 2 to 5, wherein the mature cardiomyocytes comprise positive / high expression of MLC2v and / or CTNT.

7. The composition of claim 6, wherein the mature cardiomyocytes further comprises negative / low expression of MLC2a.

8. A composition for cardiac cell therapy, the composition comprising: a plurality of cells in a physiologically acceptable medium suitable for administration to a patient in need thereof; wherein at least 50% of the plurality of cells comprise a molecular profile comprising: i) negative / low expression of one or more markers selected ANXA 7, BGN, CCDC80, CDC42EP5, CDH11, CDON. CFH, COL1A2, COL3A1, COL5A1, CRIME CTSH, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FLRT2, FN1, GATA3, GATA6, KRT19, L1MA1, LRPR2, LRRC17, MGP, MMP2, NUPR1, OGN,PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNT1, and / or UPK3B, ii) negative / low expression of one or more markers selected from: ANXA1, CCDC80, CDON, CRIM1, COLS Al, CTSH, DCN, FLRT2, KRT19, LRPR2, NPNT, NUPR1, PLAC9, PODXL, POSTN, RDH10, SBSON, S100A10, S100A11, SPARC, TMEM88, TNNT1, and / or UPK3B, iii) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, COL1A2, COL3A1, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FN1, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN, PTX3, S100A10, S100A11, SLIT2, SPARC, TFP12, TGFb, and / or THY], iv) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CDH11, COL1A2, COL3A1, COL5A1, EVA1B, FBN1, L1MA1, MGP, MMP2, OGN, PLAC9, S100A10 v) negative / low expression of one or more markers selected from COL3A1, PODXL, and / or KRT19, vi) negative / low expression of one or more markers selected from KRT19 and / or PODXL, vii) negative / low expression of one or more markers selected from COL1A2 and / or FNL, and / or viii) negative / low expression of COL3A1.

9. A composition for cardiac cell therapy, the composition comprising: a plurality of cells in a physiologically acceptable medium suitable for administration to a patient in need thereof; wherein at least 50% of the plurality of cells comprise a molecular profile characteristic of cardiomyocyte identity.

10. The composition of claim 9, wherein the molecular profile characteristic of cardiomyocytes comprises one of the following: i) negative / low expression of one or more markers selected ANXA 1, BGN, CCDC80, CDC42EP5, CDH11, CDON, CFH, COL1A2, COL3A1, COL5A1, CRIM1, CTSH, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FLRT2, FN1, GATA3, GATA6, KRT19, L1MA1, LRPR2, LRRC17, MGP, MMP2, NUPR1, OGN,PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNT1, and / or UPK3B, ii) negative / low expression of one or more markers selected from: ANXA1, CCDC80, CDON, CRIM1, COLS Al, CTSH, DCN, FLRT2, KRT19, LRPR2, NPNT, NUPR1, PLAC9, PODXL, POSTN, RDH10, SBSON, S100A10, S100A11, SPARC, TMEM88, TNNT1, and / or UPK3B, iii) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, COL1A2, COL3A1, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FN1, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN, PTX3, S100A10, S100A11, SLIT2, SPARC, TFP12, TGFb, and / or THY1, iv) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CDH11, COL1A2, COL3A1, COL5A1, EVA1B, FBN1, L1MA1, MGP, MMP2, OGN, PLAC9, S100A10 v) negative / low expression of one or more markers selected from COL3A1, PODXL, and / or KRT19, vi) negative / low expression of one or more markers selected from KRT19 and / or PODXL, vii) negative / low expression of one or more markers selected from COL1A2 and / or FNL, and / or viii) negative / low expression of COL3A1.

11. A method of preparing a cellular composition for cardiac cell therapy, the method comprising:(a) determining that a plurality of cells have a molecular profile characteristic of cardiomyocyte identity, wherein the molecular profile comprises negative / low expression of COL3A1, and(b) preparing a cellular composition for cardiac cell therapy from the plurality of cells having a molecular profile characteristic of cardiomyocyte identity.

12. The method of claim 11, wherein the plurality of cells having a molecular profile characteristic of cardiomyocyte identity represents greater than 50% of all cells used to prepare the cardiac cell therapy.

13. The method of claim 12, wherein the plurality of cells having a molecular profile characteristic of cardiomyocyte identity represents greater than 90% of all cells used to prepare the cardiac cell therapy.

14. The method of claim 12 or 13, wherein the plurality of cells having a molecular profile characteristic of cardiomyocyte identity is obtained by:(i) determining a molecular profde of a batch of cells;(ii) selecting the plurality of cells having a molecular profile characteristic of cardiomyocyte identity from the batch of cells.

15. The method of claim 14, wherein the molecular profile of the batch of cells is obtained via flow cytometry.

16. The method of claim 14, wherein the molecular profile of the batch of cells is obtained from a transcriptome of the batch of cells.

17. The method of claim 16, wherein the transcriptome is obtained via single cell ribonucleic acid sequencing (scRNA-seq).

18. The method of any one of claims 12 to 17, wherein the molecular profile characteristic of cardiomyocyte identity comprises negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CDC42EP5, CDH11, CDON. CFH, COL1A2, COLS Al, COL5A1, CRIM1, CTSH, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FLRT2, FN1, GATA3, GATA6, KRT19, L1MA1, LRPR2, LRRC17, MGP, MMP2, NUPR1, OGN, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNT1, and / or UPK3B.

19. The method of any one of claims 12 to 17, wherein the molecular profile characteristic of cardiomyocyte identity comprises negative / low expression of one or more markers selected from: COL3A1, PODXL, and / or KRT19.

20. The method of any one of claims 12 to 19, wherein the plurality of cells having a molecular profile characteristic of cardiomyocyte identity comprises mature cardiomyocytes.

21. The method of claim 20, wherein the mature cardiomyocytes are derived from pluripotent stem cells.

22. The method of claim 21, wherein the pluripotent stem cells express one or more of the following markers: SSCB3 / 4, TRA-1-60, OCT 3 / 4. NANOG. and SOX2.

23. The method of claim 20, wherein the mature cardiomyocytes are derived from embryonic stem cells.

24. The method of any one of claims 20 to 23, wherein the mature cardiomyocytes comprise positive / high expression of MLC2v and / or CTNT.

25. The method of claim 24, wherein the mature cardiomyocytes comprise negative / low expression of MLC2a.

26. The method of any one of claims 12 to 25, wherein preparing the cellular composition for cardiac cell therapy from the plurality of cells having a molecular profile characteristic of cardiomyocyte identity comprises: contacting the plurality of cells having a molecular profile characteristic of cardiomyocyte identity with a physiologically acceptable medium.

27. A method of preparing a cellular composition for cardiac cell therapy, the method comprising:(a) determining that a plurality of cells do not have a molecular profile characteristic of non- cardiomyocytes; and(b) preparing the cellular composition for cardiac cell therapy from the plurality of cells.

28. The method of claim 27, wherein the molecular profile of the plurality of cells is obtained via flow cytometry.

29. The method of claim 27, wherein the molecular profile of the plurality of cells is obtained from a transcrip tome of the plurality of cells.

30. The method of claim 29, wherein the transcriptome is obtained via single cell ribonucleic acid sequencing (scRNA-Seq).

31. The method of any one of claims 27 to 30, wherein the molecular profile characteristic of non-cardiomyocytes is a non-cardiomyocyte molecular profile.

32. The method of claim 31, wherein the molecular profile characteristic of non- cardiomyocytes comprises positive / high expression of one or more markers selected from: ANXA1, BGN, CCDC80, CDC42EP5, CDH11, CDON. CFH, COL1A2, COLS Al, COL5A1, CRIM1, CTSH, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2, FBLN5, FBN1, FLRT2, FN1, GATA3, GATA6, KRT19, L1MA1, LRPR2, LRRC17, MGP, MMP2, NUPR1, OGN, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNT1, and / or UPK3B.

33. The method of claim 32, wherein the molecular profile characteristic of non- cardiomyocytes comprises positive / high expression of COL3A1, PODXL, and / or KRT19.

34. The method of any one of claims 27 to 30, wherein the molecular profile characteristic of non-cardiomyocytes is an epicardial-like cell (EC) molecular profile.

35. The method of claim 34, wherein the EC molecular profile comprises positive / high expression of one or more markers selected from: ANXA1, CCDC80, CDON, CRIM1, COL3A1, CTSH, DCN, FLRT2, KRT19, LRPR2, NPNT, NUPR1, PLAC9, PODXL, POSTN, RDH10, SBSON, S100A10, S100A11, SPARC, TMEM88, TNNT1, and / or UPK3B.

36. The method of claim 35, wherein the EC molecular profile comprises positive / high expression of KRT19 and / or PODXL.

37. The method of any one of claims 27 to 30, wherein the molecular profile characteristic of non-cardiomyocytes is a fibroblast-like (FB) molecular profile.

38. The method of claim 37, wherein the FB molecular profile comprises positive / high expression of one or more markers selected from: ACTA2, ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, COL1A2, COL3A1, CXCL12, DCN, ELN, EMILIN 1, EVA1B, FBLN2,FBLN5, FBN1, FN1, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN. PTX3. S100A10, S100A11, SLIT2, SPARC, TFP12, TGFb, and / or THY1.

39. The method of claim 37, wherein the FB molecular profile comprises positive / high expression of COL1A2 and / or FN1.

40. The method of any one of claims 27 to 39, wherein no more than 50% of the cells in the cellular composition comprise a molecular profile characteristic of non-cardiomyocytes.

41. The method of any one of claims 27 to 38, wherein the plurality of cells comprises mature cardiomyocytes.

42. The method of claim 41, wherein the mature cardiomyocytes are derived from pluripotent stem cells.

43. The method of claim 42, wherein the pluripotent stem cells express one or more of the following markers: SSEA3 / 4, TRA-1-60, OCT3 / 4, NANOG, and SOX2.

44. The method of claim 41, wherein the mature cardiomyocytes are derived from embryonic stem cells.

45. The method of any one of claims 41 to 44 wherein the mature cardiomyocytes comprise positive / high expression of MLC2v and / or CTNT.

46. The method of claim 45, wherein the mature cardiomyocytes comprise negative / low expression of MLC2a.

47. The method of any one of claims 41 to 46, wherein preparing the cellular composition for cardiac cell therapy from the plurality of cells comprises contacting the plurality of cells with a physiologically acceptable medium.

48. A method, comprising preparing a plurality of cells for cardiac cell therapy, wherein at least 95% of the plurality cells are characterized as having a molecular profile comprising one of the following: i) negative / low expression of one or more markers selected ANXA J, BGN, CCDC80, CDC42EP5, CDH11, CDON. CFH, COLIA2, COL3AI, COL5AI, CRIME CTSH, CXCLI2, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBN1, FLRT2, FN1, GATA3, GATA6, KRT19, LIMA1, LRPR2, LRRC17, MGP, MMP2, NUPRI, OGN, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNTI, and / or UPK3B, ii) negative / low expression of one or more markers selected from: ANXA1, CCDC80, CDON, CRIM1, COL3A1, CTSH, DCN, FLRT2, KRT19, LRPR2, NPNT, NUPRI, PLAC9, PODXL, POSTN, RDH10, SBSON, SI00AI0, S100A11, SPARC, TMEM88, TNNTI, and / or UPK3B, iii) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CD90, CDC42EP5, CFH, COL1A2, COL3A1, CXCL12, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBN1, FN1, GATA4, GATA6, HAND2, LRRC17, MGP, MMP2, NPNT, OGN, OSMR, PDGFRa, PLAC9, POSTN, PTX3, S100A10, S100A11, SLIT2, SPARC, TFPI2, TGFb, and / or THY], iv) negative / low expression of one or more markers selected from: ANXA1, BGN, CCDC80, CDH11, COL1A2, COL3A1, COL5A1, EVAIB, FBNI, LIMA1, MGP, MMP2, OGN, PLAC9, S100A10 v) negative / low expression of one or more markers selected from COL3A1, PODXL, and / or KRT19, vi) negative / low expression of one or more markers selected from KRT19 and / or PODXL, vii) negative / low expression of one or more markers selected from COL1A2 and / or FNL, and / or viii) negative / low expression of COL3A149. A method of preparing a plurality of mature cardiomyocytes for cardiac cell therapy, the method comprising contacting a plurality of mature cardiomyocytes that been determined to have a molecular profile characteristic of cardiomyocyte identity with a physiologically acceptable medium suitable for administration to a subject in need of cardiac cell therapy.

50. A method of preparing a batch of cells for cardiac cell therapy, the method comprising:(a) determining that an at least first batch of cells among a plurality of batches comprises a molecular profile characteristic of cardiomyocyte identity and;(b) preparing the at least first batch of cells for cardiac cell therapy.

51. The method of claim 50, wherein the molecular profile of the at least first batch of cells is obtained via flow cytometry.

52. The method of claim 51, wherein the molecular profile of the at least first batch of cells is obtained from a transcriptome of the batch of cells.

53. The method of claim 52, wherein the transcriptome is obtained via single cell ribonucleic acid sequencing (scRNA-Seq).

54. The method of any one of claims 50 to 53, wherein the molecular profile characteristic of cardiomyocyte identity comprises negative / low expression of one or more markers selected from: ANXAI, BGN, CCDC80, CDC42EP5, CDH11, CDON. CFH, COLIA2, COL3AI, COL5AI, CRIME CTSH, CXCLI2, DCN, ELN, EMILIN 1, EVAIB, FBLN2, FBLN5, FBN1, FLRT2, FN1, GATA3, GATA6, KRT19, LIMA1, LRPR2, LRRC17, MGP, MMP2, NUPRI, OGN, PLAC9, PODXL, RDH10, S100A10, S100A11, SBSON, SEZ6L2, SPARC, SPRY1, SULF1, TMEM88, TNNTI, and / or UPK3B.

55. The method of claim 54, wherein the molecular profile characteristic of cardiomyocyte identity comprises negative / low expression of one or more markers selected from: COL3A1, PODXL, and / or KRT19.

56. The method of any one of claims 50 to 55, wherein the at least first batch of cells comprises mature cardiomyocytes.

57. The method of claim 56, wherein the mature cardiomyocytes are derived from pluripotent stem cells.

58. The method of claim 57, wherein the pluripotent stem cells express one or more of the following markers: SSCC3 / 4, TRA-1-60, OCT3 / 4, NANOG, SOX2.

59. The method of claim 56, wherein the mature cardiomyocytes are derived from embryonic stem cells.

60. The method of any one of claims 50 to 59, wherein the mature cardiomyocytes comprise positive / high expression of MLC2v n !CTNT.

61. The method of claim 60, wherein the mature cardiomyocytes further comprise negative / low expression of MLC2a.

62. The method of any one of claims 50 to 61, wherein preparing the mature cardiomyocytes for cardiac cell therapy comprises contacting the mature cardiomyocytes with a physiologically acceptable medium suitable for administration to a subject.

63. A method, comprising: determining whether a plurality of cells comprises a molecular profile characteristic of cardiomyocyte identity and; if the plurality of cells comprises a molecular profile characteristic of cardiomyocyte identity, preparing the plurality of cells for cardiac cell therapy.

64. A method of determining cardiomyocyte identity of a cell, the method comprising determining the level of COL3A1 in the cell.

65. A method of treating a subject in need of cardiac cell therapy, the method comprising administering to the subject a cardiac cell therapy comprising the composition of any one of claims 1 to 10, a composition prepared according to the method of any one of claims 11 to 49, or a batch of cells prepared according to the method of any one of claims 50 to 62.

66. A method of treating a subject in need of cardiac cell therapy, the method comprising:(a) determining that no more than 50% of cardiomyocytes in a batch of cells comprise a molecular profile characteristic of a non-cardiomyocyte identity; and(b) administering the batch of cells or a portion thereof to the subject.

67. A method of treating a subject in need of cardiac cell therapy, the method comprising administering a plurality of cells to a subject, wherein at least 50% of cells in the plurality of cells has been determined to have a molecular profile characteristic of cardiomyocyte identity.

68. A method of treating a subject in need of cardiac cell therapy, the method comprising administering a plurality of cells to a subject, wherein at least 50% of cells in the plurality of cells have a molecular profile characteristic of cardiomyocyte identity.

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